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AbstractThe aim of this study was to assess the

efficiency and practicality of combined burrowdestruction and removal of chenopods in thecontrol of a zoonotic cutaneous leishmaniasis(ZCL) focus. Karameh and Sweimeh, locatedin the southern Jordan Valley, are stable endemicfoci for ZCL. Control measures have beeninitiated in Karameh and were maintained for 2years. Sweimeh was used as the control. FiftyPsammomys obesus and 6 Meriones tristamiwere captured from various colonies. Chenopodswere uprooted and burrows destroyed to a depthof 0.5 m to 1 m within a perimeter of 2 km fromKarameh. However, man-made changes to thetopography have made the application of controlmeasures more difficult. The effect of controlwas assessed through the comparison ofleishmanin skin test (LST) positivity in childrenbelow the age of 6 years, from both foci, priorand post control. There was a significantreduction in the frequency of LST positivity afterthe application of control measures from 19.9%to 4.4% in the test focus in children aged 3

months to 2 years, compared to a nonsignificantreduction of 51.1% to 41.9% in the control focus.It was concluded that control measures bydestruction of burrows were effective in protectingcommunities from contracting ZCL due toLeishmania major.

Publications1. Kamhawi S, et al. Environmental manipulationin the control of a zoonotic cutaneous leishmaniasisfocus. Archs. Inst. Pasteur Tunis, 1993, 70 (3-4):383-390.2. Janini R, et al. Incrimination of Phlebotomuspapatasi as vector of Leishmania major in thesouthern Jordan Valley. Med Vet Entomol, 1995,9(4):420-422.3. Arbaji AK, Gradoni L, Gramiccia M. Leishmaninskin test survey in a focus of high endemicity ofLeishmania major in Jordan. Acta Trop, 1993,54(1):77-79.

BackgroundZoonotic cutaneous leishmaniasis (ZCL)

is the most prevalent and onlyepidemiologically defined form ofleishmaniasis in Jordan. The arid, semi-arid biotope of ZCL, characterized by plantsof the family chenopodiaceae, coversaround 75% of Jordan's territory, however,its endemicity varies in different locationsdepending on ecological, environmentaland socio-economic factors. The southernJordan Valley is well known for itshyperendemicity for ZCL. Leishmania majorwas isolated and characterized from bothhuman cases and Psammomys obesus.Phlebotomus papatasi was incriminated asthe vector of ZCL in Jordan. In view of thehigh endemicity of ZCL in certain areas ofJordan and the limited application of controlmeasures, the present study was carriedout to assess the efficiency of controlmeasures, through the destruction of rodentburrows in combination with the clearanceof chenopods, on minimizing the effect ofZCL in a contained area.

Materials and methodsKarameh and Sweimeh, in the southern

Jordan Valley, were chosen as the study

This study reported the success of control measures in protectingcommunities from contracting zoonotic cutaneous leishmaniasis(ZCL) due to Leishmania major by the clearance of a zone of 1.4-2 km around it from chenopods and P. obesus burrows.

The clearance of chenopods is costly and ineffective as theyregrow rapidly in the absence of a deterrent. Therefore, it is notrecommended as part of the control measures, which includes thedestruction of burrows. The latter seemes sufficient to prevent P.obesus from largely recolonizing the cleared zone. This behaviourof P. obesus to withhold from colonizing cleared zones deservesto be studied on a wider scale.

Taking into consideration the cost of Pentostam treatment, thecost of control measures is reasonable when carried out to protecta non-exposed target population.

Sustainability of control measures in endemic foci and theirfinancial feasibility depend largely on the involvement of thecommunity and the integration of local health services to ensurethe low cost and success of these measures.

Cutaneous LeishmaniasisJ o r d a n

Conclusions and implications ofthe study

ControlEvaluation of the

physical destructionof burrows in the

control of zoonoticcutaneous

leishmaniasis

Jordan Karameh and Sweimeh,southern Jordan Valley

Study period:April 1992–1994

Small GrantsScheme

(SGS) 1992 No.16

Principal InvestigatorDr Shaden Kamhawi

Department of BiologicalSciences

Yarmouk UniversityIrbid, Jordan

area as they fall within the same environmental andecological zones and are stable endemic foci for ZCL.Karameh was the test focus, it has a population of8500 and is situated east of the River Jordan. To thewest of the town there is a large farming area consistingof plots of planted crops irrigated by drip pipes formost of the year. Water outlets and small hills wereconstructed around the plots to drain water comingfrom the mountains during the winter and to drain theexcess irrigation water to protect the land from flooding.Numerous rodent burrows were observed near houseson the western side of the town and in the farmingarea, the majority being located at the top edges ofthe water outlets and hills surrounding the plots. Thecontrol measures were applied in an area extending2 km to the south, north and west of Karameh. Eightof the water outlets fall within this area in the west,while the east is a dry mountainous desert whereburrows are absent and chenopods are scarce andscattered. Sweimeh, the control town, has a populationof 2500 and has been planted with grains.

Passive case detection The number of ZCLcases registered in the health centres of Karameh andSweimeh before and after the implementation of thecontrol measures was recorded. The physicians wereinformed about the purpose of the study.

Leishmanin skin test A randomly selected sampleof preschool children under 6 years of age werescreened from both foci for past exposure to L. major.Post-control evaluation was performed on childrenborn during the control period (2 years), i.e on childrenaged 3 months to 2 years.

Application of control measures in KaramehThese measures consisted of the clearance ofchenopods and the physical destruction of burrows.Clearing the chenopds was performed manually fromwater outlets and from areas inaccessible to machines.All 7 water outlets, apart from the one furthest north,were removed. The tractor removed the rest of thechenopods, as well as colonies, by ploughing. Thephysical destruction of burrows was performed bytractors, ploughing, and by smoking in all areas withburrows inaccessible to ploughing. Although it is knownthat P. obesus burrows to a depth of 0.5 m, the softnature of the soil and the hilly topography of manysites permitted it to reach a depth of 1 m, which addedto the difficulty in the destruction of the burrows.

Main study findings

Passive case detection A total of 90 cases ofZCL from Karameh and 3 from Sweimeh were reportedfollowing the transmission season of 1992. They wereconfirmed either parasitologically or by LST and werechildren or non-immune adults.

Baseline survey by Leishmanin skin testThree hundred and sixty nine out of 1793 childrenunder 6 years of age were screened, and thepercentage of positive cases accounted for 33% ofchildren in Karameh compared to 80.2% in Sweimeh.The mapping of Karameh resulted in the identificationof several species of chenopods including: Atriplexhalimus, Suaeda vermiculata, S. fruticosa, S. aegypticaand Aellenia lancifolia. Atriplex halimus was widespreadand grew to unnatural tree-like proportions with strongdeep roots. The rest of the chenopods were abundant,forming a carpet-like cover, sometimes growingamongst crops, necessitating their clearance by handas to not damage the crops.

Regarding the captured rodents, 50 P. obesus and6 Meriones tristrami were identified, indicating therelative abundance of P. obesus in the study areacompared to other species. Eleven of the P. obesuswere mature, one had an active lesion and 2 borescars. The lesions were located on the ears, tail andnose.

Evaluation of control activities and theircost The number of reported cases was reduced to20 and 6 in 1993 and 1994, respectively, indicatingthe success of the control activities in reducing thedisease prevalence, though transmission was stillpossible through sandflies coming from areas whereno control activities were performed. There was asignificant reduction in the frequency of LST positivityafter the application of control measures from 19.9%to 4.4% in the test focus in children aged 3 months to2 years, compared to a nonsignificant reduction of51.1% to 41.9% in the control focus. The success ofcontrol measures was also assessed by the lowpercentage of leishmanin conversion in initially testednegative children in Karameh compared to Sweimeh(9.8% compared to 75.9%, respectively). Interestingly,there were no attempts by P. obesus to recolonizethe cleared zones.

Preliminary assessment of the cost of controlmeasures indicated that they could be reduced if thelocal health authorities provide training and supervision.In addition, LST was found to be a costly techniquefor the evaluation of control measures.

In conclusion, Karameh presented a focus whichclearly outlines the damaging effect man-made changesof a biotope can have on increasing the level of infectionwith ZCL. This study also provided an opportunity tothoroughly study a stable hyperendemic area of ZCLin Jordan.

The clearance of chenopods was found to be costlyand ineffective as they regrow rapidly in the absenceof a deterrent. Therefore, it was not recommended aspart of control measures, which includes the destructionof burrows. The latter seemed sufficient to prevent P.obesus from largely recolonizing the cleared one.

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AbstractThis study was conducted in order to

evaluate the impact of the modification of thebiotope of the reservoir of zoonotic cutaneousleishmaniasis (ZCL) on the transmission ofthe parasite. Different control methods wereapplied in the intervention focus (Sidi Bouzid),and the impact of intervention on diseasetransmission was evaluated in comparison toa control focus (Felta). Control activitiesconsisted of the destruction of the reservoirburrows, as well as sanitation measures toeliminate breeding places for the phlebotominesandfly vectors.

Results A surveillance system for ZCL wasimplemented through Geographic InformationSystem (GIS) software. The interventionresulted in a significant decrease in theincidence of the disease, from 1618 ZCLcases in 1991 to 339 cases in 1993. Thedecrease in incidence was also observed inprimary schools. The intervention also resultedin a decrease in the densities of the vector inthe intervention focus in comparison to thecontrol one. The squash blot technique, using2 DNA probes specific to Phlebotomuspapatasi and L. major, proved to be usefulfor the monthly follow-up of sandfly infectionrates when compared to the dissectiontechnique. There was no significant differencebetween the 2 foci regarding the populationdensities of the reservoir, Meriones shawi, asno control measures were undertakenregarding this reservoir.

Regarding the Psammomys obesuspopulation, no recolonization of the zonesoccurred in the ploughed areas. Surprisingly,the P. obesus populations disappeared in the2 foci. And there was no difference in theinfection rates between the 2 foci.

Conclusion Ecological modifications of thereservoir host's biotope had a significantimpact on disease transmission and populationdensity of the vector. On the other hand, itsimpact on the reservoir could not be evaluateddue to unexplained decrease in the reservoirpopulation in both the intervention and controlfoci.

Multicentre Collaboration• Pasteur Institute, Tunis• Governorate Council of Sidi Bouzid• Regional Division of Public Health,

and Basic Healthcare Service• Ministry of Agriculture• Ministry of Habitat and Equipment• Municipality and Public Health sector• Community participation

TrainingThe squash blot technique using DNA

probes was introduced in the Laboratoryof Epidemiology and Ecology of ParasiticDiseases of the Institute Pasteur de Tunisfor the monthly follow-up of sandflyinfection rates.

BackgroundMore than 40 000 cases of zoonotic

cutaneous leishmaniasis (ZCL) due toLeishmania major were diagnosed incentral Tunisia during the period 1983-1992. Therefore, the control of thedisease was one of the most importantpublic health priorities in the country. TheSidi Bouzid governorate was the mostaffected area, with more than 27 000recorded cases. The causative agentwas L. major; Phlebotomus papatasi wasincriminated as the vector, andPsammomys obesus followed byMeriones shawi were the reservoir hostsof the parasite. Accordingly, the controlmethods based on eco log ica lmodifications of the reservoir host's

This study reported the success of control measures in protectingcommunities from contracting zoonotic cutaneous leishmaniasis(ZCL) due to Leishmania major by the clearance of a zone of 2km around an endemic focus from chenopods and P. obesusburrows. This was demonstrated by the significant reduction inthe number of cases reported.

GIS is an ideal tool for identifying geographic clusters of diseaseand is recommended for disease surveillance in endemic countries.

The squash blot technique using two DNA probes proved to bemore reliable in the follow-up of sandfly infection rates in comparisonto the dissection technique

The results of this project indicate the possible existence of anunknown reservoir such as Rattus rattus in Sidi Bouzid and thepossible anthroponotic transmission of L. major.

Cutaneous LeishmaniasisT u n i s i a


ControlEnvironmental

changes to controlLeishmania major

cutaneousleishmaniasis in theepidemic focus of

Sidi Bouzid (Tunisia).Evaluation of the

impact of themodification of the

reservoir host'sbiotope on the

transmission of theparasite

TunisiaSidi Bouzid

Study period:June 1992–

December 1993

Small GrantsScheme

(SGS) 1992 No. 21

Principal InvestigatorDr Riadh Ben-Ismail

Laboratoire d'Epidemiologie etD'Ecologie Parasitaire

Institut Pasteur de TunisTunis, La Tunisie

biotope were proposed to control transmission in SidiBouzid. This study was conducted in order to evaluatethe impact of the modification of the reservoir host'sbiotope on the transmission of the parasite.

Materials and methodsDifferent control methods were applied in the

intervention focus (Sidi Bouzid), and the impact ofthe intervention on disease transmission wasevaluated in comparison to a control focus (Felta)where no control measures were applied.

Control activities Control activities included thedestruction of Psammomys obesus burrows bymechanical ploughing of a total area of 529 hectaresin the northern periphery of Sidi Bouzid town, coveringa zone of 2 km radius around the town. In order toavoid desertification, the regrowth of the chenopodsand the recolonization by P. obesus of the area,different species of trees, selected because of theiradaptability in poor and sandy soils, were planted inthe ploughed zone. These trees included Acaciacyanophylla (625 trees planted on 1 hectare),casuarina trees (15 000 trees planted on 14 hectares);jojoba trees (9325 trees on 0.5 hectare) with itseconomic importance as source of oil for airplaneengines were recently introduced; Eucalyptus torkota(1000 trees) was also planted, but was not successfuland was replaced by more acacias.

Other control activities were the salubrity andsanitation measures that consisted of cleaning theformer garbage outlet of Sidi Bouzid located withinthe area colonized by P. obesus and that constitutedan important potential breeding place for phlebotominesandflies.

Community participation In order to promotethe programme, the planting of the trees started on8 November 1992, which is the Feast of the TreeDay in Tunisia. This allowed the collaboration betweenlocal authorities, project participants and the wholecommunity. Health education sessions were alsoheld on that day.

Main study findingsImpact of intervention The force of infection(number of infecting bites per 1000 individuals)following each transmission season till 1991 wascalculated. A new census was carried out in thecontrol focus to update the database and locate thedwellings on aerial photographs. A surveillancesystem was implemented in the area's basic healthcentre to collect data related to ZCL (cases, newborns,deaths, emigration and immigration from the focus).A new database program was prepared usingORACLE software, followed by its introduction in aGeographic Information System (GIS), which is anideal tool for identifying geographic clusters of diseaseand analysing spatial relationships between diseaseand risk factors. From 1983 to 1992, the annualincidence of ZCL among susceptible people in thecontrol focus ranged from 0.04% to 11.2%, withmaximum peaks in 1985 and 1987. The force of

infection varied from 0.004 to 0.024. The controlfocus was hypoendemic during all the studied years,except in 1985 and 1987 where it was mesoendemic.These data showed the limits of the epidemicparameters when applied on human populationsamples residing in different geographical areasdefined by administrative levels. The introduction ofthe GIS allowed for the clearly distinction of areasthat are hyperendemic inside the sector of Felta (thecontrol focus).

The intervention resulted in a significant decreasein the incidence of the disease from 1618 ZCL casesin 1991 to 339 cases at the end of the study in 1993.The decrease in incidence was also observed inprimary schools where an active case detection wasorganized in October of each year during the period1991-1993.

Monthly measurement of population densities ofP. papatasi was determined by the sticky traps methodand compared in the 2 foci in 9 stations, 4 in SidiBouzid and 5 in Felta, during the transmission seasonsin 1992 and 1993. The intervention resulted in adecrease in the densities of the vector in theintervention focus in comparison to the control one.Regarding the vector infection rates, the squash blottechnique using two DNA probes specific to P.papatasi and L. major proved to be more useful incalculating the monthly follow-up sandfly infectionrates compared to the dissection technique. A totalof 2638 sandflies were screened by this technique.

The estimation of the population size of Merionesshawi was carried out by the tracking index methodbased on monthly measurements of rodent traceson fixed surfaces near rodent burrows. There wasno significant difference between the 2 foci regardingthe population densities of this reservoir as no controlmeasures were undertaken.

Regarding the P. obesus population, the estimationwas based on monthly estimates of the number ofactive burrows per hectare in 5 zones of 1 hectarein each focus. The results indicate that norecolonization of the zones occurred in the ploughedareas. Surprisingly, the P. obesus populationsdisappeared in the two foci, an unexplainedphenomenon that has been previously reported fromother ZCL foci in Tunisia. The infection rates of P.obesus were determined in the 2 foci, by inoculatingthe parasites from rodents' ears into BALB/c miceThere was no difference in the infection rates betweenthe 2 foci.

Conclusions and recommendationsEcological modifications of the reservoir host's biotopehad a significant impact on disease transmission andpopulation density of the vector. On the other hand,its impact on the reservoir could not be evaluateddue to unexplained decrease in the reservoirpopulation in both the intervention and control foci.Two hypotheses were postulated and recommendedfor testing: the possible existence of an unknownreservoir such as Rattus rattus in Sidi Bouzid, andthe possibility of anthroponotic transmission of L.major.

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AbstractThe study was conducted in endemic

localized cutaneous leishmaniasis (LCL) fociin south Tunisia namely, Dhibet and Rémada.The objectives of the study were to estimatethe proportion of infected children living in anendemic zone, to estimate the proportion ofnew cases of LCL among immune and non-immune children before the transmissionseason, and to determine the incidence ofsubclinical infections after the transmissionseason. Collected information includedsociodemographic variables, ecological data,spatial distribution and risk factors of LCL.Children were also subjected to clinicalexamination and leishmanin skin testing (LST)before and after the transmission season.

Results There was excellent concordancebetween the results obtained by LST and theproliferative response of the in vitro test ofcellular immunity. The proportion of positivitywas 38.5% and 51.1% in 5-15 year oldchildren and scars were detected in 76.6%and 87.5% of positive individuals in Rémadaand Dhibet, respectively, in comparison withless than 25% of LST-negative individuals.During the transmission season, surveillance

revealed that there was a significantly higherproportion of initially negative individuals whocontracted LCL infection in Rémada comparedto Dhibet. Furthermore, there was asignificantly higher proportion of LST positivityfrom a second screening test performed afterthe transmission season in Rémada comparedto Dhibet. The positivity of the test in theabsence of the characteristic LCL scars wassuggestive of subclinical infections. Thedifference between the 2 foci was also evidentconcerning the diameter of induration. Theproportion of subclinical infections was alsohigher in Dhibet than in Rémada.

Conclusion In endemic zones with lowparasite transmission, the population willacquire immunity over seasons with or withoutpatent disease. In this situation, the diseasewill affect mainly the non-immune, i.e. youngerindividuals and new settlers. The differencein LST results in different endemic foci couldbe explained by the difference in the immunereaction according to the size and number oflesions, as several infections are needed toacquire immune response to infection.

PublicationsSassi A et al. Leishmanin skin test

lymphoproliferative responses and cytokineproduction after symptomatic or asymptomaticLeishmania major infection in Tunisia. Clin ExpImmunol, 1999, 116(1):127-132.

BackgroundLocalized cutaneous leishmaniasis

(LCL) in Tunisia is caused by theLeishmania major zymodeme MON25(LON1). Field studies had revealed thatasymptomatic infection may occur inendemic areas. Although the extent ofthis phenomenon is not fully evaluated,people without a definite history of thedisease may have evidence of infectionas demonstrated by a positive delayedhypersensitivity reaction assessed by theleishmanin skin test (LST). This test isused to assess the prevalence ofle ishmania in fect ion in humanpopulations. Furthermore, epidemiologicalstudies indicate that individuals with

There was excellent concordance between the results obtainedby leishmanin skin test (LST) and the proliferative response of thein vitro test of cellular immunity.

The proportion of positivity was 38.5% and 51.1% of children,and scars were detected in 76.6% and 87.5% of LST-positivechildren in Rémada and Dhibet, respectively, in comparison withless than 25% of LST-negative individuals. Patent disease wasreported in half of schoolchildren in Rémada compared to 11.3%in Dhibet.

The positivity of the test in the absence of the characteristic CLscars is suggestive of subclinical infections.

In endemic zones with low parasite transmission, the populationwill acquire immunity over seasons with or without patent disease.In this situation, the disease will affect mainly non-immune individuals.

The difference in LST results in different endemic foci could beexplained by the difference in the immune reaction according tothe size and number of lesions, as several infections are neededto acquire immune response to infection.

Cutaneous LeishmaniasisT u n i s i a


Diagnosis

The predictivevalidity of

leishmanin skintesting in the

assessment of therisk of developing

lesion versusinfraclinic infectionby Leishmania major

TunisiaSouth Tunisia

Study period:March 1997–1999

Small GrantsScheme

(SGS) 1996 No. 28

Principal InvestigatorDr Mohamed Hechmi Louzir

Laboratoire D'ImmunologieInstitut Pasteur de Tunis

Tunis, La Tunisie

previous active cutaneous leishmaniasis or subclinicalinfection are usually resistant to a subsequent clinicalinfection.

The objectives of the study were to estimate theproportion of infected children living in an endemiczone, to estimate the proportion of new cases of LCLamong immune and non-immune children before thetransmission season, and to determine the incidenceof subclinical infections after the transmission season.

Materials and methodsThe study was conducted in 3 endemic LCL foci

in south Tunisia, namely: El-Guettar, Dhibet andRémada. All children aged between 2 and 11 yearswere included in the study. Collected informationincluded: sociodemographic variables, ecologicaldata, spatial distribution and risk factors of LCL.Children were also subjected to clinical examination.After the transmission season (March 1997), anotherclinical examination was performed in order todetermine new cases of LCL.

LST was performed by injecting 0.1 ml leishmaninin the forearm and evaluating the response 72 hourslater. A study of the concordance between the LSTand tests of cellular immunity (in vitro) was performedon young adults (18-20 year-olds) as well asindividuals who previously tested positive by LST inan earlier study.

Main study findingsThere was excellent concordance between the

results obtained by LST and the proliferative responseof the in vitro test of cellular immunity. A total of 595schoolchildren aged 5-15 years were enrolled beforethe transmission season in Rémada and Dhibet andwere examined for the LCL characteristic scars, thentested with LST. Considering induration with adiameter greater than or equal to 5 mm as a positiveresponse, and 2 to less than 5 mm as the intermediateresponse (LST-interm), the proportion of positivitywas 38.5% and 51.1% in Rémada and Dhibet,respectively. Positivity increased with age to reach60% at 14 years of age.

The LCL scar was detected in 76.6% and 87.5%of positive individuals in Rémada and Dhibet,respectively, in comparison with less than 25% ofLST-negative individuals. Interestingly, in the LST-interm group, a proportion midway between negativeand positive individuals recorded the characteristicscar (60%).

New cases were detected during biweekly schoolvisits. Diagnosis was confirmed by Giemsa stainingand culture of the parasite on NNN medium. Half ofthe schoolchildren in Rémada were affected comparedto 11.3% in Dhibet.

During the transmission season of 1997-1998,surveillance revealed that 76% of individuals initiallyLST-negative contracted LCL infection compared to

17.5% and 44% of individuals initially LST-positiveand LST-interm, respectively. In Dhibet, 18% ofinitially LST-negative individuals contracted theinfection compared to 5.2% of initially LST-positiveindividuals. The relative risks of contracting LCLinfection in LST-positive individuals revealed aprotective effect in comparison with LST-negativeindividuals.

The positivity of the test in the absence of thecharacteristic CL scars are suggestive of subclinicalinfections. However, minimal lesions could be healedwithout leaving scars. In order to test the hypothesisof the presence of subclinical infections, a secondLST was performed on all individuals. In Rémada,almost all individuals who developed the diseasebecame LST-positive, but among those who wereinitially LST-negative, more than 38% became LST-positive and 12.3% LST-interm, indicating that theseindividuals developed subclinical infections. On theother hand, those with initial LST-interm reactionsdeveloped a positive reaction with or without overtclinical disease.

By contrast, 18.2% of those initially negativebecame positive during the second LST screeningin Dhibet. These differences between the 2 foci couldbe explained by a greater immune reaction inRémada's patients as compared to Dhibet's patients,as the lesions in the latter tended to be more frequent,solitary and smaller in size than in the former focus.Another explanation was that several infections areneeded to acquire immune response to infection andthat only one will develop into patent infection mainlydue to a certain critical number of the parasite. InDhibet the proportion of subclinical infections detectedwas comparable to that of patent infection whichexceeded the proportion detected in Rémada,indicating that in foci with low parasite transmissionthe proportion of subclincal infections is higher thanin those with high parasite transmission. This latterobservation indicates that in endemic zones with lowparasite transmission, the population will acquireimmunity over seasons with or without patent diseasethat will be mostly benign (solitary lesions and ofsmall size) and that are frequently missed, and thisimmunity will be associated with resistance toreinfection during epidemics. In this situation, thedisease will affect mainly non-immune, i.e. younger,individuals and new settlers.

The difference between the 2 foci was also evidentconcerning the diameter of induration: In Rémadathe diameter of induration increased after the secondLST, while in Dhibet the increase in induration wasless significant in those initially negative, and almostnil in previously positive or diseased individuals.

The second LST tests revealed that, between May1997 and 1998, 90.3% compared to 56.4% ofschoolchildren in Rémada and Dhibet, respectively,became immune against LCL.

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AbstractThe ep idemio logy o f cu taneous

leishmaniasis (CL) was investigated in Jericho,Jordan Valley, during the period 1994-2001.Clinically suspected patients referred during1994-1999 were interviewed and subjectedto laboratory diagnosis, and plotted on twomaps for the Jordan Valley and Jericho city.This was coupled with a seroprevalencesurvey in the area (2000-2001).

A case-control phase was also conductedin 2000-2001 whereby the householdmembers of patients from Jericho city andAqbat-Jaber refugee camp (139 individuals)were compared to 108 randomly selectedindividuals living in their neighborhood. Studysubjects were interviewed regarding thedeterminants of infection.

Results More than half (52%) of suspectedand referred patients were from Jericho city,followed by Aqbat-Jaber (15%), then otherareas in the valley.

Case mapping within the Jericho cityboundaries showed clustering of cases in thesouth-eastern parts of its periphery. Thedistribution of cases in Aqbat-Jaber campwas comparable to that of the city.

Transmission of infection was associated

with periods of high rainfall, peaking betweenOctober to January.

During the period 2000-2001, a total of 471suspected individuals were referred fordiagnosis, out of whom 32% proved to bepositive by direct smear diagnosis. Thesewere mainly children, young Palestiniansoldiers camping in Jericho's outskirts, andbedouins' and farmers' wives.

The seroprevalence of CL in Jericho cityand its adjacent Aqbat-Jaber refugee campwas 26.3%.

The education level of the head of the familyand children sleeping unders nets were theonly significant determinants of infection.

Species identification showed that Leishmaniamajor coexists with L. tropica in the JerichoDistrict, Jordan Valley.

BackgroundJericho in the Jordan Valley is the

centre of a region where cutaneousleishmaniasis (CL), or "Jericho boil",caused by Leishmania major, is endemic.CL in the Jordan Valley is a vector-bornezoonotic disease, with the sand rat,Psammomys obesus, as the animalreservoir, and sandflies, Phlebotomuspapatasi, as the vector. These sandflieslive in rodent burrows in very close contactwith the sand rats. Humans are onlyincidentally infected, acting as secondaryhosts in the case of L. major. However,other species, such as L. tropica, areanthroponotic, where humans are theprimary hosts and reservoirs for newhuman infections of CL.

This study was funded in 2000 tocomplete an earlier study initiated in 1994,aiming to determine the prevalence ofCL in this area, study its risk factors, mapcases and develop a database for aDistrict Database for the Ministry ofHealth.

Materials and methods

Local database (1994-1999)Suspected referred patients were

The seroprevalence of CL in Jericho city and its adjacent Aqbat-Jaber refugee camp amounted to 26.3%.

Most of the cases exist in Jericho city and Aqbat-Jaber camp.

Cases are clustered in the south-eastern parts of the city'speriphery.

Transmission of infection is mainly during the period of Octoberto January and is associated with rainfall.

Vulnerable groups are children, young Palestinian soldierscamping in Jericho's outskirts, and bedouins' and farmers' wives.The education of the head of the family and children sleepingunder nets are the significant determinants of infection.

This study reported the coexistence of L. major together with L.tropica in the Jericho District, Jordan Valley.

A control programme based on the information provided by thisstudy is highly recommended.

Cutaneous LeishmaniasisP a l e s t i n e


Epidemiology

Epidemiology ofcutaneous

leishmaniasis in thePalestinian district

of Jericho

PalestineJericho

Study period:1994–2001

Small GrantsScheme

(SGS) 2000 No. 18

Principal InvestigatorDr Amer Omar Jawabreh

Ministry of Health,Nablus, Palestine

e-mail: [email protected]

interviewed according to a questionnaire andsubjected to diagnostic tests. The questionnaireincluded information regarding demographic data,clinical features, risk factors, laboratory diagnosis,and treatment details. Diagnosis was performed bydirect tissue smear, culture on NNN media and, onrare occasions, by histopathology.

Case-mapping (1994-1999) All suspectedreferred patients were plotted on two maps for theJordan Valley and Jericho city.

Seroprevalence study (2000-2001) Bloodsamples were collected and the CL seroprevalencewas determined using the ELISA method.

Case-control phase (2000-2001) Casesconsisted of the household members of CL patientsfrom Jericho city and Aqbat-Jaber refugee camp,totalling 139 individuals. The control group included108 individuals from 18 households, who wererandomly selected from both the city and the camp.Study subjects were interviewed regarding thedeterminants of infection such as sociodemographicfactors, presence of a nearby farm, breeding placesin the vicinity, air-conditioning use, types of roofs andwalls of the houses, cracks in walls, window screening,presence of pets, types of trees, outdoor sleepingpatterns, lifestyles such as having evening tea inyards, and children sleeping under nets.

Main study findingsDuring the period 1994-1999, a total of 114 patients

were diagnosed in the Jordan Valley. More than halfof the patients were from Jericho city (52%), followedby Aqbat-Jaber (15%), A'uja village (13%), Ein-Assultan area (6%) and Zubaidat (5%). These areasshare the common feature of neighbouringagriculturally active areas. These cases were mappedat two levels: the Jericho District level, and the cityand refugee camp level. Case-mapping within thecity boundaries showed clustering of cases. Thirtycases (45%) were clustered in the south-easternparts of the city periphery. Moreover, the distributionof cases in Aqbat-Jaber camp was comparable tothat of the city.

The peak of infection was in 1995 (31% of allcases), which coincided with the highest period ofrainfall. Moreover, about 57% of patients werediagnosed from October to January, indicating aseasonal pattern of transmission, though some casespersisted through the whole year.

During the study period, 152, out of 471 suspectedindividuals referred for diagnosis, were proved to bepositive by direct smear diagnosis (32%). More thathalf (57%) of the referred individuals were childrenunder 15 years old, of whom 32% were infected, and

63% of the infected children were under 5 years old.There was no significant difference between malesand females regarding the frequency of infectionamong clinically suspected individuals (32.6% and31.7%, respectively).

The occupations of referred adults were as follows:51% were Palestinian soldiers (20-29 years old)camping in Jericho's outskirts following the 1994"Oslo Agreement", while 13% were housewives,belonging to either bedouin or farming families.

The serosurvey conducted in Jericho city and itsadjacent Aqbat-Jaber refugee camp showed aseroprevalence of 26.3%. ELISA positive casesbelonged to 3 groups: 67% of smear-positiveindividuals, 42.9% of suspected cases (recent andold infections), and 17% of healthy individuals (noevidence of infection).

ELISA revealed a sensitivity of 67%, a positivepredictive value of 55%, a specificity of 85%, with anegative predictive value of 90%.

Clinical features More that half of the CL confirmedcases had single lesions, 22% had two lesions and22% had 3 or more lesions. Lesions were mainly onthe head and neck (52%), upper limbs (28%) andlower limbs (19%). Head lesions were mainly on thecheek or nose, and less frequently on the forehead;these sites are significantly at higher risk of gettingCL in children than in adults.

In children, the head is more frequently (61%) thesite of infection of CL than are other body sites,whereas sites of infection on adults are most frequently(78%) on the limbs. This indicates a significantassociation between age and the site of lesions.

Determinants of infection Among severalpotential risk factors for infection, the only significantdeterminants were the education of the head of thefamily and children sleeping under nets.

L. tropica in the Jericho District L. tropicawas first isolated in the Jordan Valley in the summerof 2000 and was not previously reported in the JerichoDistrict, Jordan Valley. Unlike L. major, which wasrestricted to the flat alluvial soil of the Jordan Valley,L. tropica existed in the rocky areas in the northernand southern parts of the Jericho District, JordanValley, with the sole exception of one isolate fromthe hilly areas close to Jericho city.Isolates were characterized by excreted factor (EF)serotyping and polymerase chain reaction (PCR)-based techniques.

10 11

Cutan

eous

Leish

mania

sis

AbstractThe dynamic of the leishmania life cycle in

human-infected macrophages could explainthe differences in Leishmania tropica and L.major infection in humans and could explainthe reasons for non-response to treatment.This project was conducted to study thedynamic of L. tropica and L. major amastigotegrowth within macrophages in humancutaneous leishmaniasis.

Six groups of healthy and infected subjectswith L. major or L. tropica, susceptible orresistant to treatment, were enrolled in thestudy. Peripheral blood mononuclear cells(PBMNs) were isolated from peripheral bloodby macrophages. The cells were counted,cultured, incubated in a CO2 incubator, thenexposed to metacyclic promastigotes of L.major or L. tropica. The intensity of the in vitroinfection was determined by the number ofamastigotes per 100 infected macrophagecells and the percentage of infectedmacrophage cells in different time-frames.The life cycle of the parasites was investigatedand compared among different groups.

Results There was no difference betweenL. tropica and L. major infection in human-infected macrophages. On the other hand,there was a significant difference betweenthe susceptible and resistant groups regardingthe number of infected macrophages as wellas the number of amastigotes permacrophage. The number of macrophagesand amastigotes were significantly higher inthe resistant groups in comparison to thesusceptible groups. The results also confirmedthat once the parasite is established insidethe host cell, it exhibits a life cycle consisting

of an initial stage where the proliferation ofthe parasite is inhibited for the first 3 to 4 daysafter penetrating the host cell. Proliferationresumes on the fifth day in culturedmacrophages, reaching a peak in the numberof intracellular parasite on day 7 of the in vitroinfection.

Collaborating Institutions

• Centre for Research and training in Skin Diseases and Leprosy, Tehran University of Medical Science

• School of Public Health, Tehran University of Medical Science

BackgroundPeritoneal macrophages infected in

vitro with L. donovani showed that oncethe parasite is established inside the hostcells, it exhibits a definite life cycleconsisting of a stage where theproliferation of the parasite is inhibitedduring the first 3-4 days after penetrationin the host cell, with proliferation resumedon the 5th day in the culturedmacrophages, reaching a peak on day7, with no significant difference in thenumber of amastigotes in resistant orsusceptible peritoneal macrophages 24hours after exposure to promastigotes[1]. Progression of the infection to initiallyuninvaded cells suggested that theinfection of previously unpenetrated cellsinvolved a "recruitment" into thesusceptible pool of cells due to action ofthe soluble factor produced in vivo [2]and in vitro [3] by infected macrophages.The intensity of the activation ofintramacrophages suggested that thesusceptible strain harboured moreparasites on day 7 than the peritonealcells of the resistant animals. This studywas conducted to investigate the dynamicof L. tropica and L. major amastigotegrowth within macrophages in humancutaneous leishmaniasis. The dynamicof the leishmania life cycle in human-infected macrophages could explain thedifferences in L. tropica and L. major

There is no difference between L. tropica and L. major infectionin human-infected macrophages. The only difference is betweenresistant or susceptible groups regarding the number of infectedmacrophages and the number of amastigotes per macrophage.

The life cycle of the parasite consists of an initial dormant stageof 3-4 days after infecting the cultured macrophages followed bya proliferation stage on the fifth day, then a peak in the seventhday of the in vitro infection.

Cutaneous LeishmaniasisI s l a m i c R e p u b l i c o f I r a n


Pathogenesis

The dynamic ofamastigote growthwithin macrophagesin human cutaneous

leishmaniasis

Islamic Republic ofIran

Tehran

Study period:July 1999–2001

Small GrantsScheme

(SGS) 1999 No. 16

Principal InvestigatorDr Zohreh Eslami

Center for Research andTraining in Skin Disease and

LeprosyTehran University of Medical

ScienceTehran, Islamic Republic of Iran

infection in humans and could explain the reasonsfor non-response to treatment.

Materials and methodsLife cycles of L. tropica and L. major were

investigated in healthy and infected patients. Sixgroups of 5 subjects each were selected for the study:Groups 1 and 2 consisted of healthy volunteers withno history of cutaneous leishmaniasis; Groups 3 and4 included subjects infected with L. tropica and L.major, respectively, resistant to treatment; Groups 5and 6 included subjects infected with L. tropica andL. major, respectively, susceptible to treatment.

The blood samples were taken from the patientswhen they were referred to the Centre for Researchand Training in Skin Diseases and Leprosy. PeripheralBlood Mononuclear Cells (PBMNs) were isolatedfrom peripheral blood by Ficoll gradient centrifugationand the macrophages were isolated. The cells werecounted by a haemocytometer and the number ofviable cells was determined by trypan blue. Onehundred microliter aliquots of cell suspensions (107

viable cells/ml) was placed on 18X 18 mm coverslipsand each coverslip was placed in a well of 6-wellLinbro culture plate.

The cells were incubated in CO2 incubator for 2hours prior to exposure, then the cells were exposedto 10 metacyclic promastigotes of L.major or L.tropica per each macrophage. The intensity of in vitroinfection was determined by both the number ofamastigotes per 100 infected macrophages and aspercentage of infected macrophages during certainperiod of time (0,1,3,5,7,10, 14 days). The life cycleof the parasite was investigated and compared amongdifferent groups. Statistical analysis was performedin order to compare the difference in the number ofinfected macrophages and number of amastigotesper macrophage in time frame of infection within eachgroup as well as between the different groups.

Main study findingsThere was no significant difference in the number

of infected macrophages and the number ofamastigotes per macrophage, neither between the2 healthy groups nor groups 3 and 4 resistant totreatment, nor groups 5 and 6 susceptible to treatment.On the other hand, there was a significant differencebetween the susceptible and resistant groupsregarding the number of infected macrophages aswell as the number of amastigote per macrophage.The number of macrophages and amastigotes weresignificantly higher in the resistant groups (3 and 4)in comparison to the susceptible groups. The resultsalso confirmed that once the parasite is establishedinside the host cell, it exhibits a life cycle consistingof an initial stage, where the proliferation of theparasite is inhibited for the first 3 to 4 days after

penetrating the host cell. Proliferation resumes thefifth day in cultured macrophages, reaching a peakin the number of intracellular parasite on day 7 of thein vitro infection.

References[1] Eslami Z, Tanner CE. Time course and intensity

of infection in vitro in the resident peritonealmacrophages of resistant and susceptible miceexposed to different doses of Leishmania donovanipromastigotes. Int J Parasitol,1994, 24(5):743-747.

[2] Evan TG, Smith D, Pearson RD. Humoral factorsand non specific immune suppression in Syrianhamsters infected with Leishmania donovani. JParasitology, 1990, 76: 212-217.

[3] Moore K, Turco SJ, Matlashewski G. Leishmaniadonovani infection enhances macrophage viability inthe absence of exogenous growth factor. J Leuk Biol,1994, 55: 550-555.

12 13

Cutan

eous

Leish

mania

sis

AbstractA field intervention trial to control cutaneous

leishmaniasis by using pyrethrin-impregnatedbednets was carried out in Aleppo, Syria. Fourvillages, 20 km north-east of Aleppo, wereselected as the study area. Two villages wererandomly chosen and were consideredintervention villages and the other two werethe control villages. A total of 4578 individualsin 508 households were enrolled in the study.

During May 1994, a house-to-house surveywas conducted to co l lect census,socioeconomic, and baseline epidemiologicaldata. Pyrethrin pre-impregnated bednets weredistributed to household members of theintervention villages. Bimonthly entomologicalsurveys of the vector Phlebotomous sergentiwere conducted using oily papers in 10selected households in the intervention andcontrol vil lages. Epidemiological andentomological surveys were repeated post-intervention. Furthermore, leishmanin skintests were carried out on children of all thevillages to estimate the force of infection.

ResultsThere was a 45% reduction in theincidence of cutaneous leishmaniasis in theintervention area compared to a 75% increasein the control area. These encouraging resultssuggested a high efficacy of Pyrethrin-impregnated bednets as a method for thecontrol of cutaneous leishmaniasis in endemicareas.

Second Phase A second phase was carriedout during June 1995-1996 whereby theefficacy of using re-impregnated bednets in

the intervention villages was compared to non-impregnated bednets in the control villages.This second phase was funded by WHOheadquarters.

BackgroundAleppo has been endemic for cutaneous

leishmaniasis for 2 to 3 generations, butits incidence rose sharply in the mid-1980sand reached its peak early in the 1990s.Pyrethroid residual spraying has beenused to control malaria and leishmaniasisand, in spite of the efficacy of spraying,its main disadvantages were: high cost,low sustainability, logistical and managerialproblems, high rate of nonacceptability,low community participation and the needfor trained personnel.

In spite of the limitations of residualspraying, in addition to the success ofinsecticide-impregnated bednets trials inmalaria control, it was expected thatsuccessful results with anthroponoticcutaneous leishmaniasis would beobtained. Unlike zoonotic leishmaniasis,anthroponotic cutaneous leishmaniasiscan be controlled by controlling the sandflyvector transmitt ing the disease.

This trial was conducted to evaluate theefficacy of insecticide-impregnatedbednets in controlling anthroponoticcutaneous leishmaniasis in rural Aleppo,Syria.

Materials and methodsFour villages with the highest recorded

prevalence of cutaneous leishmaniasiscases were selected. The main activitiesof the population were agricultural andindustrial. Government and private poultryfarms were scattered in the area. It alsoincluded a borrow-pit for the disposal ofsolid wastes from Aleppo city. The totalpopulation of the study area was 4578inhabitants in 508 households, and theywere mainly farmers. A pilot study wasconducted during 20-26 April 1994 to

Pyrethrin insecticide-impregnated bednets are an efficaciousmethod for the control of cutaneous leishmaniasis in endemicareas.

Non-acceptability of bednet usage was mainly associated withinadequate knowledge regarding the disease. Therefore, educatingcommunities about the new intervention method proved to be veryeffective in motivating these communities towards its use.

Ensuring the sustainability of usage of insecticide-impregnatedbednets in endemic communit ies was emphasized.

Cutaneous LeishmaniasisS y r i a n A r a b R e p u b l i c


Vector Control

Pilot project usingpyrethroid-

impregnatedbednets for the

control ofanthroponotic

cutaneousleishmaniasis in

Aleppo focus

Syrian Arab RepublicAleppo


Small GrantsScheme

(SGS) 1992 No. 7

Principal InvestigatorDr Aida Madani KhiamiParasitology Department

Faculty of MedicineDamascus, Syria

provide a rough estimate of the number of cases ineach village, and the results were as follows: 1.2%in Halisa, 3.3% in Tal Shaeer, 1.4% in Kaffar Sagheer,and 2.5% in Sheikh Najjar.

Human behaviour in respect of sleepingusually people wake up at 6:00-8:00 a.m. and sleepearly at 20:00-22:00. In summer, all householdmembers sleep in the courtyard. Some individualsuse local bed nets. There are some ceiling fans inthe bedrooms and living rooms.

Entomological survey Ten households wereselected in each of the intervention and control areasfor the entomological survey. Oily papers wereattached to nails in the four corners and the frontwalls in two bedrooms in each household. Two oilypapers were placed in the kitchen and two in thestable or animal shelter. Six oily papers were placedin the courtyard. They were kept for 3 nights in thehousehold to trap sandflies.

A baseline survey was conducted at the start ofthe project during May-June 1994 to estimate the 1-year prevalence of the disease between July 1993and June 1994. Other information was collectedincluding sociodemographic data, factors influencingdisease transmission, clinical and parasitologicaldata. Incidence of the disease was estimated betweenDecember 1994 and June 1995.

Pre and re-impregnation of bed nets In May1994, 800 Pyrethrin-impregnated bednets weredistributed to the households in the interventionvillages. These were re-impregnated in May 1995using WHO recommended techniques. Extra 400impregnated bed nets were distributed in May 1995.Similar number of non-impregnated bed nets weredistributed to households in the control villages.

Health education Villagers were educatedregarding the role of bed nets in controlling thedisease. The aim of the health education was toincrease the use of bed nets. Visits to the study areawere carried out in evening to observe the use ofbed nets.

Main study findingsThe population and households of the intervention

and control villages were comparable regarding thehousehold size, number of rooms per household,usage of local cloth bednets (11.7%), usage of fans(74.2%) and insecticide spraying of their plants inthe courtyard (31.6%).

The main reasons for not using bednets were theirsubstitution by fans, their cost, indoor sleeping, andthe absence of flies, among other reasons.

The incidence of cutaneous leishmaniasis in thepre-intervention infection season and in the post-intervention infection season was as follows: in Halisa

and Sheikh Najjar, the intervention villages, therewas a reduction in the incidence of cutaneousleishmaniasis from 4.7% to 2.4% and from 4.6% to3.2%, respectively. On the other hand, the incidenceof cutaneous leishmaniasis increased in the controlvillages, Tal Shaeer and Kaffar Sagheer, from 3.5%to 5.5% and from 1.6% to 3.1%, respectively. Theseresults indicate that there was about a 45% reductionin the incidence of cutaneous leishmaniasis in theintervention area while it increased by approximately75% in the control area.

The entomological survey demonstrated a higherdensity for both male and female P. sergenti caughtin 1994 per night/m2 of oily papers. Two peaks ofsandfly density were identified, one in mid-July andanother in October. The outdoor density wassignificantly higher than the indoor density.

ConclusionInsecticide-impregnated bednets are an efficacious

method for the control of cutaneous leishmaniasis inendemic areas.

14 15

Cutan

eous

Leish

mania

sis

AbstractLocal production of the direct agglutination

test (DAT) is imperative to the effective controlof visceral leishmaniasis (VL) in Sudan.Establishing an aseptic in vitro Leishmaniapromastigote mass culturing is experiencedas a most intricate, however, crucial step inthe production of a valid DAT antigen. Thesuccessful use of the indigenous L. donovanistrain (LD43) in this study is consideredindispensable for the continuity of local antigenproduction.

Results In spite of the sub-optimal laboratoryconditions encountered, the research groupat Ahfad University for Women succeeded inthe production of 3 DAT antigen batches. Theamount of antigen produced was sufficientfor performing 880 full-out titration tests or3666 screening doses for positive/negativeresults. The locally produced antigen batchesshowed a high level of reproducibility versusboth serum and blood samples. Bycomparison with the reference antigen, thelocal antigen revealed similar levels ofsensitivity and specificity against all 48 serumsamples tested. No cross-reactions werenoticed versus sera from endemic or non-endemic controls nor from cases with proven

malaria or onchocerciasis. The locallyproduced antigen also showed a desirabledegree of stability at ambient temperatures(28-35 °C) for at least 42 days. The DATresults obtained through blind testing of 67VL-suspected patients correlated positivelywith VL final diagnosis in 22 of these patientsand further confirmed the reliability of thelocally produced antigen. Large-scale DATproduction was successfully achievedreaching net antigen volumes of 750, 800,1150 and 1250 ml. DAT antigen specimenssufficient for performing 120-2000 tests weredistributed to 5 institutions involved in theroutine diagnosis and epidemiological studiesof VL.

Activities achieved withinthe framework of the study

A well-equipped research laboratory wasconstructed in the University. To ensure thecontinuity and dissemination of the DATtechnique, 4 post-graduates (biologists) fromthe Ahfad University for Women and theInstitute of Endemic Diseases (University ofKhartoum) were involved in all steps of antigenprocessing and in the execution of the test.Through the training of the biomedical staffand the establishment of facilities in AhfadUniversity and in the Institute of EndemicDiseases, local DAT production was achieved.This will contribute in the reduction of thecosts of kala-azar diagnosis in Sudan.

BackgroundThe imported direct agglutination test

(DAT) proved to be the most effective,though costly, method in the diagnosisof kala-azar. This prompted theinvestigators to undertake this studyaiming at establishing local production ofDAT for visceral leishmaniasis, thenevaluating its reliability in diagnosing thedisease.

Materials and methodsLeishmania cultures were kept in

locations where the ambient temperaturewas relatively low and stable (25-29 °C).An isolate of Leishmania donovani,

Local production of DAT antigen using an indigenous strain(LD43) was feasible in spite of sub-optimal conditions. Thesensitivity and specificity of the local antigen were comparable tothe reference antigen.

Three antigen batches sufficient for testing of 176-536 individualsby full-out titration or performing 733-2233 screening tests wereproduced. The reproducibility of the results obtained both in serumand blood samples was excellent.

The obtained length of shelf-life time was adequate for maintainingantigen reactivity under prolonged electric failures and for executingfield survey studies without refrigeration.

A modification in the staining procedures was performed wherebythe net volume of the antigen produced was increased.

Relative large-scale production of DAT antigen was achievedand various quantities of DAT antigen were dispatched to 5institutes at the national level to assist in the routine diagnosisand epidemiological studies of VL.

Visceral LeishmaniasisS u d a n


Diagnosis

Establishment ofproduction of DAT

antigen forLeishmania donovani

Sudan Eastern Sudan


Small GrantsScheme

(SGS) 1998 No. 31

Principal InvestigatorDr Abdallah El Harith

School of MedicineUniversity of Ahfad for Women

Omdurman, Sudan

designated as LD43, and kept in continuous cultureusing NNN or RPMI, was provided by the Universityof Khartoum. The strain was then kept in GLSH(glucose/lactalbumine/serum/haemin) medium.Promastigotes of LD43, cultured continuously in thismedium, maintained the desired elongated spindleform and suitable rate of growth. Supplementationwith gentamicin was essential to overcome thefrequent bacterial contamination. Subculturing of thestrain was performed every 4-7 days, before theappearance of the degenerative round forms. RPMI-1640 was employed for mass culturing of the L.donovani strain (LD43). RPMI-1640 wassupplemented with HEPES, fetal bovine serum andgentamicin. One litre of the ready-for-use RPMImedium was then inoculated with 20 ml GLSHmaintenance culture containing mostly the LD43elongated form of promastigotes. The inoculatedRPMI mass culture was further divided equally intotwo 1-litre flasks to allow for better aeration. Theflasks containing the cultures were manually shakenin a clockwise manner by hand for 2-3 minutes, 6-8times a day. Optimal (logarithmic) growth on the 3different occasions of mass culturing was observedbetween 7 and 10 days post inoculation with themaintenance culture.

Preparation of the DAT antigen The parasiteswere spun down in a refrigerated (4 °C) centrifugeat 3000 g for 15 minutes. Further steps for antigenprocessing were done according to Harith et al, 1995.After centrifugation, the promastigote pellet waswashed 3 times in Locke's solution and treated with2-Mercaptoethanol (2-ME) instead of trypsin. Aconcentrated promastigote suspension of 100 ml of1x108/ml was washed by centrifugation andresuspended in a similar volume of Locke's solutionto which 1.2% (v/v) 2-ME was previously added. Thetreated parasite suspension was incubated at 37 °Cfor 45 minutes. Promastigotes were then washedagain 3 times in Locke's solution and fixed in thesame medium, supplemented with 2% (w/v)formaldehyde. The fixation was for ±18 hours afterwhich the parasites were washed with citrate-saline,stained for 2 hours with Coomassie brilliant blue andfinally preserved in 1.2% (v/v) formaldehyde. Thefixed, ready-for-use antigen, so prepared, was storedat 4 °C until required. Antigen batches were produced.The reliability (sensitivity and specificity), reproducibilityand shelf-life time of the batches were assessedagainst clinical specimens (serum and blood) fromkala-azar patients and from individuals with otherconditions.

DAT execution Sera were tested against theantigen batches produced according to the proceduresdescribed by Harith et al., 1988. After dilution of thetest sera, the antigen was added and the test plateswere incubated at room temperature for ±20 hours.

The air-dried blood samples were tested by existingspots of 6 mm diameter and eluation in 760 ml salinesolution for 24 hours at 4 °C. The blood eluate soobtained was found to be equivalent to 1:100 serumdilution. Visual reading of the DAT was by localizinga clear sharp-edged blue spot, identical to the oneobserved in the control, and the preceding dilutionis considered the titre of the test serum. All test resultswere read by at least 2 observers.

Main study findingsThree antigen batches, sufficient for the testing of

176-536 individuals by full-out titration or for 733-2233 screening tests, were produced. Thereproducibility of the results obtained both in theserum and blood samples was excellent. Furtherstandardization in this procedure is expected toreduce this difference in test readings between the2 sampling methods. By comparison with thereference antigen (ITMAP-Belgium), the locallyproduced one evidenced an identical level of specificity(titres <= 1:100) versus clinical disorders other thanthe kala-azar tested.

The sensitivity of the local antigen versus the kala-azar cases tested was comparable to the referenceantigen. However, in 5 sera from kala-azar cases,the locally produced antigen was more sensitive (1-4 titre dilutions higher) by comparison with thereference. This might be due to the incorporation ofthe autochthonous Leishmania strain (LD43) fromthe same endemic area (Gedarif) in antigenprocessing. The locally produced DAT antigen showedstability at ambient room temperatures of 28-35 °Cfor at least 42 days. Although no comparison wasmade versus the reference antigen in this respect,a desirable length of shelf-life time was adequate formaintaining antigen reactivity under prolonged electricfailures and for performing field surveys withoutrefrigeration. A modification of the staining procedureswas introduced by incubating the formaldehyde-fixedpromastigote with Coomassie Brilliant Blue overnightat +4 °C leading to an increase in the dye intake by³95%, thus resulting in an increase in the net volumeof the antigen produced. The reliability of the locallyproduced DAT antigen was further confirmed bycarrying out blind testing on 67 blood specimens sentby MSF-Holland. The results obtained correlatedpositively with the disclosed final VL diagnosis in 22patients. Relative large scale production of the DATantigen was achieved on 4 different occasions,attaining volumes of 750, 800, 1150 and 1250 ml.Various quantities of the DAT antigen were dispatchedto 5 institutes at the national level to assist in VLepidemiological studies and routine diagnosis.

16 17

Viscer

al Le

ishma

niasis

AbstractThe latex agglutination test (Katex) for the

detection of leishmania antigen in the urineof patients with visceral leishmaniasis wasstudied in Gedarif State, Eastern Sudan. Theaim was to test its performance as an earlydiagnostic tool for screening populations livingin remote areas with limited accessibility tohealth services. Moreover, the fact that urinaryantigens decline quickly after treatment wasthe basis for studying the prognostic potentialof the test.

A prospective study was carried out in thekala-azar treatment centres in three villagesduring the period April 2001-February 2002,whereby urine samples from 204 patients withsuspected kala-azar were tested with Katexand the results compared to smears of lymphnode aspiration and serology (DAT, ELISA,IFAT and western blot (WB)).

A cross-sectional survey was alsoconducted in Marbata village between 11 and19 February 2002 and involved all village

inhabitants. It included clinical examinationand screening villagers with Leishmanin SkinTest, serology (DAT, ELISA, IFAT) and theKatex test.

Results Compared to microscopy, Katexhad a sensitivity of 95.2%, with goodagreement with microscopy but pooragreement with serological tests. It was alsopositive in the 2 confirmed visceralleishmaniasis patients co-infected with HIVinfection. The test recorded a specificity of94.4% in smear-negative patients subjectedto test of cure, 89% in kala-azar patientsduring their follow-up, 100% in post-kala azardermal leishamanisis (PKDL) patients whodid not show clinical features suggestive ofvisceral leishmaniasis and in smear-negativeconfirmed malaria or tuberculosis patients.The test was also negative in all confirmedpatients who had been actively followed-up3 months after completion of their treatment.

While Katex had a specificity of 100% inhealthy endemic and non-endemic controls,DAT was positive in 14% of the Katex-negativehealthy endemic controls. Cross-reaction wasnot observed with confirmed tuberculosis ormalaria patients.

Conclusion Katex is a sensitive, specific,rapid and simple addition to the diagnosticsof visceral leishmaniasis, particularly at fieldlevel. Since the test seems to be specific foractive disease, it can be particularly usefulas a complementary test for the diagnosis ofvisceral leishmaniasis in smear-negative caseswith positive DAT results and may also behelpful in the detection of teatment failure.

BackgroundTo date, there has been no reliable

diagnostic test for active infections ofv isceral le ishmaniasis. In fact ,parasitological methods record high ratesof false negative results, while serologicaltests based on the detection of anti-leishmanial antibodies are costly, needsophisticated techniques and areassociated with cross-reactivity with otherpathogens. The DAT is a sensitive,specific and simple test but its maindisadvantage is its inability to distinguish



DiagnosisField evaluation ofthe latex

agglutination test forthe detection of

urinary antigens ofvisceral

leishmaniasis inSudan

SudanGedarif State, Eastern

Sudan

Study period:April 2001–February 2002

Small GrantsScheme

(SGS) 2000 No. 32

Principal InvestigatorDr Sayda Hassan El-Safi

Faculty of Medical LaboratorySciences

Khartoum UniversityKhartoum, Sudan

Several studies using latent class analysis were conducted tryingto solve the problem of absence of a gold standard for diagnosisof visceral leishmaniasis (VL). They all recommended treatingclinical suspects in endemic areas based on positive DAT results.However, this study showed that LST was positive in 77.8% ofsmear negative VL suspects with positive DAT results while positiveLST rules out the diagnosis of active VL.

In addition, Katex could differentiate between active disease,subclinical and clinical infections. Accordingly, Katex could be asensitive, specific, and rapid diagnostic test, complementary toDAT for solving the diagnostic dilemma of VL in endemic areas.It also proved to be a valid test for the detection of treatmentfailure.

The fact that Katex was negative in all PKDL cases indicatesthat it is related to visceralization of the parasite, hence only validin diagnosis of VL.

Two leishmania zymodems were characterized in this area byIsoenzymes analysis: L. donovani Mon 18 (11) and L. archipaldiMon 82 (2).

Further studies are recommended to evaluate the performanceof the test in diagnosis of HIV co-infected cases.

between active disease, subclinical or past infections.A latex agglutination test for the detection of urinary

antigen has recently been developed. The test has100% specificity and a sensitivity of 81.4% in humanurine samples, which is comparable to bone marrowaspiration. This study was conducted to evaluate thediagnostic validity of the test and its prognosticpotential, particularly for remote areas with pooraccessibility to health services.

Materials and methodsThe study was conducted in three kala-azar

treatment centres in Gedarif state, Eastern Sudan.The study population included new visceralleishmaniasis suspects, PKDL cases, patientsinvestigated for a test of cure, and former kala-azarpatients who reported for follow-up. Confirmed visceralleishmaniasis patients under treatment were subjectedto follow-up for the evaluation of the therapeuticpotential of Katex. Non-endemic and endemic healthycontrols and cases of confirmed pulmonarytuberculosis and malaria were also included in thestudy.

Lymph gland aspiration was performed on visceralleishmaniasis suspects for the demonstration ofamastigotes in stained smears, followed by cultureon NNN and Evalus sloppy media. The strains werealso characterized by isoenzymes electrophoresis.Blood samples were collected and tested by DAT,IFAT, ELISA and WB diagnostic tests. HIV screeningwas also performed. Urine samples were collectedfrom all visceral leishmaniasis suspects, non-visceralleishmaniasis cases and healthy individuals in twosterile containers; one was used immediately for fieldtesting and the other one was kept at -20 °C.

Katex test The test was performed by mixing 50ml of the prepared latex reagent with 50 ml of theneat urine sample on a glass slide. The slide wasrotated and rocked consistently for 2 minutes.Agglutination of ++ or more was considered positive.

Epidemiological study A house-to-house surveywas conducted in Marbata village along the AtbaraRiver. The survey included screening villagers forVL by clinical examination, collection of urine andblood samples, and interviewing them regardingsociodemographic characteristics, past history ofvisceral leishmaniasis, family history and othercharacteristics. They were also screened using LSTand serological tests. The village inhabitants werestratified into those without evidence of infection(negative LST and serology), those with subclinicialinfection (negative LST and seropositive without pasthistory of visceral leishmaniasis) and those withevidence of cured past infection (positive LST andseropositive). Lymph gland aspiration was performed

for all patients with clinical features suggestive ofvisceral leishmaniasis.

Main study findingsThe diagnosis of visceral leishmaniasis was

confirmed with a positive lymph gland smear in 62out of 180 new visceral leishmaniasis suspects(34.4%) and Katex recorded a sensitivity of 95.2%.There was good agreement between the two tests(Kappa coefficient = 0.65, P = 0.0), but pooragreement with serological tests (DAT, IFAT, ELISA,WB). Unlike DAT, no false-positive cases wererecorded (100% specificity).

The follow-up of treated confirmed visceralleishmaniasis patients for 1-3 months and smear-negative ex-kala-azar cases showed conversion ofKatex results to negative. Furthermore, evaluatingits validity as a test of cure showed 100% sensitivityand 89.4% specificity. These findings are due to therapid decline of the antigen level in the urine at week12 resulting in conversion of the test before the endof the course of treatment. Other reportsrecommended the use of a competitive ELISA andrK39 for the prognostic evaluation of visceralleishmaniasis and the success of drug treatment.Obviously, due to its simplicity, the Katex may bemore appropriate in this respect. Moreover, the testwas also positive in the two parasitologically confirmedcases co-infected with HIV infection.

The field study conducted on 402 individualsscreened by LST and serological tests confirmed theprevious hospital-based study by showing goodagreement between Katex and smear-positive patientsand being negative in all patients with previousinfections including PKDL patients with no clinicalfeatures of visceral leishmaniasis. These resultsindicate that Katex, unlike the DAT, discriminatesbetween active disease, subclinical and pastinfections.

Although latent class analysis recommendedtreating clinical suspects in endemic areas based onpositive DAT results, this study showed that LST waspositive in 77.8% of smear-negative visceralleishmaniasis suspects with positive DAT results.Taking into consideration that positive LST rules outthe diagnosis of active visceral leishmaniasis,accordingly, Katex could be a sensitive, specific,rapid test that is extremely useful to complementDAT for the diagnosis of unconfirmed visceralleishmaniasis cases.

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AbstractA household survey was conducted in two

endemic villages and one non-endemic villagefor visceral leishmaniasis (VL) in South Syriawhereby 483 children, aged 6 months to 6years, were screened using the rK39 test. Theaim of the study was to test the effectivenessof this test as an early diagnostic tool for VL,especially in remote areas with limitedaccessibility to health facilities. Only 10 positivecases were subjected to microscopic detectionof amastigotes in bone marrow, while all positivecases were tested by ELISA. Screening ofdogs was perfomed on a limited scale.

Results Eighty children were positive forVL out of a total of 345 children in the twoendemic villages, recording a prevalence of23%. The prevalence was significantly higherin Assem village compared to Sour village(31.9% and 17%, respectively), while therewas no significant difference between malesand females regarding the prevalence ofinfection. All of the sera (N = 138) obtainedfrom the control village, Ottaya, were negative(single band), suggesting high specificity.

Sera from patients with acute VL were easilyidentified by rK39 testing, but patients withasymptomatic or self-healing infections hadlow or undetectable levels of anti-rK39antibodies. Only 10 out of 80 positive childrenwere symptomatic (12.5%). Symptoms includedirregular fever (for one day), cough, diarrhoea,loss of weight and hepato-splenomegaly (2-3cm). Only 22 out of the 80 positive cases byrK39 were shown to be positive by ELISA. Thiswas thought to be due to the lateseroconversion of ELISA, hence bringingevidence about early diagnosis of VL by rK39.When these positive cases were subjected tofollow-up 9 months later using rK39 dipsticks,ELISA, and clinical examination they remainedELISA-negative, and none developed the full-blown disease.

Conclusion Rapid diagnosis of VL can beperformed using rK39 in symptomatic subjectssince the test cannot differentiate betweenrecent and old infection. However, the studycould not provide evidence about rK39's rolein early diagnosis of VL.

BackgroundSerodiagnosis has been widely utilized

for the diagnosis of visceral leishmaniasis(VL) in laboratories. However, there is aneed for a simple, sensitive and specificdiagnostic test for screening infectedhumans and reservoirs in the field,especially in remote areas with pooraccessibility to health facilities. The rapidassay for the qualitative determination ofantibodies to an antigen specific (K39)caused by members of the Leishmaniadonovani complex is considered a new,effective test. The use of this recombinantantigen (rK39) as a rapid tool for controllingVL is not currently perfomed in thecountries of the Region.

The main objectives of this study wereto test the efficacy of the rK39 test forearly detection of VL cases among childrenunder 5 years old in endemic villages, aswell as testing its prognostic value in thefollow-up of the subclinical and acutecases of VL in these villages.

Screening two endemic villages for visceral leishmaniasis infectionin South Syria using rK39 revealed a prevalence of 23% in thisarea.

rK39 was positive among all confirmed cases by microscopicfinding of amastigotes in bone marrow aspirates, as well as amongsymptomatic cases, which suggests a high sensitivity. On theother hand, all the sera obtained from the non-endemic villagewere negative, suggesting high specificity.

A 9-month follow-up of rK39 positive cases with rk39, ELISA,and clinical examination refuted the hypothesis of delayedseroconversion of ELISA. Moreover, full-blown disease did notdevelop 9 months after the initial screening. Thus, this report couldnot elicit with evidence the role of rK39 in early diagnosis of thedisease.

The results of this study suggest that this test could be used asa rapid diagnostic test for screening populations in remote areaswith limited accessibility to health facilities. However, the presenceof clinical features of visceral leishmaniasis is necessary for finaldiagnosis due to the high frequency of subclinical infections inendemic areas.

Visceral LeishmaniasisS y r i a n A r a b R e p u b l i c


DiagnosisAssessment of the

rK39 test as ascreening tool for

the early detectionof visceral

leishmaniasis insouth Syria

Syrian Arab RepublicDaraa and Sweida, south

Syria

Study period:May 2000–April 2001

Small GrantsScheme

(SGS) 2000 No. 47

Principal InvestigatorDr Samar Al-NahasDepartment of Biology

Faculty of ScienceDamascus University

Damascus, Syriae-mail:

[email protected]

Materials and methodsThe study was conducted in 3 villages in Daraa,

South Syria: Sour and Assem villages are endemicfor VL, while the third one, Ottaya, was considered acontrol village. A household survey was conducted inthe 3 study villages, whereby children aged between6 months and 6 years were screened using the rK39test. Children proved to be positive by the rK39 testwere subjected to confirmed diagnosis by microscopicfinding of amastigotes in bone marrow aspirates. Thisinvestigation was performed at Damascus hospitalsbecause of the unavailability of necessary equipmentin local health facilities. Serum samples (from positivechildren by rK39 dipsticks) were also tested by theELISA method. Nine months later, positive cases wereretested by rK39, ELISA and clinical examination. Theaim of this follow-up phase was to study the prognosticpotential of the test.

Technique of the rK39 test A drop of peripheralblood was placed on the dipstick. Flooding of thebottom protein with buffer allowed blood proteins tomigrate upward. The positive reaction was obtainedin 5-10 min, and it was indicated by the presence ofan extra band "double bands" below the control band"single band".

ELISA method Breakable ELISA strips weresensitized with L. infantum soluble antigens (BordierKit). The wells were blocked with TBS-Tw. Afterremoving the blocking solution, the wells wereincubated with diluted serum samples, then washed4 times with washing solution and incubated withconjugate. After removing the conjugate and washing,the wells were then incubated with the substrate.Finally, the strips were read in the personal-lab. ELISAreader.

Main study findingsEighty children were positive for VL out of a total of

345 children in the two endemic villages, recording aprevalence of 23%. The prevalence was significantlyhigher in Assem village compared to Sour village(31.9% and 17%, respectively), and there was nosignificant difference between males and femalesregarding the prevalence of infection. On the otherhand, all the sera (N = 138) obtained from the controlvillage, Ottaya, were negative (single band).

The results of positive sera were further categorizedinto 3 groups: high, moderate, and weak. Sera frompatients with acute VL were easily identified by therK39 test but patients with asymptomatic or self-healinginfections had low or undetectable levels of anti-rK39antibodies.

Only 10 out of 80 positive children were symptomatic(12.5%), and among 3 out of these 10 cases,microscopic findings of amastigotes were positive.Symptoms included irregular fever, cough, diarrhoea,

loss of weight and hepato-splenomegaly.Microscopic finding of amastigotes in bone marrow

aspirates was only performed in 10 out of the 80positive cases due to a high refusal rate. All 10 testedcases proved to be positive for VL (sensitivity 100%).

There was poor agreement between rK39 and ELISAresults. The number of positive sera by the ELISAtechnique was 22 out of the tested 80 sera (27.5%).

Screening of dogs was not adequately conducteddue to the difficulty in catching wild dogs during visitsto the volcanic, rocky hills. Only 9 sera from domesticdogs were collected during the field survey, and allwere negative using the rK39 dipsticks. This was astudy limitation.

Nine months following the initial survey, positivechildren were assessed clinically and serologically.All the children seemed to be healthy without evidenceof full blown disease except 4 children with enlargedlymph glands.

Serologically, the data obtained after applying therK39 strips test, presented only 3 weak positive children(similar to their last results) and the rest of samples(52 sera) was negative. These results indicate thatrK39 strips is a relatively sensitive test, because it candetect even a very small quantity of antibodies resultingfrom immunological response to insect's bite. Thedisappearance of these antibodies and absence offull blown disease signifies that the child's body couldovercome the infection which was restricted to thesubclinical phase. The presence of 3 positive casesafter this nine months period could be referring toindividual specific immunological response only .However, these results also indicate that rk39 is unableto differentiate between recent and old infection, andthat the existence of clinical signs and symptoms isnecessary to diagnose VL in case of positive testresults.

ELISA test results were negative in all tested 55cases, at 9 months of follow-up, which did not varyfrom earlier results. This latter finding indicate thatELISA is more specific in diagnosis of the diseasesince it recorded a lower frequency of false positivecases compared to rK39.

In conclusion, rK39 could be a rapid, sensitive andspecific diagnostic test for visceral leishmaniasis insymptomatic cases living in remote areas with pooraccessibility to health services. In disease endemiccountries, VL could be easily misdiagnosed on clinicalbases, therefore, screening these populations usingrK39 test is recommended for early diagnosis andtreatment of the disease thereby reducing its morbidityand mortality.

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AbstractThe objectives of this study were to study

the epidemiological situation of kala-azar inYemen, including the identification of thenatural reservoir of the disease, particularlydogs. During a 2-year period, 94 cases ofkala-azar with complete data were identifiedfrom the Sana'a area. The male-to-femaleratio was 3:1 and the crude incidence ratewas 13/100 000 over 2 years.

In a cross-sectional study of the populationin 4 known endemic foci, over 285 individualswere randomly examined, and blood sampleswere taken and serologically tested for anti-leishmania antibodies by ELISA. Seropositivityrates ranged from 14% to 85.3%. There wasa high risk of seropositivity with rural residence,ownership of dogs, open refuse dumps closeto houses, stray dogs around the house andthe presence of rodents in houses. Sixteenferal dogs were captured and amastigoteswere observed in smears of livers and spleensof 4 of them. ELISA testing of their bloodindicated that 75% of animals from theBenimansour area were positive, compared

to 25% of those examined from Sharis area,giving a total prevalence of 50%.

Conclusions Kala-azar is an importantpublic health problem affecting mainly youngchildren in northern parts of Yemen andinfection is transmitted by the caninepopulation. Strengthening control activitiesthrough case-finding, vector and reservoircontrol were emphasized.

PublicationsAl-Shamahy HA. Seroprevalence of kala-azar

among humans and dogs in Yemen. Annals ofSaudi Medic ine , 1998, 18(1) :66-68.

BackgroundKala-azar has been reported from the

northern part of Yemen since 1904, butnearly 4047 cases, mostly young children,were reported throughout the countryduring the 1980s. The causativeorganisms are Leishmania donovanicomplex and L. infantum complex, andthe vectors are Phlebotomus orientalisand P. arabicus. This study wasconducted aiming to determine theincidence of kala-azar in the study area,to determine the prevalence ofseropositivity to Visceral Leishmaniasis(VL) among children in selected localitiesin Yemen, and to identify the naturalreservoir of human (VL) in the areas,particularly dogs.

Materials and methodsCases diagnosed in Althora and AlsabianHospitals, Sana'a city, during the period1992-1993 were used to study theincidence of kala-azar per 100 000 peryear among children in Sana'a, Yemen.

In order to determine the prevalenceof seropositivity to (VL) among children,three villages were selected duringDecember 1993, where cases had beenreported during the same year fromSana'a city. The study populationconsisted of schoolchildren in Benimansur(Al-Hymah, Sana'a provinces), Hooth(Sana'a provinces), Sharis (Hajjah

The incidence of kala-azar in Sana'a was 13/100 000 over 2years, with a significantly higher incidence among children under5 years old, and in males compared to females.

The human serological survey revealed a seropositivity of 34.7%,which was higher than that recorded from other endemic countriesof the region. This indicates the possible existence of subclinicalinfection, which may serve as a reservoir of infection. There wasa high risk of seropositivity with rural residence, ownership ofdogs, open refuse dumps close to houses, stray dogs around thehouses and the presence of rodents in houses.

In spite of the small sample size of the canine survey, theexistence of a reservoir of infection was confirmed and suggestsan alarming percentage, far higher than that of the Mediterraneanregion.

This study reported the failure of isolating leishmania organismsof confirmed kala azar cases on locally prepared NNN media.

It was recommended that kala-azar should be made a notifiabledisease in Yemen and that further studies should be organizedto define more clearly the foci of human kala-azar, to trace andconfirm possible reservoirs among canines and other wild animalsas well as to study the sandfly vectors.

Visceral LeishmaniasisR e p u b l i c o f Y e m e n


Epidemiology

Prevalence ofcanine and humanleishmaniasis in

Yemen

Republic of YemenSana'a area

Study period:October 1992-1993

Small GrantsScheme

(SGS) 1992 No. 26

Principal InvestigatorDr Hassan Al-Shamahy

Department of MedicalMicrobiology

Faculty of Medicine and HealthSciences

University of Sana'aSana'a, Republic of Yemen

provinces) and from Sana'a city. Informed consentwas taken, and all study participants completed aquestionnaire that included sociodemographic dataand potential risk factors for kala-azar.

Serological assay A 5 ml aliquot of venous bloodwas allowed to clot and the serum was separatedand stored at -20 °C until serologic analysis wasperformed. All sera were tested for kala-azar usingthe ELISA technique. Samples with readings under0.3 were considered negative, those with readingsabove 0.5 were positive, and readings in betweenthese levels were considered equivocal (antibodiesmay be present but at nonsignificant levels). Subjectswere divided into two groups based on their serologyto study the risk factors for the disease.

Collection and testing of canine specimensDogs were captured in two areas where cases ofhuman visceral leishmaniasis had been identified(Benimansur and Sharis). Feral dogs were abundantin the study areas, living close to humans andwandering around farm buildings and houses, causingnuisance to the local population. A tented fieldlaboratory was set up at each location for the purposeof providing a base where the animals could beexamined. Sixteen dogs were captured, examinedexternally for signs of leishmaniasis, then dissected.

Smears were prepared from any skin lesiondetected, and from the liver and spleen of each dog.These were stained with Giemsa stain and examinedmicroscopically for the presence of amastigotes.

Biopsy specimens were collected aseptically fromthe livers and spleens, inoculated into a biphasicculture medium (NNN medium), then incubated at21 °C for up to 6 weeks, and examined weekly forthe presence of amastigotes.

Blood was collected from each dog and sera wereseparated and stored at -20 °C until assayed. Serawere tested for the detection of antibodies against(VL) by the ELISA test. Sera of dogs free from thedisease were taken as reference, and 40%absorbance values of positive sera were consideredpositive.

Main study findingsDuring the 2-year period (1992-1993), a total of 94cases were diagnosed in the study area. The male-to-female ratio was 3:1. The highest percentage ofcases occurred in children under 5 years old (55%),and another 36% of cases occurred in the 5-9 year-old age group. The incidence of kala-azar in Sana'awas 13/100 000 over 2 years, with a significantlyhigher incidence in males compared to females; thehighest monthly frequency was in March and thelowest from November to January. These figures farexceeded the reported number of cases in certainNorth African countries, where incidence rates of

0.34 and 0.24 per 100 000 have been reported fromAlgeria and Tunisia, respectively. The predilectionof the disease to young children was in agreementwith several reports and was attributed to impairedacquired immunity due to immature immune systemsand inadequate nutrition. The higher incidence amongmales was attributed to increased exposures bymales due to increased outdoor activity relative tofemales.

The seroepidemiological study revealed aseropositivity of 34.7% (99/285). The seropositivitywas significantly higher in Sharis (85% of positivecases) compared to the other 3 regions that recordedcomparable rates, ranging from 14-17.6% of positivecases. The high seropositivity rates were explainedby the fact that most of individuals exposed to (VL)infection develop subclinical infections, while only afew of them will develop the clinical disease. Therewas a high risk of seropositivity with rural residence,ownership of dogs, open refuse dumps close tohouses, stray dogs around the house and thepresence of rodents in houses.

Leishmaniasis infection in feral dogs Lesionsof possible leishmania origin were observed on theears of five dogs in the Benimansur area, but noparasites were identified in the skin of these animals.Splenomegaly was observed in four andhepatomegaly in two dogs. Amastigotes wereobserved in Giemsa-stained smears of the livers andspleens of four dogs. ELISA testing of canine bloodindicated that 75% from Benimansur were positive,compared to 25% from Sharis, with a total prevalenceof 50%, which was higher than that reported fromSaudi Arabia and Tunisia (19.3% and 6.03%,respectively). No growth occurred in the cultured liverand spleen of the dogs in NNN media, even for dogsfound to be positive for amastigotes by liver andsplenic aspiration. The prevalence of antibodies notedin the canine population may have been due eitherto infection with L. infantum complex and L. donovanicomplexes, whether past infection or active disease,or to antigenic cross-reaction with cutaneousleishmaniasis.

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AbstractThe study aimed at the determination of the

seroprevalence of visceral leishmanias (VL)in northern Morocco using the directagglutination test (DAT). The study areaconsisted of 60 urban and rural clustersrandomly selected from 8 provinces in northernMorocco. All children younger than 15 yearsof age were interviewed regardingsociodemographic variables, travel history,contact with dogs, and history of VL cases inthe neighbourhood. Children were alsosubjected to clinical examination as well as toblood collection on filter paper, and bloodsamples were collected from 10% of thechildren.

Several sets of control sera were collected:from healthy subjects, confirmed VL cases,VL-positive cases with the immunofluorescentantigen (IFA) test, VL cases with negative IFAtest sera from endemic areas, patients withpositive anti-nuclear or anti-mitochondrialantibodies, and from patients with variousinfections. The DAT production was performedusing the promastigotes of the Leishmaniadonovani infantum strain L dik 263. Filter-paper blood samples were extracted andplasma eluates samples were screened withDAT. The same strain of L. donovani infantump r o v i d e d p r o m a s t i g o t e s f o r t h eimmunofluorescent antigen IFA test.

Results The seroprevalence of VL was 1.33%,

with no statistically significant differencebetween rural (1.17%) and urban areas(1.51%). There was no significant associationbetween the DAT positivity and gender, age,presence of domestic dogs at home or in theneighbourhood, and travel history. The DATwas proved to be a highly sensitive and specifictest, stable at 4 °C when stored in the dark forup to 5 months. Providing training on the DATproduction and the establishment of alaboratory for the continuous production andsupply of the DAT to the different provinceswere the main study recommendations.

Publications1. El Aouad R et al. DAT Seroprevalence of visceralleishmaniasis in the north of Morocco. EMHJ, 1999,5(5):1091.2. Mengad R et al. Production of direct agglutinationtest (DAT) antigen and seroprevalence of visceralleishmaniasis in northern Morocco. Giornale ItalianoDi Medicina Tropicale, 1998, 3(3-4):83-87.

BackgroundCentral and southern Morocco are

endemic for cutaneous leishmaniasis.Visceral leishmaniasis (VL) is found in thesubhumid zones in the north of thecountry. Since 1991, VL cases have beenincreasing in the north province. Aseroprevalence study was conducted bythe research team using commercialreagents (Behring haemagglutination tests(HA)) in Tahla where most of cases havebeen reported. The study was conductedon 1203 filter-paper blood samples andreported a prevalence of 14%. Thesealarming results motivated the group toconduct the present study using the directagglutination test (DAT) for the fielddiagnosis and determination of theseroprevalence of the disease in northernMorocco.

Materials and methodsEight provinces were randomly selected

from northern Morocco, and 60 clusterswithin these provinces were selected usingsystematic sampling in rural and urbanareas. The clusters consisted of districts

The seroprevalence of visceral leishmaniasis in northern Moroccowas low (1.33%) when compared to other regions of the country.

The DAT was proved to be a highly sensitive and specific test,stable at 4 °C when stored in the dark for up to 5 months. It wasfar more reliable than the immunofluorescent antigen IFA testwhich yielded high percentage of false positive results.

There was no significant association between the DAT positivityand gender, age, presence of domestic dogs at home or in theneighborhood, and travel history.

More attempts to improve the wide use of the DAT in field surveyswere suggested. Special emphasis was made on the importanceof providing training on DAT production and the establishment ofa laboratory for the continuous production and supply of the DATto the different provinces.

Visceral LeishmaniasisM o r o c c o


Epidemiology

DAT seroprevalenceof visceral

leishmaniasis innorthern Morocco

Morocco northern provinces

Study period:January 1996–1998

Small GrantsScheme

(SGS) 1996 No. 5

Principal InvestigatorDr Rajae El Aouad

Département D'ImmunologieInstitut National D'Hygiène

Rabat, Morocco

in urban areas, and localities in rural areas. All childrenyounger than 15 years of age residing in the studyarea were surveyed by house-to-house survey. Datawere collected according to a pre-designedquestionnaire. The collected information included:sociodemographic variables, travel history, clinicalexamination, contact with dogs, and history of VLcases in the neighbourhood. Children were alsosubjected to blood collection on filter-paper (FPB),and blood samples on dry tubes were collected from10% of the children. Blood samples were allowed toclot, and the serum was separated by centrifugation.Serum samples and air-dried FPB samples werestored at -20 °C until needed.

Control groups Several control sera were alsocollected: from healthy subjects, confirmed VL casesbefore and one month after treatment, VL-positivecases with the IFA test, VL cases with negative IFAtest sera from endemic areas, patients with positiveanti-nuclear or antimitochondrial antibodies, and frompatients with various infections (cutaneousleishmaniasis, schistosomiasis, toxoplasmosis,malaria, hydatidosis, tuberculosis, leprosy, hepatitis,cytomegalovirus, HIV and syphilis).

DAT production The promastigotes of L. donovaniinfantum strain L dik 263 were grown at 26 °C inMEM medium containing Fetal Calf Serum (FCS),penicillin, and streptomycin. Mass production wasdone in MEM supplemented by FCS. The protocolof DAT production was according to the modifiedmethod described by El-Harith [1].

Filter-paper blood samples were extracted andplasma eluates samples were screened starting at1/750 in V-shaped microtitre plates, 50 litres of antigensuspension were added, and the results visually readagainst a white background after 18 hours ofincubation at room temperature. Serum dilution of1/3000 or higher were suggestive of VL.

Immunofluorescent antigen The same strainof L. donovani infantum maintained in MEM media10/FCS provided the promastigotes for IFA test. Thepromastigotes supension was collected, washed andfixed on IFA slides with absolute methanol for 5minutes, then stored at -20 °C till used. The cut-offpoint for the control sera was 100.

Main study findingsA total of 2101 filter-paper blood samples were

collected, from which 1109 were from rural localitiesand 992 from urban districts. The IFA test wasperformed on 228 sera. All control sera were screenedby the DAT and the IFA test. Confirmed VL caseswere positive by both tests; out of 31 IFA-positiveresults, only 1 was positive with the DAT, mainlybecause of the lack of association with clinicallyproven VL; the IFA-negative sera were all negative

with the DAT; out of 15 sera from patients with auto-immune disorders, only 1 was positive with the DAT;all sera from other infectious diseases were negativewith the DAT. For the confirmed VL cases, DAT wasproved to be a sensitive test (100%) with anacceptable specificity. The DAT was also proved tobe stable at 4 °C when stored in the dark, since itwas tested at different time intervals (1 up to 5months).

The seroprevalence of VL was 1.33%, with nostatistically significant difference between rural (1.17%)and urban areas (1.51%). Higher seroprevalencerates were reported from Tetouan (3.10%) and Kenitra(2.35%). It is worth mentioning that the IFA testproved to be an insensitive test as indicated by thehigh percentages of false-positive results. There wasno significant association between the DAT positivityand gender, age, presence of domestic dogs at homeor in the neighbouhood, and travel history.

Several recommendations were suggested toimprove the quality of the DAT. Gelatin would be agood replacement for fetal calf serum in the DAT toreduce cost and improve its adaptation to fieldconditions. It was also suggested to consider thelyophylization and freezing of the antigen in order toincrease the duration of storage and facilitate itstransport to the provinces. Providing training on DATproduction and the establishment of a laboratory forthe continuous production and supply of the DAT tothe different provinces were finally recommended.

References[1] El Harith A et al. Improvement of a direct

agglutination test for field studies of visceralleishmaniasis. J Clin Microbiol, 1988, 26(7):1321-1325.

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AbstractVisceral leishmaniasis (VL) is highly

influenced by environmental factors. A studywas carried out in 2001-2002 to map thedistribution and risk of kala-azar in Sudan andinvestigate the history of infection byLeishmania donovani in Gedaref State, EasternSudan. Geographical Information Systems(GIS) were used to extract and map regressionresults for environmental variables of 190villages in Gedaref State. VL incidence in eachvillage was calculated from hospital records.Using logistic and linear multivariate regressionanalyses, models were developed to determinewhich environmental factors explain variabilityin VL presence and incidence. Average rainfalland altitude were proved to be the bestpredictors of VL incidence. The resultingmodels were mapped using GIS softwarepredicting both VL presence/absence andincidence at any locality in Gedaref State. Theresults of the models were confirmed byleishmanin skin testing carried out on 4850people living in 11 villages located in 3 differentkala-azar endemic areas in Gedaref State(Atbara, Rahad and Gedaref areas). Force ofinfection of L. donovani, varied greatly, bothin time and space, indicating that between1990 and 2000 in the Rahad area, the

incidence of L. donovani infection increasedby 4.5-fold, as compared to 1980-1990. Incontrast, the 1990-2000 incidence of infectionof L. donovani in the Atbara and Gedaref areas,dropped by 3.5- and 2.3-fold, respectively,from that observed for 1980-1990. Leishmanianskin test results also indicated a significantvariation in the exposure of different tribes,sexes and age groups. Results of anentomological survey showed that the onlyvector in the region is Phlebotomus orientalisand the village habitats are the main siteswhere people contract the infection.

PublicationsElnaiem DA, et al. Risk mapping of visceral

leishmaniasis: The role of local variation of rainfalland altitude on presence and incidence of kalaazar in eastern Sudan. Am. J. Trop. Med. Hyg.,2002, 66:6.

BackgroundThe present work describes an attempt

to develop a detailed eco-epidemiologymodel for mapping the distribution andincidence of VL at the village level inGedaref State in relation to differentenvironmental factors. In addition toproviding the first detailed map describingthe occurrence and incidence of thedisease in this important VL focus, thestudy also investigates how local variationsin environmental factors affect the diseaseburden in different endemic villages.


Study area Gedaref State is borderedin the east by Ethiopia, in the south andwest by the River Rahad, and in the north-east by the Atbara River. The region is aflat plain with almost no relief, and theprincipal soil type is vertisols. The climateis tropical continental, with an estimatedannual rainfall of 400-1400 mm. The yearis sharply divided between the rainyseason, June-October, and the dryseason, November-May. The meanminimum temperature is 21.0 °C in therainy season and 18.3 °C in the dry

Risk mapping ofvisceral

leishmaniasis inSudan

Sudan Gedaref State, Eastern

Sudan


Small GrantsScheme

(SGS) 2000 No. 55

Principal InvestigatorDr Dia El-Din Ahmed

ElNaiemDepartment of Zoology

Faculty of ScienceUniversity of Khartoum

Khartoum, Sudane-mail: [email protected]

The models developed in this study provide detailed mappingof classified incidence of VL in Gedaref State. The fact that themodels were derived from environmental variables resulted in theproduction of risk maps that could predict disease burden in areasnot covered by the initial data.

The risk maps produced from this study should be of great valuefor planning locations of treatment centres, for finding appropriateplaces for human settlement, and for deciding where to extendthe control programmes.

The produced models could be used to predict VL presence andincidence in others areas of Sudan where the disease is transmittedby the same vector.

The novel approach of this study can be used for other parts ofthe world to predict and map VL transmitted by different vectors.Such studies would provide a global understanding of the VLproblem and help prioritize control programmes.



Epidemiology

26 27

season; corresponding maxima are 37.3 °C and40.6 °C. The natural vegetation is dry savannawoodland. The main indigenous trees are Balanitisaegyptica, Acacia senegal, A. mellifera, Combretumspp., Calotropis procera, as well as some riverinevegetation. Along the river banks some fruit orchardsare found. Dura, sesame, dockhon, and groundnutsare grown as cash crops over extensive areas. Thehuman population of Gedaref State belongs to manyethnic groups, most of whom have a recent history ofsettlement in the region.

Visceral leishmaniasis cases, humanpopulation and village location data The casedata analysed in this study were obtained from detailedrecords of 2 treatment centres established andoperated by MSF-Holland in Umkra'a, situated closeto the River Rahad, and in Kassab village, situatedclose to Gedaref town. Although a few patients werealso diagnosed in Gedaref and Hawata hospitals andin other rural dispensaries, most patients have beenreferred to the 2 MSF VL treatment centres as a resultof the high cost of treatment (estimated at US$ 170per patient).

Data on cases and human populations were initiallyhandled within Excel and SPSS software to calculatethe numbers of cases of VL reported to the treatmentcentres during 1996-1999. Data were then entered ina new file containing the names of villages, theircoordinates, councils, and the human population, andanalysed to determine annual incidence (per 1000people) in different villages. Coordinates of villagelocations were obtained from readings of a Magellanglobal positioning system and from maps producedby the South Kassala Agricultural Project.

Environmental data Environmental datacorresponding to the coordinates of each of the studyvillages were extracted from a number of satellitesources and digital databases by Arcview GIS softwarewith Spatial Analyst, and the public domain softwareWINDISP 3. The U.S. Geological Survey (USGS)hydrologic data set was used to obtain a detaileddescription of the topography of the area. Informationon vegetation status was obtained from data archivesof the vegetation sensor on board the French satellitesystem, SPOT. Ten daily images of 10 daily rainfallestimates for the years 1996-1998 were obtained fromthe Africa Data Dissemination Service and analyzedby Windisp 3 GIS software to obtain the averageannual rainfall for each village. Soil types of differentvillages were read from a map produced by the SouthKassala Agricultural Project and classified into 9classes. Through the use of Arcview GIS software,the distance of each village from each of the 2 treatmentcentres and the two seasonal rivers was calculated.

Stat ist ical and GIS analysis ofenvironmental and VL incidence dataCorrelation analysis was performed to determine therelationship between the incidence of the disease anddifferent environmental variables. Stepwise multivariateanalysis was then carried out by binary logistic andlinear regressions to determine predictor variablesaffecting the presence and incidence of VL,respectively. The produced models were then enteredinto the map calculator module of Spatial Analyst andused to create maps of probability of disease presenceand incidence.

Main study findingsThe mean incidence of VL per 1000 people was

6.91, with marked variation between different villages,ranging from 0 to 53.21. Clear clustering of highincidence villages and areas of low altitude and highrainfall zones was noticeable on the maps.

The results showed that distance from the river, thetopography, rainfall, and the minimum NormalizedDifference Vegetation Index (NDVI) are the mainenvironmental variables independently associatedwith the distribution and incidence of VL in GedarefState. These variables influence the populations ofthe vector and reservoir hosts of L. donovani byaffecting other microclimatic factors in the area.Phlebotomus orientalis is known to thrive in habitatscharacterized by the presence of B. aegyptica trees,A.senegal trees, and vertisols. The vector was alsofound to inhabit a "climate space" of rainfall of 400-1200 mm and of annual mean maximum dailytemperatures of 34-38 °C. Because most of the regionis covered by vertisol, it is not surprising that this factordid not seem to affect the distribution of disease withinthe region.

In all the analysis carried out in this study, annualrainfall appeared to be the most important predictivevariable affecting both the probability of presence andincidence of the disease. Rainfall may affect the vectorand reservoir hosts by affecting the vegetation, thetemperature, and the relative humidity.

Conclusions and recommendationsThe models developed in this study provide detailed

mapping of classified incidence of VL in Gedaref State.The fact that the 2 models were derived fromenvironmental variables resulted in the production ofrisk maps that could predict disease burden in areasnot covered by the intial data. It is suggested that thenovel approach of this study can be used for otherparts of the world to predict and map VL.

Viscer

al Le

ishma

niasis

AbstractThere have been contradictory reports

regarding the types of visceral leishmaniasisstrains prevalent in Iraq; and this variabilitymight explain the observed differences in theseverity of the disease and its response tochemotherapy. The present study wasconducted to identify the new isolated visceralstocks of leishmania, and its relation to thevisceral stocks from Ethiopia and theMediterranean region. Seven human visceralstocks isolated during the period 1988-1990and obtained from the cryobank weresubjected to isoenzymes characterization.During the period 1993-1994, primary isolationof leishmania was attempted from bonemarrow aspirates taken from 15 childrendiagnosed clinically and/or by indirectimmunofluorescent antibody test (IFAT).Primary isolation was attempted again duringthe period November 1994-May 1995. Bonemarrow cultures were done for 33 patients(4-28 months) whom were proven to havekala-azar by IFAT and/or bone marrow smear.Several manipulations were done trying toisolate the parasites.

Results According to the zymogramsobtained, the 7 isolates were divided into 3groups (African, Mediterranean and mixed).These results confirmed the coexistence ofthe Mediterranean Leishmania donovaniinfantum and the African L. donovani in Iraq.In comparison with earlier results, there wasa change in parasite type, which may accountfor the recent difficulty in parasite isolation.

Eighty percent of cases isolated during theperiod 1993-1994 showed positive primarycultures. There was some difficulty in primaryisolation, which was explained by straindifferences. When primary isolation wasattempted again, few parasites were detectedin the primary cultures of 3 out of the 33aspirates incubated in the semisolid medium.None of the manipulation improved parasiterecovery. Aspirates from 30 cases givenintraperitoneally into mice revealed parasitesin liver smears when the animals weresacrificed 3-6 weeks later, but the cultureswere negative. No stock was thus obtainedfrom these 33 cases of kala-azar.

Conclusion The African L . donovani coexistswith the Mediterranean L. donovani infantumin Iraq. Furthermore, there was evidence ofa change in the parasite towards strains thatare difficult to isolate.

BackgroundThe clinical picture of kala-azar in Iraq

is of the infantile type, which is primarilyseen in the Mediterranean area. It hasbeen noticed by many investigators thatchildren with visceral leishmaniasis showdifferences in the severity of the diseaseas well as in their response tochemotherapy. An earlier study conductedin Iraq, using isoenzyme electrophoresis,reported that only 8% of the humanvisceral stocks were identical to a WHOreference stock (MHOM/ET/67/HU3) fromEthiopia, while 92% of the stocks revealeda new GPI enzyme variant [1]. Otherstudies reported the heterogeneity ofvisceral stocks in the country and theirdissimilarity from Sudanese, Iranian, andTunisian stocks, and their similarity toreference stocks from Ethiopia [2]. In viewof the controversy between differentreports, there was a need to conduct thepresent study in order to identify the newisolated visceral stocks of leishmania,and its relation to the visceral stocks fromEthiopia and the Mediterranean region(MHOM/ET/67/HU3 and MHOM/TN/80/IPT1, respectively).

Identification of theaetiological agent of

visceralleishmaniasis in Iraq

IraqBaghdad


Small GrantsScheme

(SGS) 1992 No. 30

Principal InvestigatorDr Maysoon B Rassam

Department of BiochemistrySaddam College of Medicine

Baghdad, Iraq

Visceral LeishmaniasisI r a q


Molecular Epidemiology

Isoenzyme characterization confirmed the coexistence of theAfrican L. donovani together with the Mediterranean L. donovaniinfantum in Iraq.

Bone marrow aspirates of children diagnosed clinically and/orby indirect immunofluorescent antibody test (IFAT) showed a highfrequency of positive primary cultures. However, there was difficultyin the primary isolation, which was explained by strain differences.

Bone marrow cultures done for children proven to have kala-azar by IFAT and/or bone marrow smear yielded negative results.In comparison with earlier results, there was a change in parasitetype, which may account for the recent difficulty in parasite isolation.

28 29

Materials and methodsSeven human visceral stocks isolated during theperiod 1988-1990 and obtained from the cryobankof the Medical Research Centre at Saddam Collegeof Medicine were subjected to gel electrophoresis of5 enzymes: PGM, SPGDH, MDH, GPI and ME,together with the reference stocks from Ethiopia andTunisia: MHOM/ET/67/HU3 and MHOM/TN/80/IPT1.

During the period 1993-1994, primary isolation ofleishmania was attempted from the bone marrowaspirates of 15 children diagnosed clinically and/orby indirect immunofluorescent antibody test (IFAT).A few drops of the aspirate were usually introducedinto a semisolid medium or biphasic medium (brainheart infusion agar containing 0.8% glucose and 10%defibrinated rabbit blood as the solid phase withLocke's overlay). The cultures were incubated at26 °C and examined every 3 days. If no parasiteswere observed during the 3-week incubation period,they were discarded. The first subcultures were notas good as the primary cultures, and on the thirdsubcultures the promastigotes disappeared. In anattempt to rescue the primary cultures, 2 stocks wereinoculated intraperitoneally in mice. After 1 monththe mice were dissected and the parasites wererecovered in cultures of the liver and spleen after 5days. No evidence of growth was noted in the thirdsubculture.

Primary isolation was attempted again during theperiod November 1994-May 1995. Bone marrowcultures were done for 33 cases (4-28 months) provento have kala-azar by IFAT and/or bone marrow smear.The following manipulations were done to try to isolatethe parasites:

1- A semisolid medium containing 20% defibrinatedrabbit blood was used.

2- Eight aspirates were introduced into the mediumas well as on the same medium but containing 20%hamster liver or spleen extract. The extract wasprepared by adding Locke's solution to the tissue(1/5, v/v) followed by homogenization andcentrifugation.

3- Eight bone marrow aspirates were introducedinto EDTA-containing tubes to prevent bloodcoagulation. The samples were centrifuged andwashed twice with PBS. The pellets were re-suspended in Locke's and inoculated into thesemisol id medium as well as introducedintraperitoneally into mice (2 per sample).

4- Four bone marrow aspirates were diluted withPBS containing EDTA as anticoagulant, centrifuged,washed and resuspended in PBS, then injectedintraperitoneally into mice.

5- Glass syringes were used in 6 cases to excludethe possibility of monocyte sticking to the plasticsurface of the syringe.

Main study findings

Isolates obtained during 1988-1990 Accordingto the zymograms obtained, the 7 isolates weredivided into 3 groups:

Group 1 included the stocks RTC7, RTC8, RRLL45and the reference stock IPT1.

Group 2 included only one stock (RRLL7) whichwas identical to the reference stock HU3.

Group 3 included the stocks RRLL34, RRLL36 andRRLL37. These stocks had zymograms identical tothat for IPT1, except for GPI which was identical toHU3.

These results confirm the coexistence of theMediterranean L. donovani infantum and the AfricanL. donovani. The former was undetected in parasiteisolations made during the period 1978-1984. Thischange in parasite type may account for the recentdifficulty in parasite isolation. Group 3 may representhybridized strains evolved as a result of thecoexistence of the parent parasites in the vector orthe host.

Recent results During the period 1993-1994, 12out of 15 bone marrow aspirates (80%) showedpositive primary cultures. There was difficulty in theprimary isolation, which was explained by straindifferences.

Primary isolation was attempted again during theperiod November 1994-May 1995 and the resultsshowed the presence of few parasites in the primarycultures of 3 out of the 33 aspirates incubated in thesemi-solid medium. None of the manipulationsimproved parasite recovery. Aspirates from 30 casesgiven intraperitoneally into mice revealed parasitesin liver smears when the animals were sacrificed 3-6 weeks later, but the cultures were negative. Nostock was thus obtained from these 33 cases of kala-azar.

These results indicate a change in parasitestowards strains difficult to isolate. It has to bementioned that isolation of the parasite from thesandfly vector could not be performed because ofdifficulties in conducting field visits created by thesanctions.

References[1] Rassam MB, Al-Mudhaffar SA, Chance ML.

Isoenzyme characterization of Leishmania speciesfrom Iraq. Ann Trop Med Parasitol, 1979, 73(6):527-534.

[2] Al-Hussayni NK et al. Numerical taxonomy ofsome Old World Leishmania species. Trans R SocTrop Med Hyg, 1987, 81(4):581-586.

Viscer

al Le

ishma

niasis

AbstractThe present study aimed at identifying the

possible role of humans as a reservoir ofleishmaniasis and its implication in theepidemiology and control of the disease.

Study area: Three groups of populationsliving in different areas were selected; thefirst group consisted of the entire populationof a village in Karshola Town, Nuba Mountainsarea, western Sudan; the second groupconsisted of 89 high-risk individuals from ahighly endemic area in eastern Sudan, calledDinder Park; the third comparison group wasfrom Salala Village, eastern Sudan.

Data were collected from the 3 studiedgroups by leishmanin skin test, serology andclinical examination of the population. Theinitial visit was followed by 2 subsequentvisits, before and after the rainy season, andfollow-up data were collected. The presenceof parasite DNA was detected by PCR andsubsequent southern blotting. The kDNA wasamplified using species specific primers.

ResultsThe leishmanin skin test was positivein 50%, 53% and 70.5% of the populationsof Karshola, Salala and Dinder, respectively.Serology was positive in 0.3%, 9.6% and44.4%, respectively. PCR was positive in28.6% of the Dinder Park population only andthere was a significantly higher proportion ofclinical kala-azar cases in Dinder Park (15.8%)as compared to Karshola and Salala (0.13%and 3.85%, respectively). This study allowedthe identification of new foci of visceralleishmaniasis in the Nuba Mountains, western

Sudan, where low transmission is takingplace. Among the high-risk groups living inendemic areas, the geographical origin andethnicity of the subjects were superaddedfactors.

Conclusion These results confirm thegeneral preponderance of subclinical infectionto clinical disease, but pointed as well to thesusceptibi l i ty of certain ethnic andgeographical populations to kala-azar.

Publications1. Ibrahim ME et al. Kala-azar in a high transmissionfocus: an ethnic and geographic dimension. Am JTrop Med Hyg, 1999, 61(6):941-944.

2. Ibrahim ME et al. Molecular and immuno-epidemiology of low and high endemicity of visceralLeishmaniasis in Sudan. First World Congress onLeishmaniasis. Istanbul, 1997.

TrainingDr Nazar M Abdalla received a WHO/TDR

research fellowship in Dr Douglas Barker'slaboratory in Cambridge. He learned severaltechniques such as labelling of probes andhybridization that he introduced in 2 institutionsin Sudan: the Unit of Genetics and MolecularParasitology, National Health Laboratory,Khartoum, and the University of Gezira, hishome institution.

BackgroundLeishmaniasis is one of the major

health problems in the Sudan. Severalstudies have been conducted, mainly inthe eastern and southern Sudan. Inwestern Sudan, foci of leishmaniasiswere recognized during the 1970s.Studies performed in the differentendemic areas have reported that themajority of the inhabitants of these areascontract infection but do not develop theclinical disease, i.e. remain subclinical.However, there was evidence that aconsiderable proportion of theseindividuals harbour the leishmaniaparasite in their peripheral blood [1]. Thisfinding was the basis of conducting thepresent study that aimed at identifying

This study allowed the identification of new foci of visceralleishmaniasis in the Nuba Mountains in western Sudan, wherelow transmission is taking place.

These results confirmed the general preponderance of subclinicalinfection to clinical disease, thereby indicating that the onset ofleishmaniasis outbreaks depends on a critical mass of susceptibleindividuals and the availability of human carriers (reservoir).

Results obtained from high-risk individuals living in endemicareas pointed to the ethnic and geographical determinants ofvisceral leishmaniasis.




The polymerasechain reaction, skintesting and serologyin the establishment

of humans as apossible reservoir ofleishmania infection

Sudanwestern and eastern

Sudan


Small GrantsScheme

(SGS)1996 No. 25

Principal InvestigatorDr Muntaser E Ibrahim

Unit of Genetics and MolecularParasitology

National Health LaboratoryMinistry of HealthKhartoum, Sudan

30 31

the possible role of humans as a reservoir ofleishmaniasis and its implication in the epidemiologyand control of the disease.

Materials and methodsThree population groups living in different areas

were selected: the first group consisted of the entirepopulation of a village in western Sudan, the secondgroup consisted of high-risk individuals living in ahighly endemic area in eastern Sudan, and the thirdgroup was from Salala Village in eastern Sudan.

The first study site was selected from the healthrecords of the Nuba Mountains area. Accordingly,an accessible village with the highest prevalence ofleishmaniasis with a population of 1000 individualswas selected. The village, El Mougaful near KarsholaTown, western Sudan, was visited in order to takethe consent of the population, perform the census,and collect baseline information in a pre-designedquestionnaire. Leishmanin skin test, serology andclinical examination of the population were alsocarried out. The initial visit was followed by 2subsequent visits, before and after the rainy season,and follow-up data were collected.

Another group consisting of 89 high-risk individualswere from a highly endemic area in eastern Sudan.These individuals were mainly healthy game wardensand army soldiers, and they were subjected to PCR,leishmanin skin test and serology to determine theincidence of kala-azar and sub-clinical infection amongthem. The presence of parasite DNA was detectedby PCR and subsequent southern blotting. The kDNAwas amplified using species-specific primersdeveloped by the group of Dr DC Barker inCambridge. PCR products of 800 bp were obtainedin positive samples. Comparison was also made withdata obtained from Salala Village in eastern Sudanwhere a 6-year longitudinal study on kala-azar wasjust completed during the conduction of the presentstudy.

Main study findingsThe incidence of the clinical disease (kala-azar) in

El-Mougaful village, Karshola, was around 0.1%, andthe leishmanin rate was high (50%), although therewas no evidence of cutaneous leishmaniasis. Thedirect agglutination test (DAT) was positive in lessthan 0.3% of the villagers and positive cases recordeda very high titre (>25 000). PCR was performed forthe entire village. And despite the suboptimal qualityof the DNA extracted, positive samples of Leishmaniadonovani were obtained and confirmed by southernblotting technique.

Preliminary PCR screening in Khartoum wassuccessful in some samples, indicating the presenceof the leishmania parasite. There were difficulties inanalysing the larger sample in Cambridge mainly

due to the low parasitaemia of the area andinadequate sampling methods.

In Dinder Park and Karshola Village, 2 sets ofserological tests were used, DAT and k39. The latterwas more sensitive as evidenced by its picking upsubtle cases of infection with Leishmania donovani.

This study allowed the identification of new foci ofvisceral leishmaniasis in the Nuba Mountains inwestern Sudan, where low transmission is takingplace. The 3 different comparison foci were situatedwithin the same Acacia belt running through central,eastern, and western Sudan. The Leishmania-positiverates were comparable in the 3 studied areas. Itappears that the onset of outbreaks depends on acritical mass of susceptible individuals and theavailability of human carriers (reservoir). Amonggame wardens of Dinder Park, the geographical originand ethnicity of the subjects were additional factors.These results pointed to the susceptibility of certaingroups to the disease. It was recommended to confirmthis observation by future in-depth studies intensivelyinvestigating the role of the human host in theepidemiology of the disease.

References[1] Ibrahim et al. Visceral leishmaniasis in a national

game reserve: infection and exposure in a high riskgroup. First European Congress of Tropical Medicine,Hamburg, Germany, 1995.

Viscer

al Le

ishma

niasis

AbstractThe objectives of this study were to

characterize parasite isolates from visceralleishmaniasis (VL), post kala-azar dermalleishmaniasis (PKDL) patients and pairedisolates from patients who developed VLfollowed by PKDL, as well as the parasitedeterminants of PKDL.

A total of 270 confirmed VL patients, aged3-12 years old, and residing villages inGedaref State, Eastern Sudan, were includedin the study. Lymph node aspirates werecultured on NNN media.

Two methods of DNA extraction were used:direct lysis of promastigotes and the Chelexextraction method. The DNA band profilesand restriction fragment length polymorphismwere determined using PCR. Electrophoresiswas done on promastigotes, growing VL andPKDL were lysed and the lysate separated,then the protein bands were stained. ELISAwas used for the measurement of IgM, IgGand IgG subclasses in both VL and PKDLsera samples.

Results The culture of the parasites on theNNN medium revealed that 44.8% of parasitesisolated from VL grew on this mediumcompared to 26.7% of those isolated fromPKDL patients. The Chelex method provedto be superior to the direct lysis method inDNA extraction and amplification. All isolates

typed by PCR were identified as Leishmaniadonovani complex with amplification of thecharacteristic 800 bp band as compared withthe Ld 2S reference strain.

The PKDL restriction profiles wereheterogenous, indicating that PKDL is causedby more than one Leishmania donovani strain(clone). This finding has not been previouslyreported and has an important epidemiologicalimplication.

Two protein bands were invariably detectedin VL and PKDL isolates, but the 65 000dalton band was more intense in PKDLisolates.

Anti-leishmania IgM titres were significantlyhigher in VL patients compared to normalcontrols.

Anti-leishmania IgG antibodies were alsosignificantly higher in VL and PKDL samplescompared to resistant VL patients; on theother hand, there was no significant differencebetween VL and PKDL regarding IgG levels.There were differences between VL, PKDL,or resistant VL patients regarding the levelsof anti-leishmania IgG subclasses antibodies.

TrainingTwo postgraduate students were trained

on each of the following techniques: parasitei so la t i on , cu l tu re , PCR pa ras i techaracterization and immunoblotting and onELISA monitoring of antibody classes andsubclasses in VL and PKDL sera.

A laboratory technician was trained onparasite culture and ELISA.

BackgroundPost kala-azar dermal leishmaniasis

(PKDL) is a serious complication ofvisceral leishmaniasis (VL), and ischaracterized by skin lesions (papules,macules, and/or nodules) on the exposedparts of the body. Cases were alsoreported in individuals without a historyof clinical VL. PKDL is considered ofepidemiological importance as casesprovide a rich source of infection forsandflies. Furthermore, it is often clinicallymisdiagnosed as leprosy, therebydelaying proper management of thecondition.

Culture of the parasites on NNN medium was successful in44.8% of parasites isolated from VL compared to 26.7% in PKDLpatients.

The Chelex method was proved to be superior to the direct lysismethod in DNA extraction and amplification.

All isolates typed by PCR were identified as L. donovani complex.Furthermore, PKDL restriction profiles were heterogenous indicatingthat PKDL is caused by more than one Leishmania donovanistrain (clone).

Anti-leishmanial IgM titres were significantly higher in VL patientscompared to normal controls and there was no significant differencebetween VL and PKDL regarding IgG levels, but they weresignificantly higher in these patients compared to resistant VLpatients. IgG1, and IgG3 subclasses were significantly higher inall VL and PKDL compared to IgG2 and IgG4.




Parasite and hostimmune factors

associated with postkala-azar dermal

leishmaniasis(PKDL) in Sudan

Sudan Gedaref State, Eastern

Sudan

Study period:November 1998–December 2000

Small GrantsScheme

(SGS) 1998 No. 28

Principal InvestigatorDr Maowia M MukhtarDepartment of Molecular

BiologyInstitute of Endemic Diseases

University of KhartoumKhartoum, Sudan

32 33

The pathogenesis of PKDL is not known and severalhypotheses have been proposed including a uniqueparasite species or strain, differential expression ofpeculiar genes in the parasite during the pathogenesisof PKDL, and the involvement of host immuneresponse. The existence of a peculiar parasite wasruled out. Parasites isolated from PKDL patients hadan identical isoenzyme pattern of VL isolates,indicating that PKDL is caused by a typical L. donovaniparasite. However, the cloning of a DNA minicirclesequence of 560 bp from a Leishmania donovaniPKDL isolate was proved to be specific for PKDL.

Regarding the role of the host immune response,immunological investigaton of PKDL lesions showedmarginal to massive infiltration of mononuclear cellsdepending on the duration of illness and lesion type.Analysis of the humoral immune response in VL andPKDL patients showed the induction of leishmaniaspecific antibodies of IgG class with the dominationof IgG1, followed by IgG2, IgG3 and IgG4. Thesubclasses have different specificity to variableleishmania antigens. Furthermore, Interleukin IL 10has been identified as an immunological predictor ofPKDL.

The objectives of this study were to characterizeparasite isolates from VL, PKDL patients and pairedisolates from patients who developed VL followed byPKDL, as well as the parasite determinants of PKDL.

Materials and methodsDuring 12 field visits, 270 individuals out of 342

suspected VL patients had a confirmed VL diagnosisusing the direct agglutination test (DAT). The majorityof patients (90%) were aged 3-12 years, and theywere residing in villages in Gedaref State, easternSudan. All patients were treated with Pentostamunder medical supervision.

Parasite cultures Lymph node aspirates fromthe 270 confirmed VL patients and 30 PKDL patientswere cultured on NNN media as a primary isolationmedium. Mass cultures were done using RPMI 1640media supplemented with 20% heat-inactivated fetalcalf serum (FCS) and antibiotics. Growth of theparasite was detected by examining the culture underan inverted microscope.

Characterization of the isolates Two methodsof DNA extraction were used: direct lysis ofpromastigotes and the Chelex extraction method.

Polymerase chain reaction Two sets of primerwere used: AJS3/BDYand RH1/RH2. This wasfollowed by PCR amplification, then the amplifiedDNA fragments were separated, stained andvisualized to determine the DNA band profiles.

Restriction fragment length polymorphism(RFLP) A minicircle of selected L. donovani isolates

was amplified by PCR using AJS3/DBY primers. Theproduct was digested with a restriction enzyme andthe restriction profile examined.

SDS PAGE Sodium dodocyl sulfate polycramidegel electrophoresis was done using the Lamellaemethod. Whole promastigotes of growing VL andPKDL were lysed and the lysate separated, then theprotein bands were stained.

Analysis of antibody profiles of visceraland PKDL patients ELISA was used for themeasurement of IgM, IgG and IgG subclasses inboth VL and PKDL sera samples.

Main study findingsCulture of the parasites on NNN medium revealed

that 44.8% (121/270) of parasites isolated from VLgrew on this medium compared to 26.7% (8/30) ofthose isolated from PKDL patients, and two isolatesgrew directly from skin snips of PKDL lesions. TheChelex method was proved to be superior to thedirect lysis method in DNA extraction from 121 VLand 8 PKDL isolates and their amplification. Moreover,the amplified bands obtained by this method wereintense and sharp. All isolates typed by PCR wereidentified as L. donovani complex with amplificationof the characteristic 800 bp band as compared withthe Ld 2S reference strain.

The PKDL restriction profiles were heterogenous,indicating that PKDL is caused by more than oneLeishmania donovani strain (clone). This finding hasnot been previously reported and has an importantepidemiological implication.

More than 5 protein bands were identified in all theisolates; out of these bands 2 were invariably detectedin VL and PKDL isolates. The 65 000 dalton bandwas more intense in PKDL compared to the VLisolates.

Regarding the antibody profile of patients, anti-leishmania IgM titres were significantly higher in VLpatients compared to normal controls.

Anti-leishmania IgG antibodies were significantlyhigher in VL and PKDL samples compared to resistantVL patients; on the other hand, there was no significantdifference between VL and PKDL regarding IgGlevels.

Study of the lgG subclasses revealed the following:IgG1 was significantly elevated in all VL and PKDLpatients and in 90% of resistant VL (RVL) patients;IgG2 in all PKDL patients, 91% of VL and 70% ofRVL patients; IgG3 in all VL, 93% PKDL patients andin 70% of RVL patients; and IgG4 in 89% of VL and50% of RVL patients.

Viscer

al Le

ishma

niasis

AbstractIn this study, 406 dogs with owners and with

no anti-Leishmania antibodies from Parikhanvillage in Meshkin-Shahr district were physicallyexamined. Three hundred and fifteen healthyseronegative dogs with no response toleishmanin were selected and randomlyinjected by either alum-precipitated autoclavedLeishmania major (ALM) vaccine (200 µg) ofLeishmania protein precipitated in roughly 620µg of aluminum hydroxide mixed with BCG(roughly each dog received 2 million CFUs)or injected with PBS as a control. Bloodsamples were taken from the dogs 4 monthspost injection and were skin tested withleishmanin (LST). The injected dogs werefollowed-up for 16 months.

Results The examination of the site of injectionshowed that the vaccine is well tolerated andno severe reaction was observed. The LSTconversion rate was significantly higher in thevaccinated group compared to the placebogroup. The efficacy evaluation performed at16-month post vaccination indicated thatadministration of this vaccine plus BCG had54.6% protective fraction effects against caninevisceral leishmaniasis.

BackgroundHuman visceral leishmaniasis (VL)

caused by Leishmania infantum isendemic throughout the north-westernand southern parts of Iran. Dogs are themain reservoir of infection and manyseropositive dogs exist in endemic areas.However, treatment of infected dogs with

antimonials proved to be noneffective. Inview of the growing public healthimportance of zoonot ic v isceralleishmaniasis (ZVL) and problemsencountered in its control, new preventivestrategies seemed necessary. Severalstudies performed on dogs experimentallyand naturally infected with L. chagasi andL. infantum indicated that many animalssurvive the infection and develop a cellularimmune response that probably resultedin resistance. These findings suggestedthat a vaccine against canine visceralleishmaniasis is quite feasible. This studywas conducted to test the efficacy of asingle injection of aluminium hydroxide-autoclaved Leishmania major (Alum-ALM)vaccine mixed with BCG against caninevisceral leishmaniasis, in Meshkin-Shahr,Iran.


Study site Parikhan village is located5 km west of Meshkin-Shahr, Ardebilprovince, north-western Iran. The villagehas a population of 8000, and has morethan 800 leashed dogs. The selection ofthe study site was based upon previousreports on the seropositivity of its childrenand dogs reservoir to infection.

Vaccine The alum-precipi tatedLeishmania major vaccine (Alum-ALM)was produced at Razi Institute, Hesarak,Iran. Autoclaved Leishmania majorvaccine (ALM) was produced frompromastigotes of L. major (MRHO/IR/76/ER) from a seed bank. Thepromastigotes were grown in RPMIsupplemented with 20% FCS at 25 °C ina roux bottle. The promastigotes wereharvested at the stationary phase, andwere washed 5 times with phospate-buffered saline (PBS) and stored at -70 °C.

Aluminium hydroxide containing 22.22mg aluminium ion per millilitre wasproduced and autoclaved at 121 °C for

Field efficacyvaccine trial withalum-precipitatedLeishmania majorantigen againstcanine visceralleishmaniasis inMeshkin Shahr

district, north-westIran


Meshkin-Shahr district,North-west Iran

Study period:January 1999–July 2001

Small GrantsScheme

(SGS) 1998 No. 38

Principal InvestigatorDr Mehdi Mohebali

Dept. of Medical ParasitologySchool of Public Health and

Institute of Public HealthResearch

Tehran University of MedicalSciences

Tehran, Islamic Republic of Iran

This study reported the preliminary results in the developmentof a vaccine for canine visceral leishmaniasis. The leishmaninskin conversion rate among vaccinated dogs was higher than forthe placebo group.

The vaccine was well tolerated and no severe reaction wasobserved.

Administration of this vaccine to canids provided a protectiveeffect of 54.6% against canine visceral leishmaniasis.

Further evaluation of this vaccine trial is recommended.

Visceral LeishmaniasisI s l a m i c R e p u b l i c o f I r a n


Vaccine trial

34 35

15 minutes. The alum solution was mixed with anequal volume of ALM and aliquoted 0.9 ml in eachvial of small vials of 9 ml capacity. The vials wereautoclaved under similar conditions, the finalconcentration of Leishmania protein is 3.6 mg/ml.Freshly re-suspended BCG was added and mixedwell just prior to injection, and each dog received 200µg of Leishmania protein precipitated in roughly 620µg of aluminium hydroxide.

BCG and the leishmanin skin test (LST) wereproduced at the Pasteur Institute, Tehran, Iran.Leishmanin was prepared from the same isolate ofL. major that was used for the vaccine preparation.

Experimental design This study was a double-blind randomized trial. Eligibility criteria for the dogswere: age older than 1 month, ownership of the dogduring the study period (at least 2 years); freedomfrom current disease based on veterinary examination,and negative antibody titre against L. infantum.

A total of 406 dogs were physically checked by avet, and blood samples were collected from 360dogs. LST indicated that 325 dogs had no antibodytitre against L . infantum antigens. Based on physicalexamination and serological results, a total of 320dogs were randomly allocated to either of 2 vaccines:0.1 ml of Alum-ALM vaccine (200 µg protein perdose) mixed with BCG (2x106 colony forming units(CFU) per dose) or with 0.1 ml of PBS alone. A singleinjection was administered intradermally in the forearmof the dogs. A safety follow-up was performed 30-45days post vaccine injection. The injected dogs werechecked by a vet and the site of inoculation wasexamined. The type and size of the lesion inducedby the injection was recorded. Blood samples werecollected from 242 dogs 4 and 16 months postvaccination to determine the humoral immuneresponse induced by the vaccine.

The control group were dogs that were vaccinatedand did not develop the disease and non-vaccinateddogs (with or without disease).

Main study findings

Post-vaccination follow-up A total of 246 injecteddogs were checked after vaccination, and in morethan half of injected dogs, no lesion was detected atthe injection site. However, an ulcer or an indurationwere induced by the injection in 45.3% and 2.9% ofdogs, respectively. The size of the lesion (ulcer orinduration) ranged from 2-10 mm in diameter.

Immunological studies The antibody responsemeasured by ELISA post-vaccination revealed that95.4% of dogs were negative compared to 95.9%when tested by the DAT. Leishmanin skin testingpost vaccination assessed the cell mediated immunityresponse induced by vaccination. The LST revealedthat in 246 tested dogs, 191 were negative (77.6%),

while the remainder showed a response ranging from5-10 mm in diameter. Blood samples collected fromevery injected dog 16 months post vaccination wereused to detect anti-Leishmaniasis antibodies by DATand ELISA techniques. The results showed that 5.9%of dogs injected with ALM mixed with BCG and 13% of dogs receiving PBS alone, were seroconverted.

Conclusions and recommendationsAlum ALM mixed with BCG had 54.6% protective

fraction against canine VL. Furthermore, the vaccineprepared proved to be a safe vaccine. These resultsprovide an initial step in the vaccine preparation fordogs.

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AbstractField collected sandflies were tested for

susceptibility to different insecticides usingWHO test kits. The susceptibility ofPhlebotomus papatasi adults to differentinsecticides was tested in 2 successive years,the summers of 1994 and 1995. Tests werecarried out under field conditions. Insecticidestested were DDT, dieldrin, malathion,propoxur, permethrin and deltamethrin,representing different insecticide groups.

ResultsThe sandflies which were tested not only

showed the highest susceptibility toinsecticides among all other Egyptiansandflies, but also among sandfly populationstested in several other parts of the world. Thehigh susceptibility level of the adult P. papatasipopulation in this study was attributed to thefact that this area had no history of insecticideapplications. The fact that the adult P. papatasipopulation tested in this study was still highlysusceptible to DDT and dieldrin raised thepossibility of using these 2 compounds insandfly control programmes.

PublicationsFahmy AR et al. Insecticide susceptibility

status of field populations of sandfly Phlebotomuspapatasi in the Sinai peninsula, Egypt. Geneva,World Health Organization, 1996 (unpublished

document WHO/LEISH/96.38; available onrequest from Division of Control of TropicalDiseases, World Health Organization, 1211Geneva 27, Switzerland).

BackgroundIn Egypt, phlebotomine sandflies are

important vectors of the phlebotomusfever (sandfly fever) virus and 2 forms ofLeishmania, Leishmania major and L.infantum [1]. L. major is transmitted bythe sandfly Phlebotomus papatasi, thevector of cutaneous leishmaniasis (CL)in North Sinai, and L. infantum istransmitted by P. langeroni, the vector ofvisceral leishmaniasis (VL) in Alexandria.An outbreak of a CL epidemic in NorthSinai was documented in 1991 [1].Application of residual insecticidesremains the principal method for thecontrol of sandflies, especially indeveloping countries. Since sandflieshave not been principally targeted incontrol operations, the control of sandflieshas been often a by-product ofantimalarial spraying. In addition to DDT,malathion, fenitrothion, propoxur, dieldrin,lindane and synthetic pyrethroids havebeen successfully used for the control ofsandflies. However, the spectrum ofsusceptibility of sandflies to a range ofinsecticides has not yet been studied.The objective of this work was todetermine and monitor the susceptibilitylevel of sandfly field populations in theSinai peninsula to different insecticides.

Materials and methodsAdult sandflies were collected from Sad

El-Rawafaa, North Sinai. CDC light trapswere used for overnight insect collection,and insects were allowed to settle for afew hours for conditioning together with10% sucrose solution. Only active healthyinsects were tested against a series ofinsecticide concentrations and differentexposure times. Tested insects wereidentified as Phlebotomus papatasi

Susceptibity ofPhlebotomus major,

P. papatasi and P.sergenti to various

insecticides inNekhel, Sinai, Egypt

EgyptNorth Sinai


September 1995

Small GrantsScheme

(SGS) 1992 No. 9

Principal InvestigatorDr Magdi Shehata

Leishmania UnitResearch and Training Centre

on Vectors of DiseasesAin Ahams University

Cairo, Egypt

Sandfly collection revealed that Phlebotomus papatasi was theonly sandfly species identified in the study area.

The Phlebotomus papatasi population was highly susceptibleto all insecticides: DDT (chlorinated hydrocarbons), dieldrin(cyclodiene), malathion (5%) (organophosphate), propoxur (0.1%)(carbamate), deltamethrin (0.025%) and permethrin (0.25%)(pyrethroids).

The high susceptibility level of the adult Phlebotomus papatasipopulation in this study could be attributed to the fact that thisarea had no history of insecticide applications.

The fact that the adult Phlebotomus papatasi population testedin this study was still highly susceptible to DDT and dieldrin raisedthe possibility of using these 2 compounds in sandfly controlprogrammes.

Visceral LeishmaniasisE g y p t


Vector control

36 37

according to Lane, 1986 [2].

Insecticides Impregnated papers with differentconcentrations of DDT (chlorinated hydrocarbons),d i e l d r i n ( cyc lod iene ) , ma la th i on (5%)(organophosphate), propoxur (0.1%) (carbamate),deltamethrin (0.025%) and permethrin (0.25%)(pyrethroids) were supplied by the WHO. For controltests, insects were exposed to impregnated papersprepared in the laboratory: resiella oil (fororganochlorines and pyrethroids) and olive oil (fororganophosphates and carbamates).

Exposure techniques WHO standard testtechniques were used [3]. The sandflies weresubjected to different concentrations, or differentexposure times at the same concentration. At least3 replicates of 20 to 25 insects were tested for eachconcentration or exposure time. Mortality counts weretaken after a 24-hour recovery period. Control insectswere exposed at the maximum exposure time usedfor the tested insects.

Statistical analysis Test mortality percentageswere corrected using Abbott's formula (controlmortality percentages >5%) and subsequentlyanalysed by probit analysis. Goodness of fit ofregression lines was measured by the Chi-squaretest. LC50 or LT50 values were estimated and theslope values of the regression lines were calculated.

Experimental conditions Testing was performedduring the summer (May-October) in the same localityfor 2 consecutive days, in 1994 and 1995. Seasonalpeaks of sandfly abundance in this area were reportedto occur between June and September. However, inthe 1994 season, the adult density was unexpectedlylow due to floods in the study area so that tests withDDT and dieldrin could not be performed. Also, thenumber of insects used for every test was relativelysmaller than that used in the 1995 season.Susceptibility tests were carried out in a room whichwas specially prepared and free of insecticide. Duringthe test, the temperature was maintained at 27-30°C and the relative humidity (RH) at 80-90%.

Main study findingsThe results obtained in this study revealed that the

tested population was highly susceptible to allinsecticides as evidenced by the discriminatingconcentrations or times. They also indicated that thesusceptibility of adult sandflies to different insecticidesdid not show any significant difference between 1994and 1995. However, little difference (less than 3-fold)in the LT50 values was detected with malathion andpropoxur between the 1994 and 1995 tests. On theother hand, there was no remarkable differencebetween the LT95 values of the same insecticides.The high susceptibility level of the adult P. papatasi

population in this study could be attributed to the factthat this area had no history of insecticide applications,unlike other Egyptian regions that were subjected tomosquito control measures such as the El-Agamyarea, or to insecticide applications for the control ofagricultural pests as in the Nile Delta and GizaGovernorate. The sandflies which were tested notonly showed the highest susceptibility to insecticidesamong all other Egyptian sandflies, but also amongsandfly populations tested in several other parts ofthe world. The fact that the adult P. papatasi populationtested in this study was still highly susceptible toDDT and dieldrin raised the possibility of using these2 compounds in sandfly control programmes. Theslight differences in susceptibility based on LC50 andLT50 reported in this study do not reflect a realresistance difference because there were nosignificant differences among LC95 or LT95 values.This slight difference may have been due tofluctuations in tolerance and seasonal variations inthe population.

References[1] Mansour NS et al. Cutaneous leishmaniasis in

the peace keeping forces in East Sinai. J Egypt SocParasitol, 1989, 19:725-733.

[2] Lane RP. The sandf l ies of Egypt(Diptera:Psychodidae), Bull Br Mus Nat Hist, 1986,52:1-35.

[3] Instructions for determining the susceptibility orresistance of blackflies, sandflies and biting midgesto insecticides. Geneva, World Health Organization,1981 (unpublished document WHO/VBC/81.810 ;available on request from World Health Organization,1211 Geneva 27, Switzerland), p.1-6.

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AbstractPrevious studies have confirmed the efficacy

of Scalibor dog collars in protecting againstsandfly bites. This justified the same researchteam to carry out a community-basedintervention trial using deltamethrin-impregnated dog collars during thetransmission season in the endemic zone ofzoonotic visceral leishmaniasis. Anintervention study was conducted in Kalaybarand Meshkin-Shahr, north-west Iran, wherebychildren aged 1-10 years were surveyed usingthe DAT and leishmanin skin test (LST). Alldomestic dogs were also screened by theDAT. Villages were matched for diseaseprevalence and villages in each pair wererandomly allocated as either an interventionor control village.

Reults There was a significant difference inthe seroconversion rate among dogs in theintervention compared to the control villages,indicating that the collars had reduced therisk of dog seroconversion.Both the DATseroconversion rate and LST conversion ratein children were lower in the intervention thanin the control villages, suggesting that collarsmay have provided protection for childrenagainst the risk of zoonotic visceralleishmaniasis.

PublicationsGavgani et al. Effect of insecticide-impregnated

dog collars on incidence of zoonotic visceralleishmaniasis in Iranian children: a matched-cluster randomised trial. Lancet, 2002,360(9330):374-379.

BackgroundIn many foci of zoonotic visceral

leishmaniasis (ZVL), domestic dogs areimportant reservoir hosts of the causativeleishmania parasites transmitted byphlebotomine sandfl ies (Diptera:Psychodidae). Experimental trials carriedout under field conditions in Iran haveconfirmed the efficacy of Scalibor dogcollars in protecting against sandfly bites[1]. These results justified the sameresearch team in carrying out acommunity-based intervention trial usingdeltamethrin-impregnated dog collarsduring the transmission season in theendemic zone of zoonotic visceralleishmaniasis in Kalaybar and Meshkin-Shahr in north-west Iran.


Study area The study area was 21villages in the districts of Kalaybar andMeshkin-Shahr in north-west Iran.

Design An intervention study wasconducted whereby children aged 1-10years were surveyed using the DAT andleishmanin skin test (LST). All domesticdogs were diagnosed by the DAT, whileall stray dogs were poisoned withstrychnine by the village health workers.Within each district, the trial villages wereranked and ordered in pairs withcomparable transmission rates. Villagesin each pair were randomly allocated asthe intervention or control group, wherebya matched community design wasconstructed. Prior to each village survey,a meeting was held with local communityleaders to explain the objectives andlogistics of the intervention study.

For testing with the direct agglutinationtest (DAT), finger-prick blood sampleswere spotted on filter-paper and the DATantigen was prepared by the standardmethod from Leishmania infantumpromastigotes as described by El-Harith[2]. The cut-off point for positive infections

A village interventiontrial for reducing

Leishmaniasisinfantum

transmission in Iranwith insecticide-impregnated dog

collars


Meshkin-Shahr andKalayabar,

North - West Iran

Study period:December 1999–

January 2001

Small GrantsScheme

(SGS) 1999 No. 25

Principal InvestigatorDr Abdosamad Mazloumi

GavganiDepartment of Parasitology and

ImmunologyFaculty of Medicine

Tabriz University of MedicalSciences

Tabriz, Islamic Republic of Irane-mail :

[email protected]



Vector control

The use of deltamethrin-impregnated dog collars was successfulin the interruption of transmission in dogs, which are reservoirhosts for Leishmania parasites as evidenced by the reduction inthe DAT seroconversion rates after application of this deviceduring the transmission season.

Both the DAT seroconversion rate and LST conversion ratewere lower in the intervention than in the control villages, suggestingthat collars may have provided protection for children against therisk of zoonotic visceral leishmaniasis.

38 39

in humans was 1/1600, and in dogs was 1/800.Children with a high DAT response ³1/3200 receivedparticular clinical attention and were treated for VL.

The LST was derived from L. major and preparedat the Pasteur Institute of Iran. The volar surface ofone forearm was injected intradermally with 0.1 mlantigen (at a concentration of 107 promastigotes/ml)and a control solution (0.1 ml sterile pyrogen-freePBS with 0.01% thimersal) injected into the arm. TheLST response was measured 48 hours later, and aninduration width of 5 mm or greater was taken as apositive response.

Main study findings

Pre-intervention baseline survey The resultsof this survey confirmed the matching factor of thestudy design, as there was no significant differencebetween the control and intervention villages regardingthe seroprevalence rates of VL in humans tested bythe DAT and LST, as well as in dogs tested by theDAT.

In the 9 control villages, out of the 84% (1225/1493)of children tested by the DAT, 92 were positive (7.5%;range: 3.5-10.5%). In the 9 intervention villages, outof the 86% (1369/1593) of children tested, 107 werepositive (7.8%; range: 5.4-11.7%).

Testing with the LST indicated that in control villages,200/294 children were positive (21.6%; range: 12-26%), compared to 223/967 (23.1%; range:12-27%)in the intervention villages.

The seroprevalence in dogs was 64/562 (11.4%;range: 8-13%) in the control villages compared to56/426 (13.1%; range: 6-19%) in the interventionvillages.

Intervention All 18 villages were revisited in March2000 prior to the sandfly season (May-September),during which the Scalibor collars were applied to alldomestic dogs aged at least 7 weeks in theintervention villages. Spare collars were left for villagehealth workers for replacing lost ones or for applicationon puppies turning 7 weeks old. Health workersvisited each household in all 18 villages every 2weeks to keep a record of changes in the dogpopulation or any problems with the collars.

Post-intervention survey (December 2000-January 2001) During the first year, the rate ofdog loss and replacement was similar in the controland intervention villages, though it was significantlyhigher amongst the DAT-positive than the DAT-negative dogs. There was a significant difference inthe seroconversion rate amongst dogs in theintervention villages compared to the control villages(3.2% and 6.2%, respectively), indicating that thecollars had reduced the risk of dog seroconversionby 49%.

Regarding infection rates in children, there werecomparable sero-recovery rates in the interventionand control villages (12.9% and 12.4%, respectively).However, the seroconversion rate in the interventionwas lower than in the control villages (1.6% and2.8%, respectively).

The LST conversion rate in the intervention villageswas 1.2% compared to 2.0% in the control villages,suggesting that collars may have provided 40%protection for children against the risk of LSTconversion.

The difference in conversion rates obtained by theDAT and LST could be explained by the fact thatLST conversion has a longer latency thanseroconversion. Therefore, it would be interesting toconfirm the consistency between the results of bothtests by another LST survey carried out 1 year later.

The results of the present study were consideredsufficiently encouraging to justify a community-widefield trial of deltamethrin-impregnated dog collarsagainst ZVL vector sandflies in Iran. This was thebasis of another study funded by SGG2000 entitled:"A matched community intervention trial for reducingLeishmania infantum transmission in Iran withinsecticide impregnated dog collars." Furthermore,a third study on the insecticide-impregnated dogcollars was funded in 2001 entitled: "Could infectionsin wild canids limit the potential effectiveness ofinsecticide-impregnated dog collars for reducingLeishmania infantum transmission in Iran?"

References[1] Halbig P et al. Further evidence that deltamethrin-

impregnated collars protect domestic dogs fromsandfly bites. Med Vet Entomol, 2000, 14(2):223-226.

[2] El Harith A et al. Improvement of a directagglutination test for field studies of visceralleishmaniasis. Clin Microbiol, 1988, 26(7):1321-1325.

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AbstractThis study aimed at evaluating the impact

of deltamethrin-impregnated dog collars onLeishmania infantum transmission in theIslamic Republic of Iran. Eighteen villageswere paired and matched by pre-interventionchild prevalence of L. infantum infection. Withinpairs, villages were assigned randomly ascontrol or intervention. All domestic dogs inintervention villages were provided with collarsthroughout 2 transmission seasons. Childrenand dogs were surveyed on three consecutiveoccasions at yearly intervals, i.e. pre-intervention in 2000, year 1 post-interventionin 2001, and year 2 post-intervention in 2002.The incidence of L. infantum infection wasmeasured by seroconversion and leishmaninskin test (LST) conversion in children, and byseroconversion in dogs. Significant protectionagainst seroconversion was detected in bothdogs (collars causing an average reduction inincidence of 66%) and children (reduction of53%). The impact of dog collars was alsoevaluated on both bloodfeeding and mortalityrates of local sandfly vectors.

A survey on the knowledge and attitudes ofthe community regarding visceral leishmaniasiswas conducted in 50 villages in the endemicregion. The questionnaire also includedinformation about the role of domestic dogsin the region, details of care delivered by thedog owners to their dogs and their attitudestowards the collars.

Results Deltamethrin-impregnated dog collarssignificantly reduced the risk of L. infantum

infection in dogs and hence its transmissionto children. However, 2 years of collaring dogsfailed to completely eliminate transmission invillages. This could be because transmissioncontinues in sylvatic reservoirs that visit villagesfrequently (e.g. foxes and jackals). The trialdescribed in this report only measured theimpact on infection. In order to detect theimpact of collars on the disease, their cost-effectiveness, and hence, to draw conclusionsand recommendations about their use as acontrol measure for visceral leishmaniasis, amuch larger population study was undertakenin a wide-scale trial that will end in 2004.

Publications1. Gavgani et al. Domestic dog ownership in Iranis a risk factor for human infection with Leishmaniainfantum. Am J Trop Med Hyg, 2002, 67(5):511-515.2. Gavgani et al. Effect of insecticide-impregnateddog collars on incidence of zoonotic visceralleishmaniasis in Iranian children: a matched-cluster randomised trial. The Lancet, 2002,360:374-379.

BackgroundThis study was a follow-up of a small

grants scheme project implemented in1999. It was recommended to continuethe study for another year in order toimprove the accuracy of measurementsof the epidemiological effectiveness ofthe intervention, to carry out sandflysurveys in each village for vectoridentification and test the entomologicaleffectiveness of collars in field conditions,as well as to investigate the attitudes ofthe local populations concerning visceralle ishmaniasis and dog col lars.

Materials and methodsEighteen villages were ordered into

pairs matched by pre-intervention childprevalence of L. infantum infection. Withinpairs, villages were assigned randomlyas control or intervention. All domesticdogs in intervention villages were providedwith Scalibor collars (Intervet®, IntervetInternat ional of Boxmeer, The

Deltamethrin-impregnated dog collars could clearly reduce therisk of L. infantum infection in dogs, and hence its transmissionto children.

There was inadequate knowledge of the population regardingthe role played by the vector or reservoirs regarding diseasetransmission. On the other hand, there was a high acceptabilityfor using dog collars and evidence favouring the sustainability oftheir use in the future.

The main sandfly species identified in Meshkin-Shahr andKalayabar are Phlebotomus major and P. chinensis, followed byP. kandelaki.



Vector Control

A matchedcommunity

intervention trial forreducing Leishmania

infantumtransmission in

Islamic Republic ofIran with

deltamethrin-impregnated dog

collars


Meshkin-Shahr andKalayabar,

North - West Iran


Small GrantsScheme

(SGS) 2000 No. 80

Principal InvestigatorDr Abdolsamad Mazloumi

GavganiLeishmania Research Unit,

Drug Applied Research CentreFaculty of Medicine

Tabriz University of MedicalSciences

Tabriz, Islamic Republic of Irane-mail: [email protected]

40 41

Netherlands) through 2 transmission seasons. Childrenand dogs were surveyed on 3 consecutive occasionsat yearly intervals.

Baseline survey in the control villages,January-March 2000 A total of 1237 childrenwere tested by DAT, and 615 by LST. Four hundredand eighty seven dogs were also tested by DAT inthese villages during the period March-December2000. A second survey was repeated in 2001 and2002.

Intervention: Insecticide-Impregnated DogCollars (IIDG) Following a baseline survey (January-March 2000) of children in the intervention villagesby DAT and LST and of dogs by DAT, dogs receivednew collars during March-April 2000, and again inMay-June 2001. Each dog was treated with ananthelminthic and antibiotic and wore the IIDC for atleast 2 weeks prior to the entomological experimentsand for the remainder of the sandfly season. Childrenwere re-tested by DAT and LST, and dogs were re-tested by DAT in January-March 2001, and again inApril-May 2002.

Entomological study, June-August 2001 inthe Kalayabar area After identifying villages withhigh sandfly populations, specimens were caughtusing sticky traps and mouth aspirators.

Experimental study to test the efficacy ofIIDG Dogs were caged in 2 cages and put into 2tents within the compounds of their owners. Sandflieswere caught between 20:00 and 23:30 by handaspirator, and placed into cups until a total of between150 and 200 were caught. At hourly intervals, the 75-100 sandflies were released into each of the 2 tents.Seven hours later the tents were entered and theremaining flies were collected into prepared plasticcups and offered a glucose meal. They were recheckedafter 20 hours for delayed mortality, then killed usingsmoke or permethrin and the dead sandflies wereplaced into Lacto phenol and stored for speciesidentification.

Main study findings

Sandfly identification The main species identifiedin this area were P. major and P. chinensis, followedby P. kandelaki. However, other species were alsoidentified: P. keshishiani, P. sergenti, P. papatasi,Genus Sergentomyia. The majority of each specieswere females except for P. kandelaki, which weremainly males.

Impact of IIDC on sandflies There was asignificant difference in sandfly blood feeding ratesbetween the collared and the control dogs. A significantrise in dead flies was reported for collared dogs at 7hours, but not at 20 hours.

Impact of IIDG on children and dogs in thestudied villages The use of impregnated dogcollars halved the risk of DAT conversion in childrenbut had less impact on the LST conversion rate (21%).Greater (66%) protection was provided to dogs.

Post-intervention data in children During the2-year trial, a total of 115 DAT conversions wasdetected amongst the 4324 followed-up tests in initiallyDAT-negative children. There was a significant effectof collars on DAT conversion per year. A total of 66LST conversions were detected among the 2562followed-up tests in initially LST-negative children. Asignificant effect of treatment on the risk of LSTconversion per year was reported.

Post-intervention data in dogs During the 2-year trial, a total of 67 DAT conversions were detectedamongst the 1622 followed-up tests in initially DAT-negative dogs. There was a significant effect oftreatment on the risk of DAT conversion per year.The impact in the second year (71% protection) wasgreater than in the first year (54% protection),suggesting a cumulative effect.

KAP study Out of 1872 villagers interviewed, 727were dog owners (39%). The survey was conductedamong these dog owners indicate that they are willingto spend considerable money for dog care, that 66%of dogs are in good health, but that only 22% werevaccinated.

Many observations favoured the potential forsustained collar use throughout the transmissionseason such as: the low dog population turnover rate,the low rate of collar loss, the fact that most guarddogs (71%) nearly always stay at home, the fact thatmost dogs have long hair (97%) which presumablyhelps the effectiveness of the collars, the fact thatmost dogs already use metal collars (70%) forprotection against bites by other dogs or wolves, andthat dogs are not irritated by the new Scalibor collars.

Only 55% had heard of kala-azar and 39% believedthat kala-azar was related to dogs. Half therespondents believed that children got sick with kala-azar due to the bite of an insect, but only 10%mentioned sandflies specifically. Most of the dogowners were satisfied with dog collars but only 42%thought that they could prevent kala-azar. Respondentshave seen stray dogs and foxes in their village, and42% saw jackals and wolves. Almost all villagersrecognized that people get sick from dogs, with themost common disease mentioned being rabies. Theyalso reported that their dogs suffer from fleas or ticks.So the collars should be helpful for dog healthirrespective of their impact on sandflies. The majorityof dog owners were satisfied with dog collars. However,only 42% of respondents thought that collars could

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AbstractThe AMRAD-ICT Filariasis Test (ICT-Fil) is

a new, rapid-format card test for the detectionof bancroftian antigenaemia in human blood.The performance of the test was evaluatedunder field conditions in Egypt among 1813subjects residing in known filariasis endemicfoci and 102 participants from non-endemicfilariasis areas. Participants from endemicfoci were tested for microfilaraemia (thicksmear and membrane filtration) and serumantigenaemia (ELISA).

Results The infection rates detected were2.8% by thick smear, 3.5% by membranefiltration, 8.8% by ELISA and 9.0% by ICT-Fil. The card test detected antigenaemia in98.0% and 95.3% of microfilaraemia carrierstesting positive by thick smear and bloodfiltration, respectively. Participants from non-endemic foci were ICT-Fil-negative. Identicalresults were obtained for 173 out of 184participants (94%) from endemic foci testedby the serum and whole blood ICT-Filversions.

PublicationsRamzy RM et al. Field evaluation of a rapid-

format kit for the diagnosis of bancroftian filariasisin Egypt. EMHJ, 2000, 5(5):880-887.

BackgroundNocturnally periodic bancroftian filariasis

is endemic in several foci in the Nile deltain Egypt where it represents a majorpubl ic health problem. Rout inesurveillance of the disease relies ondetection of microfilaraemia in thick bloodfilms, but this method was proved to beinsensitive and impractical due to thenecessity of blood collection at night. Theresearch group has shown that filarialantigen detection by a monoclonal (AD12)ant ibody-based, enzyme- l inkedimmunosorbent assay (ELISA) issensitive and specific for the diagnosisof active Wuchereria bancrofti infections[1]. This method allowed for the detectionof parasite antigenaemia in finger-prickblood collected during the day. However,the system required sophisticatedequipment and cannot be performeddirectly in the field. Recently, a filariasiscard-ki t was developed by theImmunochromatographic DiagnosticTests (AMRAD ICT) Company,Balgowlah, Australia. This ICT Filariasis(ICT-Fil) test is an antigen capture formatthat uses the AD12 monoclonal antibodyand a polyclonal antibody attached tocolloidal gold to detect filarial antigens insera or plasma. A new version of thefilariasis card-kit test was then developedthat was based on the detection ofcirculating W. bancrofti antigens in finger-prick blood directly in the field. This studywas conducted aiming at evaluating theperformance of the whole blood ICT-Filcard test under field conditions in filariasis-endemic areas.

Materials and methodsThe study was conducted in 4 villages

in the Governorate of Qaliubiya, locatedin an area known to be endemic with W.bancrofti. A house-to-house survey wascarried out in the village of Kafr Tahoriaon all members of the households over9 years of age. In the other villages,

The ICT-Fil test is highly sensitive and specific, with excellentnegative predictive values but low positive predictive values asit detects antigenaemia in amicrofilaraemic individuals.

The overall filaria prevalence rates for the ICT-Fil test and themonoclonal antibody-based ELISA test were fairly close (8.8%and 9.0%, respectively)

The test has tremendous technical and practical advantagesover microfilaria detection for routine surveys. It can be performedduring the day and results are obtained quickly, directly in thefield. The test does not require any laboratory facilities orexperienced microscopists and can be easily read by individualsin rural clinic setting.

This test could play a major role in the assessment of filariasisendemicity in affected communities, which is the basis of successfulimplemention of control activities.

Lymphatic FilariasisE g y p t


Diagnosis

Evaluation of animmunodiagnosticcard-kit test for therapid diagnosis of

Wuchereria bancroftiinfection in the field

EgyptQaliubiya Governorate

Study period:August 1998–1999

Small GrantsScheme

(SGS) 1997 No. 8

Principal InvestigatorDr Reda Ramzy

Research and Training Centreon Vectors of DiseasesAin Shams University

Cairo, Egypt

42 43

participants (9 years and over) were selected froma longitudinal study designed to follow a populationsubset, based on index children from Tahoria middleschool which serves nearby villages. After obtainingdemographic information, household members werephysically examined. Four diagnostic tests wereperformed on a total of 1813 subjects from endemicfoci (924 females and 888 males). Another 102 controlsubjects from a non-endemic area in Cairo weretested by the ICT-Fil test and ELISA.

The ICT-Fil test was carried out using a finger-prickof blood drawn onto the card and the results visuallyread after 15 minutes by 3 examiners, then confirmedafter being left overnight. Individuals were consideredpositive only upon agreement between the 3 readings.

Blood samples were obtained at night. Thick filmsand membrane filters were Giemsa-stained andmicroscopically examined for the presence ofmicrofilariae.

Filarial antigen was detected in plasma by AD12monclonal antibody-based ELISA [1]. The evaluationwas based on rigorous comparison tests; membranefiltration of blood collected during the peak hours ofmicrofilaraemia was used as the gold standard, plusmicrofilarial detection by thick blood smears, as thisis the method currently used for routine surveys.

Main study findingsOf the 1813 subjects studied by the 4 tests, 64

(3.5%) had microfilaremia by membrane filtration.The mean ± SD of microfilaria (MF) count in thesesubjects was 123 ± 158 MF/ml (range 1-755, median81 MF/ml). Of these, 50 (67.1%) were microfilaremicby thick smears, and 61 (95.3%) had positive ICT-Fil test. In addition, one case had 1 MF by thick filmexamination and positive ICT-Fil tets, but was negativefor microfilaraemia by membrane filtration. The mean± SD MF count by thick smears was 13 ± 12 MF /50ul (range 1-54, median 9MF/50ul).

The ICT-Fil test successfully identified 98% ofmicrofilaraemic participants detected by thick smearand 95.3% identified by membrane filtration, whereascontrol subjects were uniformly negative. Theseresults showed that the ICT-Fil test is highly sensitiveand specific, and is highly recommended as areplacement for the microfilaria detection testscurrently used in the routine surveillance of bancroftianfilariasis. The test had excellent negative predictivevalues (99.9% and 99.8%), but its positive predictivevalues were low (30.7% and 37.4%) when comparedto microfilaria detection by thick smear and membranefiltration, respectively. This could be explained by thefact that the ICT-Fil test detects antigenaemia inamicrofilaraemic individuals, the latter being actuallyinfected with W. bancrofti.

Although the overall filaria prevalence rates for theICT-fil test and the AD12-ELISA were fairly close

(8.8% and 9.0%, respectively), these tests did notdetect the same antigen-positive endemic normalindividuals. This was attributed to the differentdetection systems use in these assays. In this study,however, antigen levels were not quantitativelymeasured.

The whole blood version of the ICT-Fil test showedslightly higher sensitivity than the serum version.

Twenty subjects (1.1%) in the study population hadclinical filariasis: 11 with lymphedema, 6 with hydroceleand 3 with elephantiasis. All were amicrofilaraemic,ICT-Fil and ELISA negative, except for two caseswith hydrocele that had MF (4 and 65 MF/ml,respectively) and were ICT-Fil and ELISA positive.

ConclusionsThis second generation whole blood ICT-Fil test

has tremendous technical and practical advantagesover microfilaria detection tests currently used forroutine surveys. It can be performed during the dayand results are obtained quickly, directly in the field.The test does not require any laboratory facilities orexperienced microscopists. Because less than 1%of the ICT-Fil cards were questionnable, this indicatesthat, given adequate training, the test can beperformed and easily read by individuals in rural clinicsettings.

References[1] Ramzy MR et al. Evaluation of a monoclonal-

antibody based antigen assay for diagnosis ofWuchereria bancrofti infection in Egypt. Am J TropMed Hyg, 1991, 44(6):691-695.

Lymph

atic F

ilaria

sis

AbstractThe present study was conducted in 8

laboratories in Khartoum State during theperiod September 1995-December 1996 toevaluate the accuracy of diagnosis of malariain selected public and private laboratories. Allpatients referred for fever were examined. Ablood sample was taken and 2 slides weremade for each patient; one for routine bloodfilm and the other was processed using thestandard Giemsa stain as a referencetechnique in the National Health Laboratories.

Results The standard Giemsa techniqueshowed that 13% of cases with fever werepositive for malaria. Plasmodium falciparumwas the main species identified (96.2%), andthe remaining proportion was P. vivax. Thesensitivity of the light microscope ranged from50.0% in some laboratories to up to 75.0% inothers. Similarly, its specificity ranged from52.6% up to 95.7%. These results indicatethat the performance of the field laboratorieswas strikingly variable. The low sensitivity ofthe light microscope reveals that many positivecases will remain undiagnosed. And the lowspecificity in other laboratories reveals that aconsiderable percentage of individuals will bewrongly diagnosed as having malaria. In 4laboratories, the results were absolutelyunreliable. Suboptimal performance wasattributed to the high workload and poorsupervision.

Conclusion This study emphasizes the need

to direct limited health care resources to theprovision of laboratory facilities, continuingmedical and technical education of healthprofessionals, and to organizing trainingworkshops for the technical personnel.

BackgroundThe prevalence of malaria in Sudan

remains constantly high due to thepresence of favourable environmentalconditions and the progressive expansionand cultivation of agricultural land. Sincelight microscopy is the most reliablediagnostic method of parasitaemia [1],prompt diagnosis of malaria cases isdependent upon the accuracy of laboratoryinvestigations, provided that results couldbe rapidly communicated to the healthcareproviders. The aim of this study was toevaluate the accuracy of diagnosis andstaining techniques of malaria in publicand private laboratories.

Materials and methodsEight public and private laboratories

were selected from the 3 administrativeKhartoum areas. The personnel of theselaboratories were briefed about the study.All patients referred for fever wereexamined. A blood sample was takenfrom each patient and 2 slides made, onefor routine blood film (Field stain) [2] andthe other processed using the standardGiemsa stain as a reference technique(Reference stain) in the National HealthLaboratories (NHL).

A patient was considered negative if noparasites were seen in 100 microscopicfields of each slide. After examination andidentification of the parasite species andstage, the parasite density was determinedby counting the number of parasitesagainst 300 leucocytes i.e the number ofparasites per microliter of blood in a thickfilm is counted in relation to a standardnumber of leucocytes [3]. .

All the slides were examined by theprincipal investigator. Then, all positive

Decisive assessmentof diagnostic

staining methods ofmalaria in eight

public and privatelaboratories,

Khartoum area

SudanKhartoum State

Study period:September1995–December 1996

Small GrantsScheme

(SGS) 1995 No. 12

Principal InvestigatorMs Samia Mamoun Ibrahim

Department of ParasitologyNational Health Laboratories

Khartoum, Sudane-mail: [email protected]

The results of the different laboratories were strikingly variable,sometimes unreliable, and in many instances, irrelevant. Suboptimalperformance was attributed to the high workload and poorsupervision.

Diagnosis of malaria is more reliable with the Giemsa than withthe Field stain. It is therefore recommended to abstain from theuse of the Field stain for malaria diagnosis.

This study emphasizes the need to direct the limited health careresources to the provision of laboratory facilities, continuing medicaland technical education of health professionals, and to the tightsupervision and training of the technical personnel. The need fora quality assurance system is also emphasized.

MalariaS u d a n


Diagnosis

44 45

slides and 20% of negative slides were re-examined(blindly cross-checked) by a senior haematologist.The two sets of results were then compared and incase of discrepancy, the final diagnosis wasestablished after a third examination by a co-investigator. Data collected from the 8 laboratorieswas recorded in special data collection forms. Fieldstain results obtained by the light microscope werethen compared to the results of the reference stainobtained from the NHL.

Statistical analysis The validity of diagnosingmalaria with the light microscope was computedusing the SPSS statistical package.

Main study findingsThe standard Giemsa technique indicated that 13%

of patients presenting with fever were positive formalaria. P. falciparum was the main species identified(96.2%), and the remaining proportion was P. vivax.

The sensitivity of the light microscope ranged from50.0% in some laboratories to up to 75.0% in others.Similarly, its specificity ranged from 52.63% up to95.65%. These results indicate that the performanceof the field laboratories was strikingly variable. Thelow sensitivity of the light microscope reveals thatmany positive cases will remain undiagnosed (highfrequency of false-negative cases). This low specificityalso reveals that a considerable percentage ofindividuals will be wrongly diagnosed as havingmalaria (false positive). In 4 laboratories, the resultswere absolutely unreliable. This suboptimalperformance was attributed to the high workload andpoor supervision. Individual technicians do notaccurately follow the steps of the staining technique.They never adjust their time according to thediminishing concentration of stain throughout theday. The same amount of diluted stain might be usedfor several days without filtration. In other instances,they use water, which might be contaminated, insteadof buffer. As a result, the chromatin of the malariaparasites is seldom poorly differentiated and themicroscopic field contains a lot of micro-organismsand dirt. As a rule, 100 microscopic fields of eachslide should be examined. This is rarely being done.Usually, after coming across the first few parasites,the technicians declare the slide positive and decidewhich species is involved. On the other hand, if theslide is too poor to be examined, because the bloodhas been washed or otherwise destroyed and noparasites are in sight, the slide is always declarednegative. These laboratory errors, in addition to themalfunctioning of the available light microscopes,indicate that the diagnosis of malaria should bedetermined with Giemsa rather than with Field stain.

Conclusions and recommendationsThe results of this study indicate the necessity to

abstain from the use of Field stain for malariadiagnosis. The study also emphasizes the need todirect the limited health care resources to the provisionof laboratory facilities such as equipment and theregular supply of reagents. Continuing medical andtechnical education of health professionals, togetherwith training and tight supervision of the technicalpersonnel, is warranted. The importance ofestablishing a quality assurance system is alsoemphasized.

References[1] Omer, AHS. Species prevalence of malaria in

Northern and Southern Sudan and control by masschemoprophylaxis. Am J Trop Med Hyg, 1978,27:858-863.

[2] Shute, GT. The microscopic diagnosis of malaria.In: Wernsdorfer WH et al., eds. Malaria, principlesand practice of malariology. Edinburgh, et al., ChurchillLivingstone, 1988,781-814.

[3] World Health Organization. Basic malariamicroscopy, Part 1, Learners' Guide 1991, 67-68.

Malar

ia

AbstractMalaria transmission in Gezira State, an

endemic area in Sudan, is mainly seasonal,with two annual peaks in October and January.Questionnaire surveys were conducted for a4-week period of October 1995. A sample of400 households (3062 persons), including200 rural and 200 urban households, wasstudied. Malaria management was assessedin terms of diagnosis, types of antimalarialdrugs used, self-medication and compliance.

Results One-quarter of the rural and 35.6%of the urban population received at least onecourse of antimalarial drugs during the 4weeks . D iagnos is was conf i rmedmicroscopically in 81.7% and 34.3% of treatedpersons in urban and rural communities,respectively. Although chloroquine was themost frequently used antimalarial drug in bothcommunities, there were significant differencesin the pattern of its utilization in the two studiedcommunities. Overuse of chloroquine injectionwas notable in rural patients and in thosemanaged by paramedical health workers. Bycontrast, oral chloroquine was more commonlyprescribed by medical doctors or taken asself-medication in the urban community. Self-

prescribed medication was more common inthe rural than in the urban population (40.2%and 23.9%, respectively). Compliance withthe standard therapeutic doses was poorestwith quinine and best with sulfadoxine/pyrimethamine.

Conclusion Improving antimalarial treatmentpractices requires the dissemination of recenttherapeutic guidelines and national healthpolicies, improvement of diagnostic services,and health education. It is also concluded thatself-medication is an important component ofmalaria management that needs to bestreamlined in endemic areas.

PublicationsAdeel AAH et al. Antimalarial prescribing

patterns in Gezira State: precepts and practices.EMHJ, 2000, 6(5-6):939-947.

BackgroundRational use of antimalarial drugs is a

key element for the control of the diseaseand for coping with the emergence andspread of drug resistance. In communitieswith limited access to health care facilities,most patients resort to self-medication[1]. Over-the-counter use of drugs andnon-compliance are major factors leadingto drug resistance. In areas where malariais endemic, the home is the setting wherediagnosis and treatment of most casesof malaria take place [2]. Early diagnosisand adequate health care eventuallyaffect the prognosis of the disease. InSudan, strains of Plasmodium falciparumresistant to chloroquine are alreadyprevalent [3] and resistance topyrimethamine/sulfadoxine has beenreported [4], attributed mainly to irrationaluse of antimalarial drugs such aschloroquine, quinine, pyrimethamine/sulfadoxine and mefloquine. The presentstudy was conducted to evaluate thepattern of use of antimalarial drugs at thecommunity level.

Materials and methodsGezira State is the main crop-producing

agricultural area in Sudan. Malaria due

There was a significantly higher rate of utilization of laboratoryservices in urban compared to rural areas. In addition, empiricaldiagnosis and treatment of fever with antimalarial drugs in therural population was comparable to other endemic countries.

There was over-the-counter use of antimalarial drugs. However,self-medication could be beneficial if streamlined by appropriateinstructions by health workers in health facilities. Abuse of limitedhealth resources was manifest in the use of costly injections,mainly by rural residents.

Compliance with therapeutic doses of antimalarial drugs waspoor with quinine but optimal with chloroquine.

The repeated intake/administration of antimalarial drugs wasattributed to low therapeutic efficacy, use of sub-therapeutic doses,or erroneous primary diagnosis.

Improvement of diagnostic services, testing the therapeuticefficacy of drugs, and health education regarding the rational useof antimalarials are highly recommended.

MalariaS u d a n


Drug Studies

Community use ofantimalarial drugs in

Gezira, Sudan

SudanGezira State

Study period:May 1995–June 1998

Small GrantsScheme

(SGS) 1995 No. 15

Principal InvestigatorDr Ahmed Adeel

Blue Nile Centre for Researchand Training

University of GeziraWad Medani, Sudan

46 47

to Plasmodium falciparum is highly prevalent, with aseasonal peak in October following the rainy season.Four communities were involved in the study, 2 urbanand 2 rural, with different socioeconomic levels andaccessibility to healthcare facilities. The urbancommunities were residents of 2 blocks in Wad-Medani town, the capital of Gezira State, while therural communities were residents of Ganneb andShukkaba villages, south of Wad-Medani. Randomselection of the households within each locality wasperformed. Household occupants were theninterviewed by trained field workers for a 4-weekperiod during October 1995, using a structuredquestionnaire

Main study findingsThere was a marked discrepancy between urban

and rural communities regarding laboratory diagnosisof cases. The rate of utilization of these facilities washigher in urban areas, indicating good awareness ofthe disease and the tendency to obtain a confirmedlaboratory diagnosis, even among those who did notseek medical consultation. The scenario of empiricaldiagnosis and treatment of fever with antimalarialsin the rural population is comparable with otherendemic countries.

There was over-the-counter use of antimalarialdrugs. However, self-medication could be beneficialif streamlined by the appropriate instruction of healthworkers in health facilities. In the present study, abuseof injections was more notable in cases treated byparamedical personnel than by medical doctors. It isinteresting to note that self-medication in rural areas,where most of the health care is delivered byparamedical personnel, is characterized by greateruse of injections. Conversely self-medication in urbanareas, where most patients seek medical consultation,was predominantly by oral medications. It seems thatpatients are influenced by the practices of theirattending clinicians. In this area, the paramedicalpersonnel are generally underpaid and their incomedepends, to a great extent, on dressing wounds andgiving injections, which explains the higher frequencyof injections in rural areas. However, cultural beliefsthat injections are more potent than tablets cannotbe ruled out. Overuse of injections is an unnecessaryburden to the meagre health resources. At the timeof this survey, a course of oral chloroquine for anadult in Sudan cost the equivalent of US$ 0.45,whereas a course of 5 chloroqine injections cost atleast US$ 3.06, including the price of the drugs, feesfor the nurse giving the injections and expenses forthe transportation to the health facility where theinjections are given. Therefore, the unnecessary useof injections increases the cost of treatment by atleast 500%.

Compliance with therapeutic doses of antimalarialdrugs was poor with quinine, but optimal with

chloroquine owing to the Blue Nile Health Project,an extensive control programme undertaken in Geziraduring the period 1979-1989. This programme hasraised the general public awareness of malaria in theGezira area.

Of the treated urban population 56.6%, a highproportion, had taken antimalarial drugs within the 2weeks prior to the survey and 16.0% repeatedtreatment during the 4 weeks of observation. Therepeated use of drugs by the same patient may beexplained by many factors: treatment failure due tolow therapeutic efficacy of antimalarials, use of sub-therapeutic doses, and erroneous primary diagnosis.Finally, the results of this study indicate the need toassess the validity of laboratory diagnosis andtherapeutic efficacy of antimalarials in the study area.

Recommendations1. Improve the existing laboratory diagnostic

services, and extend these services to ruralcommunities.

2. Test the therapeutic efficacy of antimalarial drugsand communicate this information to health policy-makers.

3. Educate drug dispensers on the recenttherapeutic guidelines, with special emphasis on self-medication. Rational use of injections is alsorecommended.

References[1] Breman JG, Campbell CC. Combating severe

malaria in African children. Bull WHO, 1988, 66:611-620.

[2] Gomes M, et al. Symptomatic identification ofmalaria in the home and in the primary health careclinic. Bull WHO, 1994, 72:383-390.

[3] Bayoumi RA, et al. Chloroquine-resistantPlasmodium falciparum in Eastern Sudan. ActaTropica, 1989, 46:157-156.

[4] Ibrahim ME, et al. A case of Plasmodiumfalciparum malaria sensitive to chloroquine butresistant to pyrimethamine/sulfadoxine in Sennar,Sudan. Trans R Soc Trop Med Hyg, 1991, 85:446.

Malar

ia

AbstractThis study was conducted to assess the

therapeutic efficacy of chloroquine againstPlasmodium falciparum malaria in Sudan. Anin vivo test was implemented in 5 sentinelposts in areas of unstable malaria during thetransmission season. A standard dose of oralchloroquine was administered to a randomsample of patients with uncomplicatedfalciparum malaria attending primary healthcare units and were followed-up for clinicaland parasitological response for 14 days.Designations of "early treatment failure", "latetreatment failure" and "adequate response"were based on clinical and parasitologicalcriteria. Data analysis for prevalence ofresistance was done for each individualsentinel post, using 2-stage Lot QualityAssurance Sampling.

Results At 95% confidence level and 80%power, the prevalence of chloroquineresistance was found to be 25% in all 5 posts.

Conclusion It is concluded that the protocolwas simple and easily applicable and couldbe the basis of future sentinel posts for

continuous monitoring of malaria drugresistance for the whole country.

BackgroundThe ultimate goal of malaria treatment

policies is to ensure prompt, effectiveand safe treatment of malaria, which isone of the basic technical elements ofthe WHO Global Malaria Control Strategy.The standard in vivo test for theassessment of the therapeutic efficacyof antimalarial drugs was developed andstandardized by the WHO . However, thetest in its original form was too demandingto be applied as a routine field test inareas with limited resources. Accordingly,in certain studies, blood film examinationswere performed on 3 occasions onlyinstead of daily. This study wasundertaken to evaluate the therapeuticefficacy of antimalarial drugs in Sudan.


Study area Five sentinel posts wereselected from northern and central Sudan.Selection of these posts was based onthe availabil i ty of well-equippedlaboratories, the possibility of crosschecking the results, the accessibility tohospitals for case referral and adequatecommunity participation. The selectedposts were health centres in Wad Medani,Kenana, Kassala, El-Obied and Dilling.

Clinical examination The clinicalexamination of symptomatic patients wasconfirmed with laboratory investigations.Patients were instructed to presentthemselves at the clinic during days 0-14 in case of development of side-effects.The inclusion criteria were a history offever during the past 48 hours, mono-infection with uncomplicated P.falciparum, and adherence to follow-upvisits. Informed consent was obtainedfrom the patients or the child's caregiver.Individuals with other febrile diseases

MalariaS u d a n


Drug Studies

The proportion of clinical failures was unacceptably high in allsentinel posts. This emphasizes the need for revision of the currentmalaria treatment policies in Sudan, which consider chloroquineas the first-line drug for treatment of uncomplicated P. falciparummalaria.

The relatively high prevalence of drug resistance in 2 out of the5 posts was mainly attributed to the spread of resistant strainsfrom neighbouring African countries as well as to displacedpopulations from southern Sudan.

Both early and late treatment failures were significantly higherin children than in adults. Moreover, children had a significantlyhigher parasite count compared to adults. This could reflect lowerimmunity in children. It also indicates that assessment of therapeuticfailure should consider children as a subgroup with a higher riskof therapeutic failure.

The study was conducted under different epidemiologicalconditions (inter-area comparison), which makes it feasible inother African countries. It is finally recommended to develop othersentinel posts as an initial step for a nationwide plan for malariacontrol.

A system formonitoring the

therapeutic efficacyof antimalarial drugsagainst Plasmodiumfalciparum infections

in the Sudan

Sudannorth and central Sudan


Small GrantsScheme

(SGS) 1997 No. 10

Principal InvestigatorDr Ahmed Adeel

Blue Nile Centre for Researchand Training

University of GeziraWad Medani, Sudan

48 49

were excluded from the study.

Laboratory diagnosis Preparation and stainingof the blood slides were performed according to thestandard WHO procedures.

Treatment All cases with uncomplicated malariawere treated with oral chloroquine tablets in a 3-daycourse with the following doses: Day 0: 10 mg/kg,Day 1: 10 mg/kg and Day 2: 5mg/kg body weight.For children, the appropriate number and fraction oftablet (to the nearest 1/4 of a tablet) were crushedand administered with water in a spoon.

Patients recording treatment failure were treatedwith either appropriate doses of sulfadoxine/pyrimethamine or with quinine.

The administration of paracetamol on D-0, D-1and D-2 was permitted when the patient's conditionwarranted such medication.

Follow-up For each patient there was a form onwhich clinicians were asked to record any prescribedmedications during the next 14 days.

Therapeutic response Three categories ofresponse to therapy were considered: adequateresponse (AR), early treatment failure (ETF), andlate treatment failure (LTF). These are defined asfollows:

AR: Parasitaemia on D-3 < 25% count of D-0 andno parasitaemia on D-7 and D-14.

ETF: Parasite count on D-3 ( 25% of count on D-0, or development of danger signs or other criteriaof severe malaria OR complicated malaria on D-3 orbefore, in the presence of parasitaemia.

LTF: Parasite counts on D-3 < 25 % of count onD-0, and parasites present on scheduled visits on D-7 or D-14 OR unscheduled presentation after D-3due to the development of danger signs or othersigns of severe and complicated malaria or failure toimprove, in the presence of parasitaemia.

Individuals with ETF or LTF were given alternativetreatment.

The laboratory equipment, supplies and themicroscopic examination of slides were subjected toquality control measures.

The sample size was estimated using the Lot QualityAssurance Sampling (LQAS) method.

Main study findingsOut of a total of 242 patients initially enrolled, there

were 9 (3.7%) lost to follow-up.The proportion of treatment failures in all posts was

not significantly less than 25% with a higher proportionin Dilling (72%) and Kassala (77%). The proportionof clinical failures was unacceptably high in all thesentinel posts covered. This calls for revision ofcurrent malaria treatment policies in Sudan, whichstill consider chloroquine as the first-line drug for

treatment of uncomplicated Plasmodium falciparummalaria.

The relatively high prevalence of drug resistancein Kassala was expected because this post is closeto the eastern borders of Sudan where importationof drug-resistant strains from neighbouring Eritreahad long been suspected. The high prevalence ofresistance in Dilling was probably due to the samplepopulation that included displaced populations fromsouthern Sudan where chloroquine-resistant P.falciparum is suspected to have been imported thoughcontinuous population movements from neighbouringAfrican states of Uganda, Kenya and Congo, wheredrug resistance has been reported.

Both early and late treatment failures weresignificantly higher in children than in adults, with61.2% of children recording treatment failurecompared to 33.9% of adults. Moreover, children hada significantly higher parasite count compared toadults. This could reflect lower immunity in children.More importantly it indicates that even in areas oflow transmission, the assessment of therapeuticfailures should concentrate on children as a subgroupwith a higher risk of therapeutic failure. Several reportsdemonstrated the widespread chloroquine resistanceof P. falciparum in Sudan. However, the presentstudy assessed both the clinical and parasitologicalresponses of the patient while previous studiesconsidered only the clinical response in evaluatingthe overall response to treatment. This led to thedesignation of "treatment success" in cases that showclinical improvement in spite of persistentparasitaemia.

Furthermore, the study was conducted underdifferent epidemiological conditions (inter-areacomparison), which makes it feasible for other Africancountries. This study also provides the essentialinformation needed for policy-makers. It gives a fairlyaccurate estimate of chloroquine resistance, whichshould influence treatment policies in the studiedarea. It is finally recommended to develop othersentinel posts as an initial step for a nationwide planfor malaria control.

Malar

ia

AbstractMalaria in Saudi Arabia is localized to the

southern and south-western parts of thecountry, with the highest frequency of casesreported from Jezan and Asir regions. A recentepidemic in Jezan region was associated withhigh mortality and severe morbidity suspectedto be related to the emergence of chloroquine-resistant malaria falciparum. This study wasundertaken to assess the efficacy ofchloroquine therapy for P. falciparum malaria,in Al-Khishil, Jezan, south-western SaudiArabia. A total of 291 laboratory-confirmedmalaria patients were enrolled in the study.The patients were given a standard 3-dayregimen of chloroquine and were examinedclinically and parasitologically for malaria onthe fourth day. Positive cases were givensulfadoxine pyrimethamine and checked formalaria parasites on the seventh day oftreatment.

Results The cure rate of malaria with thestandard chloroquine therapy was 87.6%.Chloroquine-resistant patients weresuccessfully treated with sulfadoxinepyrimethamine.

Conclusion Chloroquine is still an efficaciousdrug for the treatment of malaria in the region.The treatment failure is acceptable (12.4%)compared to other parts of the world. However,the level of drug resistance should be carefullymonitored and analysed in relation to changingdynamics of malaria transmission in the region.

PublicationsGhalib HW, Al-Ghamdi S, Akood M, Haridi

AE, Ageel AA, Abdalla RE. Therapeutic efficacyof chloroquine against uncomplicated,Plasmodium falciparum malaria in south-westernSaudi Arabia. Ann Trop Med Parasitol, 2001Dec, 95(8):773-9.

BackgroundMalaria is endemic in Asir and Jezan

Lowlands ("Tihama") region in south-western Saudi Arabia. There are 2 yearlypeaks of transmission, both associatedwith rainy seasons, one in winter(December to April) and the other insummer (June to July). In 1998, anepidemic was reported from Asir andJezan regions. In the next winter season,the disease became highly endemic inJezan whilst declining significantly in Asir.It was associated with high mortality andsevere morbidity in Jezan, whereby theemergence of chloroquine-resistantmalaria falciparum was suspected. Thisstudy was undertaken to assess theefficacy of a standard regimen ofchloroquine (CQ) therapy in regard toPlasmodium falciparum malaria, in Al-Khisil, Jezan, south-western Saudi Arabia.


Study setting Based on the recordsof the regional Ministry of Health, aprimary health care centre recording ahigh prevalence of malaria transmissionin Al-Khishil in Jezan region was selected.

Study population The studypopulation consisted of individuals withclinical manifestations and confirmedlaboratory diagnosis of malaria, whilepatients suffering from severe orcomplicated malaria were excluded fromthe study.

Study design All patients enrolledin the study were examined clinically andparasitologically on presentation and werethen given a standard, 3-day regimen of

Chloroquine therapyof P. falciparum in

South-western SaudiArabia

Saudi ArabiaJezan, south-western

Saudi Arabia

Study period:October 1998–1999

Small GrantsScheme

(SGS) 1998 No. 35

Principal InvestigatorDr Hashim Warsama Ghalib

College of MedicineKing Saud UniversityAbha, Saudi Arabia

Jezan region, south-western Saudi Arabia, a highly endemicmalaria region, recorded high cure rates for the antimalarial drug,chloroquine.

Sulfadoxine/pyrimethamine is considered a safe and efficaciousantimalarial for the management of patients not responding tochloroquine in this region.

The lack of response to chloroquine was significantly correlatedwith high body temperature on init ial presentation.

The emergence of drug resistance should be carefully monitoredand analysed in relation to the changing dynamics of malariatransmission.

MalariaS a u d i A r a b i a


Drug Studies

50 51

oral CQ (i.e 10, 10 and 5 mg CQ base/kg body weighton days 0, 1 and 2, respectively).

Fingerprick blood samples were collected on days0, 3 and 7, used to make thick smears, stained with10% Giemsa stain for 10 min, and then checked formalaria parasites. The level of parasitaemia(parasites/µl blood) was estimated by countingparasites against 500 leucocytes and assuming thateach patient had 8000 leucocytes / µl blood.

Each patient was assessed clinically on days 0, 3,7 and day 14. During the complete physicalexamination given on each of these occasions, specialattention was given to the malaria-attributablesymptoms of fever, rigors, vomiting, diarrhoea,dehydration, cough and jaundice. Body temperature,taken as a marker of illness or clinical cure, wasrecorded carefully over 3 min, on days 0, 3, 7 and14, with the standard, graduated, Mediturfthermometer (Great New Life, Suzhou, China) usedroutinely by the primary healthcare facilities in theregion; this thermometer has a temperature rangeof 35-42°C graduated in intervals of 0.1 °C.

Any individual found to have a malarial infectionon day 3 was then given replacement therapy- asingle dose of Fansidar (Roche), representing 25 mgsulfadoxine and 1.25 mg pyrimethamine/kg.Theprotocol was carried out according to the WHOguidelines for Good Clinical Practice [1]

Data analysis The demographic, clinical,parasitological and treatment data collected wererecorded and analysed using version 7.5 of the SPSSsoftware package (SPSS Inc., Chicago, IL).

Main study findings A total of 291 patients, out of 350 parasitologically

positive clinical cases, completed the study.Chloroquine was found to be a safe and efficaciousdrug in the treatment of malaria in this region as87.6% of treated patients achieved a clinical andparasitological cure with the standard regimen ofchloroquine therapy.

Treatment failure accounted for 12.4%. The non-responders had clinical symptoms and wereparasitologically positive on the fourth day posttreatment. All patients who failed to respond tochloroquine therapy, responded well to a singlestandard regimen of sulfadoxine/pyrimethamine. Thelack of response to chloroquine correlated significantlywith high body temperature on initial presentation.The mean temperature of non-responders was 39.1°C ± 0.14 °C compared to 38.7 °C ± 0.05 °C inresponders. On the other hand, there was nocorrelation between the non-reponse to chloroquinetherapy and the age of the patient or the degree ofparasitaemia at initial presentation. The factorsunderlying the treatment failure could be related tothe parasite and/or host factors. The possibility of

cross-border importation of resistant strains fromneighbouring countries such as Yemen was alsohighlighted.

Conclusions and recommendationsThis study confirms the efficacy of chloroquine for

the initial management of malaria. Sulfadoxine/pyrimethamine is a safe and efficacious antimalarialfor the management of patients not responding tochloroquine in this region.

The level of treatment failure was within anacceptable range compared to other parts of theworld. However, the emergence of drug resistanceshould be carefully monitored and analysed in relationto the changing dynamics of malaria transmission inthe region.

References[1] World Health Organization. Guidelines for Good

Clinical Practice for trials on pharmaceutical products.Technical Report Series, 1995, No. 850, Geneva,WHO. Ma

laria

AbstractThis study aimed at exploring the current

epidemiological profile of malaria in the post-war period. Fifty major hospitals wererandomly selected from all private hospitalsin Lebanon. Laboratory records were reviewedfor diagnosed cases of malaria during a 3-year period (1 September 1994 to 31 August1997). Details related to the frequency ofrequesting malaria smears, diagnostictechniques and case reporting were recorded.

Results There was a high frequency ofrequesting malaria smears, mainly performedas a routine investigation for travellingpurposes. A total of 228 malaria cases weredetected during the study period (1.3% ofrequested smears). Among the minority ofcases subjected to species identification,Plasmodium falciparum proved to be thepredominant type (88.9%), while P. vivax wasrarely identified (5.6%). Neither the laboratoryinvestigations nor the case reportingtechniques were standardized over thecountry.

Conclusion It was suggested that theresurgence of malaria in Lebanon in recent

years could be attributed to emigrants fromAfrica due the high relative frequency of P.falciparum compared to P. vivax, as the latterproved to be of an endogenous origin.Diligence in case detection, and betterefficiency and standardization of the casereporting system and laboratory investigationsare finally recommended.

BackgroundDuring the war, there were no statistics

on diagnosed cases of malaria inLebanon, aside from the interrupted andincomplete case reporting to the malariaoffice, Ministry of Public Health.Furthermore, due to improving methodsof transportation, ease of travel, andclimatic changes, the WHO has warnedagainst the spread of malaria around theworld, especially in the 'Middle East'.These factors prompted researchers inLebanon to s tudy the currentepidemiological profile of malaria throughconducting a survey on laboratoryrecords.

Materials and methodsAs a result of the collapse of public

health services after the war, the healthcare system in Lebanon became confinedto private health services partially fundedby the Ministry of Public Health. Fiftymajor hospitals were randomly selectedfrom all private hospitals distributed overthe 5 Lebanese Governorates. Letterswere sent to the administrators andlaboratory physicians in the selectedhospitals and were contacted by phone.Interviewers visited the laboratories ofthese hopsitals and reviewed laboratoryrecords for diagnosed cases of malariaduring the 3-year period (1 September1994 to 31 August 1997).

Data were not available for the wholestudy period in all laboratories. Therewere some missing months for 1994-1995, and only few missing months for1996, while the records for 1997 were

Malaria in Lebanon:the current state

Lebanon all governorates

Study period:September 1994–

August 1997

Small GrantsScheme

(SGS) 1995 No. 4

Principal InvestigatorDr Ghassan AI-Awar

Department of Internal MedicineAmerican University of Beirut

Beirut, Lebanon

Thousands of malaria smears were requested per year mainlyas routine investigations, and the frequency of positive casesaccounted for 1.3% of requested smears.

Though malaria species were seldom identified, P. falciparumwas the predominant type diagnosed (88.9%), which could beexplained by the return of Lebanese emigrants from Africa insummer. On the other hand, P. vivax cases (5.6%) were ofendogenous origin. Diligent surveillance and chemoprophylaxisof malaria cases are therefore recommended.

There was no standardization or quality assurance for laboratorydiagnosis of malaria over the country. Furthermore, there wasalso a significant difference in the case reporting of the differentlaboratories. A need for better efficiency and standardization ofthe laboratory techniques and case reporting system is emphasized.

By exploring the epidemiological profile of malaria in the post-war period, a foundation is provided upon which to build a qualityimprovement programme using the parameters in this study asan initial framework.

MalariaL e b a n o n


Epidemiology

52 53

complete for all surveyed hospitals. Collectedinformation included: the frequency of requests formalaria smears, the frequency of positive and negativecases, the technique of diagnosis and the method ofreporting to the Ministry of Public Health. Theinformation gathered from these laboratories wasthen compared to that provided by the Ministry ofPublic Health, and the rate of case reporting and itsdelay for each hospital were computed.

Main study findingsThere was a high frequency of requesting malaria

smears per year (mean 5817 smears). These weremainly performed as a routine investigation for thecompletion of administrative requirements for travellingto or from the country. A total of 228 malaria caseswere detected during the study period (1.3% ofrequested smears). The frequency of positive caseswas 1.0%, 1.7% and 1.4% for the periods 1994-1995(16 months), 1996 (12 months), and 1997(8 months),respectively. The species of malaria was seldomidentified (36/228); of those identified, P. falciparumwas the predominant diagnosed type (88.9%), andonly 2 cases were P. vivax ( 5.6%). The highestfrequency of cases was in summer, which coincideswith the return of Lebanese emigrants from Africa,and explains the predominance of P. falciparummalaria as imported from Africa. On the other hand,P. vivax cases were reported to be endogenous.

The thin blood film stained with Wright and Giemsastain was the most commonly used method fordiagnosis in 37 laboratories, May-Gruenwald (MCG)in 12 laboratories, and Field stain in 1. There weresome variations within the laboratories regarding theincubation time of the different reagents. Thick bloodfilms were only performed in 13 laboratories, withvariable methods of preparation and staining. Bloodfilms were primarily read by laboratory techniciansand cross-checked by a specialized microbiologist.

Nevertheless, there was significant different in casereporting between the different laboratories. For themajority of hospitals this was carried out by a Ministryof Public Health employee who regularly visits thesehospitals for data collection. On the other hand, somecentres sent their positive results directly to theepidemiology unit of the Ministry of Public Health orthe malaria office of the Ministry. Reporting seemedto improve over the study period, ranging from 34%in 1994-95, to 54% in 1997, with an average 9.15-days' delay in reporting. Verification of availablesmears for reported cases was routinely done in themalaria office. However, there was somedisagreement between the lists of reported cases atthe malaria office and epidemiology unit in the Ministryof Public Health which improved with time due tobetter communication.

RecommendationsAs the resurgence of malaria in Lebanon in the

recent years is mainly attributed to emigrants fromAfrica, diligent surveillance and chemoprophylaxisare considered one of the public health priorities formalaria control. The scarcity of preparation of thickfilms and the variability in staining procedure indicatethe need for standardization of laboratory techniquesaccording to the recent WHO guidelines for malariadiagnosis. Quality assurance for laboratory diagnosisof malaria is therefore recommended.

Furthermore, a need for a better efficiency andstandardization of the case reporting system is alsoenvisaged.

By exploring the epidemiological profile of malariain the post-war period, a foundation is provided uponwhich to build a quality improvement programmeusing the parameters in this study as an initialframework.

Malar

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AbstractThis study aimed at analysing the

epidemiological factors causing the persistenceof malaria in Fayoum Governorate, the onlyremaining malaria focus in Egypt. The studytook place in a village in Fayoum Governorate.The collected information included: housecharacteristics, presence of domestic animals,location of houses in relation to agriculturalfields, water streams, swamps and brickfactories. Monthly larval and adult mosquitosurveys were carried out over a 1-year period.Relevant information on the breeding placesand collected larvae or mosquitoes wasrecorded. Samples of the aquatic and semi-aquatic vegetation were collected and identified.Meteorological data collected included theaverage daily temperature, relative humidity,rainfall and wind speed during 1995-1996. Bloodslides were prepared from persons with fever,from those who had a fever in the recent pastor from those referred for blood examinationfor malaria parasites. Mass blood examinationwas performed by house-to-house visits.

ResultsThe factors causing persistence ofmalaria included: the hydraulic problem ofsubsoi l water, swamps, favourablemeteorological conditions, land excavation forthe brick industry, house location in the vicinityof water streams, the shift from spraying cottonwith insecticides to the use of perhormonesthat attract insect males, and increased rice

cultivation. The results of the larval and adultmosquito surveys showed that Anophelessergenti was the prevailing species, followedby An. multicolor. There was a significantseasonal variation in the total number ofcollected larvae, which was influenced by themean monthly temperature. The highestfrequency of adult mosquitoes was recordedin animal sheds rather than bedrooms. Thefemale indoor resting density showed significantseasonal variation, peaking from October toDecember. The transmission season ofPlasmodium falciparum extended to more than8 months per year, from the end of March tothe end of November. The parasite density ofthe study population was highest in youngerage groups.

Conclusion The study uncovered severalfactors causing the persistence of malariatransmission in Fayoum, thereby providingtargets for planning future intervention strategies.

PublicationsBassiouny HK, et al. Determination of

epidemiological factors causing persistence ofmalaria, P. falciparum transmission in Fayoumgovernorate, Egypt. EMHJ 2002:8(2) in press.

Early project outcomeThe results of the study motivated government

officials to undertake vector control measuresthrough effective environmental manipulation.Intersectoral collaboration of authorities involvedin control activities was achieved. Theseactivities included filling the low-lying excavatedland, drainage of swamps and other watercollections, clearance of vegetation fromaccessible water streams, spray painting mostof the houses with malathion, larvicidingbreeding places with pyremethrin, as well asactive reporting.

BackgroundSince 1946, there has been a dramatic

decline in the malaria incidence in Egyptdue to the successful application of DDT.By the late 1970s, the disease had becomelocalized to 2 of the 5 districts of FayoumGovernorate. Despite the similar bio-environmental and climatological conditionsfavouring malaria transmission throughout

Determination ofepidemiologicalfactors causingpersistence of

malaria, P. falciparumtransmission in

FayoumGovernorate, Egypt

EgyptFayoum Governoratesouth-west of Cairo

Study period:January–December 1996

Small GrantsScheme

(SGS) 1995 No. 22

Principal InvestigatorDr Hassan Kamel BassiounyHigh Institute of Public Health

Alexandria UniversityAlexandria, Egypt

e-mail:[email protected]

An. sergenti and An. multicolor are the prevalent species in thisarea, with variable frequencies depending on the season andmean monthly temperature. The female indoor resting densitywas highest from October to December and the transmissionseason of P. falciparum extended from March to November.

The dynamics of P. falciparum transmission in this district indicatea prevalence of hypoendemicity that is unstable and subject toepidemic exacerbation every 3-5 years.

The persistence of malaria was attributed to the hydraulic problemof subsoil water, swamps, favourable meteorological conditions,land excavation, house locations near water collections, agriculturalfactors (diminished use of pesticides and increased cultivation ofrice), in addition to the restriction of malaria health services tomalaria units.

MalariaE g y p t


Epidemiology

the governorate, the disease was eradicated from 3districts (Tamiyia, Itsa and Ebshway) while stillpersisting in the 2 others (Sinnuris and Fayoum). Thisstudy sought to determine the epidemiological factorscausing the persistence of malaria in FayoumGovernorate.


Study area Since most reported malaria patientswere residing in Kafr Fazara village in the central partof Sinnuris district, this village was selected as a studyarea. A census of all households was performed. Thecollected information included: house characteristics,presence of domestic animals, location of houses inrelation to agricultural fields, and details of nearbywater streams, swamps and brick factories.

Meteorological data Meteorological data collectedincluded the average daily temperature, relativehumidity, rainfall and wind speed during 1995-1996.

Larval surveys Monthly larval surveys were carriedout over a 1-year period from randomly selectedpermanent water sources and sites with high larvaldensity. Larvae were collected by the dipping andnetting methods. Relevant information on breedingplaces and collected larvae was also recorded.Samples of the aquatic and semi-aquatic vegetationwere collected and identified.

Monthly adult mosquito survey A monthly adultmosquito survey was performed over the year fromrandomly selected houses and animal sheds to obtainseasonal baseline data on malaria transmission.Information collected included vector determination,abdominal appearance, seasonal vector behaviourand abundance. Mosquitoes were collected in theearly morning by the spray sheet collection methodusing pyrethrum solution and placed in small labelledboxes for counting and identification. Identification ofthe collected larvae and adult mosquitoes was carriedout.

Parasitological survey Blood slides were preparedfrom villagers presenting with fever, with a recenthistory of fever, or from those referred for bloodexamination for malaria. Mass blood examination wasperformed by house-to-house visits and from theneighbours of positive patients. Duplicate slides wereprepared from the peripheral blood by finger-prickingusing disposable lancets. Giemsa stain was used asthe reference technique. Parasite densities wererecorded and classified into low class (up to 10 parasitesper 10 fields) or high class (1 or more parasite per 1field). Slides were primarily examined by technicians(field test), then cross-checked blindly by a seniormicroscopist (reference test 1), and the two resultscompared. All positive slides and 20% of randomlyselected negatives slides were re-examined by the PI

(reference level-2). This cross-checking at differentlevels increased the chances of detecting low-levelparasitaemia.

Main study findingsThe majority of the land is cultivated and most of

the houses are located in the vicinity of water streams,are made of mud bricks and contain enclosed animalsheds. Other houses were modern, built with red bricksand concrete and were provided with electricity andwater supply. The high level of subsoil water led tothe formation of pools, seepage water collections andswamps. A big swamp (Abou-Naoura) was formed asa result of intensive excavation of agricultural land toobtain the clay necessary for the brick industry. In oneof the houses, the bursting of subsoil water led to theformation of a small spring inside the house.

The results of the larval and adult mosquito surveysindicated that An. sergenti was the prevailing speciesfollowed by An. multicolor, while An. pharoensis wasrarely encountered. An. sergenti recorded the highestpercentage for gravidity in females while An. pharoensisrecorded the highest percentage of fed mosquitoes.

There was a significant seasonal variation in thetotal number of collected larvae, which was influencedby the mean monthly temperature. The highestfrequency of adult mosquitoes was recorded in animalsheds rather than bedrooms. The female indoor restingdensity showed significant seasonal variation, peakingfrom October to December.

Relating vector behaviour to meteorological datarevealed that the transmission season of P. falciparumextends to more than 8 months per year, from the endof March to the end of November. The parasite densityof the study population was 0.57%, with higher densityin younger age groups. The dynamics of P. falciparumtransmission in this district indicate a situation ofprevalent hypoendemicity, but is unstable and subjectto epidemic exacerbation every 3-5 years. The factorscausing persistence of malaria include: the problemof subsoil water, swamps, favourable meteorologicalconditions, land excavation for the brick industry,location of houses in the vicinity of water streams,shifting from spraying cotton with insecticides to theuse of perhormone that attract insect males, andincreasing rice cultivation. Furthermore, the restrictionof malaria health services to malaria units resulted ina decrease in the number of detected cases and alack of reliable data concerning morbidity and mortalityfrom the disease. Strengthening of the malaria controlstrategy as well as integrating the malaria controlactivities into the primary health care system are highlyrecommended.

54 55

Malar

ia

AbstractThe importance of Anopheles stephensi in

the maintenance of malaria transmission inrural Pakistan needed further clarification.This project was conducted to clarify the roleof An. stephensi in malaria transmission inareas near Lahore where An. stephensi isthe dominant anopheline. Four villages wereselected in Punjab province near Lahore. Thepopulation of the 4 villages was checked forthe malaria parasite in the peripheral bloodonce per month from September 1995 to June1996. Mosquito sampling was performedfortnightly from all villages. Humans andbuffaloes were made available as bait oncea month. Two types of dissections were carriedout, immediate and late. For late dissections,mosquitoes were allowed to feed on infectedblood and the gut and glands of mosquitoeswere examined for the presence of the malariaparasite.

Results Only 4 out of 1937 dissectedmosquitoes were found positive at the oocyststage. Delayed dissection of mosquitoes afterfeeding on infected blood revealed thedevelopment of oocysts and sporozoites onthe 4th and 8th day, respectively, forPlasmodium vivax and on the 8th and 14-17th day, respectively, for Plasmodiumfalciparum.

Conclusion The results suggest that An.stephensi could be a vector for P. falciparum.However, these results need to be confirmedwith further studies due to the failure of

previous attempts to find such an associationin this area.

BackgroundAn. culicifacies Gilles is recognized as

the primary vector of rural malaria, [1]and is uniformly susceptible to malathion,the insecticide used for control by residualspraying, while in some areas of Punjabincipient resistance to this insecticide wasreported [2]. Among the remaining 24Anopheles species existing in Pakistan[3], only Anopheles stephensi Liston hasbeen widely incriminated in malariatransmission and has also developedresistance to malathion [4]. This specieswas considered the primary vector oftransmission of urban malaria in Karachi.The importance of An. stephensi in themaintenance of malaria transmission inrural Pakistan needed further clarification.In Punjab, comparative dissections ofAn. culicifacies and An. stephensi overan 18-month period failed to revealsporozoite-positive An. stephensi, whilepositive An. culicifacies were detectedfrom August to November [5]. Theresistance of An. stephensi to malathionwas also reported, and transmission hasbeen successfully interrupted by thecombination of malathion spraying anddrug administration. This project wasconducted with the aim of studying therole of An. stephensi in malariatransmission in areas near Lahore whereAn. stephensi is the dominant anopheline,and to study the entomologicalparameters of this species.


Study area Four villages were selectedin Punjab province near Lahore, 2 indistrict Kasur (Rohewal, Dera Islampura)and 2 in district Lahore (Billianwala andMehdipur). The selection of villages wasprimarily based on elevated malaria

Role of Anophelesstephensi in

transmission ofmalaria in Pakistan

PakistanPunjab province

Study period:September 1995–1996

Small GrantsScheme

(SGS) 1995 No. 23

Principal InvestigatorMrs Ghazala Toqir Nadeem

National Institute of MalariaResearch and Training

Lahore, Pakistan

A negligible proportion (4/1937) of immediately dissectedmosquitoes were proved to be positive at the oocyst stage.

Delayed dissection of mosquitoes after feeding on infected bloodrevealed the development of oocysts and sporozoites on the 4thand 8th day, respectively, for P. vivax, and on the 8th and 14-17thday, respectively, for P. falciparum.

Mosquitoes infected with P. falciparum develop a higher numberof oocysts and sporozoites compared to P. vivax, suggesting apossible association between An. stephensi and P. falciparum.However, further confirmation of these results was recommended.

MalariaP a k i s t a n


Epidemiology

56 57

incidence and on the abundance of An. stephensi.

Methods Maps and population censuses of thevillages were completed along with house numbering.

Malaria case detection The population of the4 villages was checked for malaria by blood testingfor the parasite once per month from September1995 to June 1996.

Mosquito sampling Collection of An. stephensiwas performed fortnightly from all villages. Nightbiting collections from humans and buffaloes weremade once each month. Field collected mosquitoeswere kept in the insectary at constant temperature(27-28 °C) and humidity. Two types of dissectionswere carried out, immediate and late. For latedissections mosquitoes were allowed to feed oninfected blood and to lay eggs, and were given bloodmeals from mice, and they were dissected 4 daysafter feeding. The gut and glands of the mosquitoeswere examined for the presence of the malariaparasite.

Major study findingsOnly 4 out of 1937 immediately dissected

mosquitoes were proved to be positive at the oocyststage. Reared An. stephensi dissected 4 days afterfeeding on infected blood revealed the developmentof oocysts of P. vivax, whereas sporozoites wereseen on the 8th day after the blood meal in thesalivary gland. In P. falciparum, the oocysts andsporozoites developed on the 8th and up to the 17thday, respectively, after the blood meal. Furthermore,there was a higher number of oocysts and sporozoitesin P. falciparum compared to P. vivax.

When 43 mosquitoes infected with P. vivax wereobserved, it was noticed that 23 survived up to day9 and none developed the parasite, while 20mosquitoes developed oocysts on the 4th day (46.5%)whereas sporozoites were detected in only 1 mosquitoon the 8th day (2.3%).

On the other hand, 31 out of 41 mosquitoes fed onblood infected with P. falciparum survived till the 17thday. Out of 31 dissected mosquitoes, 25 (80.6%)were positive for oocysts and 15 (48.4%) forsporozoites that were detected on the 14th day.

These results suggested that An. stephensi wasassociated with P. falciparum. However, it wasconcluded that these results should be taken withdue caution and needed to be confirmed with furtherstudies due to the failure of previous attempts to findsuch an association in this area.

References[1] Mahmood F, Sakai R K, Akhtar K. Vector

incrimination studies and observations of species Aand B of the Taxon Anopheles culicifacies in Pakistan.Trans Roy Soc Trop Med Hyg, 1984, 78: 607-616.

[2] Rathor H, Toqir G, Rashid S. Pattern of Malathioncross-resistance to Femitrothion in malaria vectormosquitoes An. stephensi and An. culicifacies. PakJ Med Res, 1985, 24 (2): 115-120.

[3] Aslam K. Mosquitoes of Pakistan, I.A. Checklist.Mosquito Sytematic Newsletter, 1971, 3: 147-159.

[4] Rathor H, Toqir G. Malathion resistance inAn.stephensi Liston in Lahore, Pakistan. Mosq News,1980, 40: 526-531.

[5] Mahmood F, Macdonald MB. Ecology of malariatransmission and vectorial capacity of Anophelesculicifacies species in rural Punjab Province, Pakistan.Pak J Med Res, 1985, 24:95-106.

Malar

ia

AbstractThis project was conducted in order tounderstand and evaluate the possible effectof displaced people in the transmissiondynamics of the malaria parasite in rural areasaround Khartoum. The study was carried outin 3 camps in Khartoum state: Dar Al Salam,El Baraka and Jebel Awlia. Duplicate thin andthick blood smears were collected from eachsuspected malaria case referred to the healthfacilities of the camps who were alsointerviewed to obtain information about socio-demographic variables and determinants ofmalaria infection. Malaria diagnosis andparasite species were confirmed by PCR. Thelevel of parasitaemia in the area was alsodetermined. An entomological survey wasalso carried out in the area to determine thevector species.

Results The majority of inhabitants in thedisplaced camps belong to the Nuba, Dinkaand Shuluk tribes, from Southern and WesternSudan.

Malaria infection was confirmed bymicroscopy in 73% of individuals referred to

health facilities for suspected malaria, but thesensitivity of microscopic examination of bloodwas only 77%. Al Salam camp showed thehighest infection rate of malaria, followed byJebel Awlia and El Baraka camps. The levelof parasitaemia in these areas rangedbetween 400 and 6960 parasites per microlitreof blood. Plasmodium falciparum infectionconstituted 63.3% of species identified,followed by mixed P. falciparum and P.malariae, and a few cases of P. malariae andmixed P. falciparum and P. vivax. No ovalecases were identif ied in this area.

The relative frequency of different malariaspecies did not vary significantly between thedisplaced people and those residing in theneighbouring villages. Mixed infections weresignificantly higher in people who stayed formore than 6 years, those with a negativehistory of malaria infection, and in people fromSouthern and Western tribes. Anophelesarabiensis is the only species in this area butno sporozoite-positive mosquitoes wereidentified.

Conclusion Malaria infection does not varysignificantly between people living inside oroutside camps in rural areas aroundKhartoum.

BackgroundAccurate knowledge of the geographical

distribution of the 4 Plasmodium speciesinfecting humans is very crucial fordesigning proper control measures.These species differ greatly with respectto their biology, clinical manifestationsand response to antimalarial drugs. Whenparasite levels are very low, the sensitivityof microscopy in detecting mixed infectionis reduced. In these cases, PCR offersa great advantage. The detection of mixedinfection is not only important forsuccessful medical treatment, but alsofor ascertaining the relative frequency ofeach species and consequently itstransmission potential. P. falciparumrepresents the main species foundthroughout Sudan. However, war, droughtand population movement contributed to

Malaria in rural areasaround Khartoum:

the role of displacedpeople in thegeographicaldistribution of

different malariaspecies

Sudan Dar El Salam, El Barakaand Jebel Awlia camps,

rural Khartoum


December 2002

Small GrantsScheme

(SGS) 2000 No. 49

Principal InvestigatorDr Mohamed Ziada Satti

Department of Immunology andBiotechnology

Tropical Medicine ResearchInstitute

Khartoum, Sudan.e-mail: [email protected]

MalariaS u d a n


Epidemiology

Malaria infection and parasite species do not vary significantlybetween people living inside or outside camps in rural areasaround Khartoum, mainly because villages are new residentialareas established almost at the same time as the camps, andboth populations have similar social and economic conditions.

P. falciparum infection constituted 63.3% of species identified,followed by mixed P. falciparum and P. malariae, a few cases ofP. malariae and mixed P. falciparum and P. vivax. No ovale caseswere identified in this area.

Al Salam camp showed the highest infection rate of malaria,followed by Jebel Awlia and El Baraka camps. Factors reducingsensitivity of microscopic examination in the latter two campsshould be investigated, such as the low level of infection orinadequate laboratory services.

Mixed infection was significantly higher in people who stayedfor more than 6 years, those with a negative history of malariainfection, and in people from southern and western tribes.

A. arabiensis is the only species identified. No sporozoite-positivemosquitoes were detected, suggesting a low malaria transmissionin rural areas around Khartoum.

58 59

the settlement of displaced people from differentregions into Khartoum. It was expected that thesenew settlers had modified the relative frequency ofvarious species, or introduced new ones. This wasconfirmed by recent reports of the increasingfrequency of P. vivax in some villages aroundKhartoum.

Therefore, this project was conducted in order tounderstand and evaluate the possible effect ofdisplaced people in the transmission dynamics of themalaria parasite in areas around Khartoum.

Materials and methodsThe study was carried out in 3 selected camps of

displaced people in Khartoum state: Dar Al Salam,El Baraka and Jebel Awlia. Al Salam and Jebel Awliacamps were established in 1991, while El Barakacamp was established in 1998.

The study area has a semi-arid climate, with a rainyseason from August to October and a dry seasonfrom November to May. The mean temperature variesbetween 31.1 °C and 32.6 °C. Humidity is highest inAugust and September. Al Salam and El Barakacamps have a sandy soil, while Jebel Awlia has aclay soil. The vegetation in all camps consists ofmainly semi-desert varieties.

A questionnaire was designed to collectsociodemographic and clinical information.

Duplicate thin and thick blood smears were collectedfrom each suspected malaria case referred to thehealth facilities of the camps. Blood smears werestained with Giemsa according to standard methods.Confirmation of the parasite species was based onmorphologic characteristics of the parasite in thinblood films. The level of parasitaemia was alsodetermined.

Filter paper blood samples were collected forsubsequent DNA extraction, PCR analysis andidentification of species using species-specific primers.

Entomological survey Random collection of 875indoor-resting adult mosquitoes was performed usingan aspirator in unsprayed rooms in the 3 study sites.Identification of the Anopheline mosquito wasaccomplished as described by Gilles and De Meillon.

A group of 10 mosquitoes were crushed on 1 filterpaper and kept at 4 °C before using them for DNAextraction and subsequently PCR.

Main study findingsThe majority of inhabitants in the displaced camps

belong to the Nuba, Dinka and Shuluk tribes, whomigrated from southern and western Sudan, and afew tribes were from central and northern Sudan.These displaced people began settling in camps in1991. The settlers are employed as labourers,soldiers, teachers, domestic servants, vegetable

retailers and brick-makers. Some work as seasonalagricultural workers. Many of the males are employedoutside the village and return to their homes late inthe evening or on weekends.

Most of the houses in the study sites are made ofmud or brick, and a few are constructed from woodor are simple shelters of old jute, plastic cartons ordried grass. The majority of the houses are not paintedand have no windows.

Malaria infection was confirmed by microscopy in107 out of 147 individuals referred to health facilitiesfor suspected malaria (73%). On the other hand,malaria was confirmed by PCR in 94.5% of theseindividuals. Therefore, the sensitivity of microscopyin these areas was only 77%. Al Salam camp showedthe highest infection rate of malaria, followed by JebelAwlia and El Baraka, which was statistically significantby microscopy but not by PCR.

The level of parasitaemia in these areas rangedbetween 400 and 6960 parasites per microlitre ofblood.

Using PCR, P. falciparum infection constituted63.3% of the species identified, followed by mixedP. falciparum and P. malariae (25.9%), and a fewcases of P. malariae (9.4%), and mixed P. falciparumand P. vivax (1.4%). No ovale cases were identifiedby PCR. Several false-positive results were obtainedby microscopy which proved to be of low sensitivityin species identification.

The relative frequency of different malaria speciesdid not vary significantly between the displaced peopleand those residing the neighbouring villages. Thismight be due to the fact that these villages are newresidential areas established almost at the same timeof the camps. Another factor may be that these campsand neighbouring villages have similar tribal, socialand economic situations.

Mixed infections were significantly higher in peoplewho stayed for more than 6 years, and for those witha negative history of malaria infection. In particular,the rate of mixed infection of P. falciparum and P.malariae varied significantly between tribes. In general,mixed infection was higher in people from southernand western tribes (50% and 47.1%, respectively).Few cases of mixed infection were detected amongcentral tribes (2.9%), and none among the northerntribes.

The entomological survey showed that A. arabiensisis the only species in this area with no significantvariation between the three studied camps regardingits indoor resting density. No sporozoite-positivemosquitoes were detected, suggesting low malariatransmission in rural areas around Khartoum.

Malar

ia

AbstractA cluster sample of 2168 housewives was

randomly selected from the urban and ruralareas of 5 districts, and interviewed using aquestionnaire based on the Health BeliefModel (HBM).

Results The majority of subjects (67.5%)were illiterate. One-third of the respondentsconsidered malaria as an important diseaseand 58.4% identified mosquitoes as vectors.There was poor knowledge regarding malaria;subjects recorded a total knowledge score of15 and the mean scores in rural and urbanareas were 5.5 and 4.7, respectively, with asignificantly higher awareness regarding thedisease among rural housewives. Similarly,the perceived susceptibility and severity ofmalaria were higher in rural residents. Therewas an association between the perceivedrole of mosquitoes in the transmission of thedisease and the use of bednets although netswere primarily used to avoid mosquito stings.One-third of individuals used bednets. Indoorsleeping and fans were the main reasons fornot using bednets and less than one-quarterof houses had window and door screens. Upto 73.8% of individuals received their firsttreatment from the local health house and

health centre, and Behvarz was the mainsource of information pertaining to the disease,while the role of physicians was minimal.

Conclusion This study showed that theindividual perception of risk is the maindeterminant of adherence to malaria controlprocedures. Strengthening of the perceptionof risk by health education is thereforerecommended


A seminar was held whereby medicalstudents were instructed regarding thesocioeconomic level of the population, designof the study, sampling technique, items of thequestionnaire and interviewing techniques.

BackgroundThe Health Belief Model (HBM) is a

psychological model that attempts toexplain and predict health behaviour byfocusing on attitudes and beliefs ofindividuals [1]. The key variables are thefollowing [2]:

1. Perceived threat: entailing perceivedsusceptibility or perception of risk andperceived severity or perception of theseriousness of illness and possibleconsequences.

2. Perceived benefits: the effectivenessof strategies to control the disease.

3. Perceived barriers: the physical,psychological and financial factorsundermining a negative health action.

4. Cues to action: events that motivatepeople to take an action.

5. Other demographic, socio-psychological and structural variablesthat influence health-related behaviour.

6. Self-efficacy: the beliefs in being ableto successfully execute the behaviourrequired and produce the desiredoutcome.

According to the HBM, healthfulbehaviour is more likely if an individualfeels susceptible to a condition, perceivesa benefit to the behaviour while perceiving

Study of theperception and

practice towardsmalaria among

housewives in Sistanand Baluchestanprovince, IslamicRepublic of Iran


Sistan and Baluchestanprovince

Study period:October 1998 –1999

Small GrantsScheme

(SGS) 1998 No. 17

Principal InvestigatorDr Fatemeh RakhshaniZahedan School of Public

HealthZahedan, Islamic Republic of

Iran

This study uncovered marked deficiencies in the level of educationdelivered to housewives. However, increased knowledge aboutthe benefits of bednets increased their usage. Health educationprogrammes aimed at enhancing community participation in controlstrategies are warranted.

The majority of individuals used bednets to avoid outdoormosquito stings and abandon them during indoor sleeping. Thehealth hazards of mosquitoes should therefore be communicatedto the community.

Physicians provided very low participation to the level ofinformation delivered to villagers related to malaria. Only 50% ofvillagers had ever been taught by health care providers.

A control programme could be successfully achieved ifimplemented by the village headman or party officials rather thanby relying mainly on enhancing individuals' knowledge of theperceived risks. This concept could be applied in countries witha comparable epidemiological profile.

MalariaI s l a m i c R e p u b l i c o f I r a n


Knowledge Attitudes and Practice

60 61

relatively few barriers, and has cues to perform theaction. Perceived severity and SES can also influencebehaviour. Health beliefs are complex constructswith no reference standard for their assessment [3].The components of the HBM hypothesize thatbehaviour is a function of 2 factors: the value anindividual places on health (goal orientation) and theindividual's belief that specific preventive actions willachieve the goal. The HBM has a direct implicationfor intervention programmes as each of itscomponents can be modified using traditional healtheducation strategies; strategies for enhancing bednetusage that have been identified by an educator mightbe used in the HBM to reduce the perceived barriersto bednet usage.

The aim of this study was to use the HBM to predictmalaria preventive behaviour.

Materials and methods A structured questionnaire was developed to

measure perceived susceptibility, seriousness, barriersto action, knowledge regarding the disease and itsmode of transmission and cues to action. The validityof the items was tested during the pilot study. Acluster sample of 2168 housewives was randomlyselected from the urban and rural areas of 5 districts(Kash, Saravan, Iranshahr, Nikshahr and Chabahar).Interviews were conducted by trained medicalstudents.

Major study findingsThe majority of the housewives (67.5%) and half

of their husbands were illiterate. About one-third ofsubjects recognized malaria as an important diseasebut comparable to diarrhoeal illnesses, and only 50%identified mosquitoes as the vector responsible fordisease transmission in spite of the high prevalenceof malaria in the area. There was a relatively highfrequency of misconception manifested by the beliefthat polluted water is the mode of transmission ofthe disease (21.3%). Such misconception has anadverse impact on preventive behaviour. There wasa low frequency of bednet use (31%), mainly at night,while most of the mosquitoes' activity is at sunset.Less than half of the people attempted to dry wateraccumulations; it would therefore be beneficial toeducate them in this context. The economic burdenwas the main reason for not screening the doors andwindows in this area. The study indicated thatadequate knowledge concerning the mode oftransmission of the disease and the health benefitsof bednet use resulted in an increase of bednet useas a preventive measure, while the perceivedprobability of acquiring malaria did not. By contrast,a considerable proportion of those who had adequateknowledge regarding the mode of transmission ofthe disease did not use bednets, indicating a need

for a control programme to change public behaviour.However, the social context of malaria should betaken into consideration as the Western philosophyin educating individuals to take responsibility for theirown health might not to succeed in changing theindividual's behaviour [4]. Therefore, a controlprogramme could be more successfully achieved ifimplemented by the village headman or party officialsthan it can be by relying mainly on enhancing eachindividual's knowledge of the perceived risks.

RecommendationsEffective prevention programmes for malaria require

the mobilization of community networks rather thaneducation at the individual level. Communityparticipation in the planning, implementation,monitoring and evaluation of a control programme istherefore recommended.

References[1] Rosenstock I, Strecher V, Becker M. The Health

Belief Model and HIV risk behavior change. NewYork, Plenum Press, 1994:5-24.

[2] Badura A. Perceived self-efficacy in the exerciseof control over AIDS infection, primary prevention ofAIDS: Psychological approaches. London, SagePublications, 1989:128-141.

[3] Petosa R and Jackson K. Using the HealthBelief Model to predict safer sex intentions amongadolescents. Health Education Quarterly, 1991, 18(4):465.

[4] Ager A. Perception of risk for malaria andschistosomiasis in rural Malawi. Trop Med Parasitol,1992, 43.

Malar

ia

AbstractThis study aimed at applying different

molecular techniques and cytogenetics toinvestigate the genetic variation betweenpopulations of the Anopheles fluviatiliscomplex belonging to different geographicalareas of the south-eastern provinces with thehighest burden of malaria in Iran. Mosquitosampling was performed during the seasonalactivity of mosquitoes, and sibling species ofthe complex were identified. A modifiedprotocol of Balinger-Crabtree yielded goodDNA for PCR amplification. Eight RAPDprimers were prepared, 3 were tested, andthe RAPD-PCR reactions were carried outusing 1 of these primers.

Results Three populations of the Anophelesfluviatilis complex were identified in this region,and they belonged to Kazeron, Bandar-Abbasand Iran-Shahr. One hundred specimens wereamplified for each population.

The results of the RAPD-PCR using eachof the primers were as follows:

UBC-306: This primer reveals differencesat the level of intra- and inter-population. Atthe level of intra-population, it differentiatedthe 3 populations of Iran-Shahr, Bandar-Abbasand Kazeron.

UBC-304: This primer produced a series ofbands for each population in which there wasa single common band (690 bp) in allpopulations. As in the products of the UBC-306 primer, the products of the UBC-304 forBandar-Abbas samples were mostly similarto the Karezon samples. The last band (360bp) was not detected in the Iran-Shahrsamples. As this primer also detects intra-population variations, within the populationof Kazeron, 2-3 distinct patterns could beidentified.

M13: This primer produced a series of bandsfor each population in which there were twocommon bands (600 bp and 360 bp) in allpopulations.

Conclusion Molecular techniques identified3 populations of the Anopheles fluviatiliscomplex in south-east Iran that belonged todifferent geographical areas. The UBC-306primer reveals differences at the level of inter- and intra-population, UBC-304 could onlydetect intra-population variations, while M13could not detect differences betweenpopulations or within the same population.This characterization of the speciescomposition of the local population of a vectorcould be used in planning vector controlprogammes and controlling insecticideresistance.

BackgroundMalaria is the most important health

problem in Iran, with up to 98 000 newcases diagnosed each year, with themajority (70%) occurring in the south-eastern (SE) provinces of Sistan-Baluchestan, Kerman, and Hormosgan.The Anopheles fluviatilis has been oneof the incriminated vectors in this region.Distinct differences have been observedbetween the populations of this speciesin their feeding preferences, restingbehaviour and infection rates. Theidentification of sibling species (S, T andU) was based on the banding pattern ofpolytene chromosomes, which is acumbersome technique, time consuming,and necessitates skilled personnel and

Investigation ofsibling species of

Anopheles fluviatiliscomplex in Iran


south-eastern provinces:Sistan and Baluchestan,Kerman and Hormosgan

Study period:1999-2001

Small GrantsScheme

(SCS) 1999 No. 56

Principal InvestigatorDr Hassan Vatandoost

Department of MedicalEntomology

School of Public HealthUniversity of Tehran Medical

SciencesTehran, Islamic Republic of Iran

Molecular techniques identified 3 populations of the Anophelesfluviatilis complex belonging to different geographical areas of thesouth-eastern provinces of Iran. These genetic variants were fromKazeron, Bandar-Abbas and Iran-Shahr.

Eight primers were prepared and 3 were tested: UBC-304,UBC-306, and M13.

UBC-306 reveals differences at the level of inter- and intra-population. At the level of intra-population it differentiated the 3populations of Iran-Shahr, Bandar-Abbas and Kazeron. UBC-304could only detect intra-population variations, while M13 could notdetect differences between populations or within the samepopulation.

Characterization of the species composition of a local populationof a vector could be used in planning vector control progammesand controlling insecticide resistance.

MalariaI s l a m i c R e p u b l i c o f I r a n



62 63

a certain stage of development for the samples.Therefore, there is a need for an alternative rapidand sensitive method that could be applied on anystage of the mosquito. Previous studies on a limitedsample using demonstrated DNA variations withinpopulations of this species. This study aimed atapplying different molecular techniques andcytogenetics to investigate the genetic variationbetween populations of the An. fluviatilis complexbelonging to different geographical areas of south-east Iran.

Materials and methodsMosquito sampling was performed during the

seasonal activity of mosquitoes in the south-easternprovinces. Indoor and outdoor catching of adults wascarried out and larvae were collected from breedingplaces. Identification of the sibling species of thecomplex was performed by examination of the bandingpattern of polytene chromosomes present in theovarian nurse cells of the adult. Reagents for thePCR and new primers (RAPD primers) were preparedin the laboratory.

DNA extraction: A modified protocol of Balinger-Crabtree [1] yielded a good DNA for PCR amplification.

RAPD-PCR: Eight RAPD primers were preparedand 3 were tested: UBC-304, UBC-306, and M13.Optimizing PCR reactions and troubleshooting wereperformed to produce good PCR products. TheRAPD-PCR reactions were carried out using 1 of thepreviously mentioned primers.

Main study findingsThree populations of the Anopheles fluviatilis

complex were identified in this region; they belongedto Kazeron, Bandar-Abbas and Iran-Shahr. Onehundred specimens were amplified for eachpopulation.

The results of RAPD-PCR using each of the primerswere as follows:

UBC-306: This primers reveals differences at thelevel of intra- and inter-population. At the level ofintra-population, it differentiated the 3 populations ofIran-Shahr, Bandar-Abbas and Kazeron. Specimensof Iran-Shahr showed a sharp band at about 1115bp, which was specific for this population. There wereother bands which were not very clear, but amongthem there were 2 bands (at 400 and 370 bp) whichdivided the Iran-Shahr samples into 2 groups.Samples of Bandar-Abbas had 3 specific bands, atabout 1320, 690 and 210 bp. At the level of intra-population, this primer revealed variations within thispopulation. There were 2 different patterns within thepopulation in which 1 group had a band at about 495bp, whereas the other group had a band at about470 bp. Most of Kazeron samples were similar to

Iran-Shahr, however, there was a 495 bp band thatwas shared with Bandar-Abbas samples, and wasnot detected in those of Iran-Shahr.

UBC-304: This primer produced a series of bandsfor each population in which there was a singlecommon band (690 bp) in all populations. As withthe products of UBC-306 primer, the products ofUBC-304 for the Bandar-Abbas samples were mostlysimilar to the Karezon samples. These 2 populationshad a series of common bands such as the one atabout 600 bp and another one at about 360 bp. Thislatter band (360 bp) was not detected in the Iran-shahr samples. As this primer also detects intra-population variations, within the population of Kazeron,2-3 distinct patterns could be identified.

M13: This primer produced a series of bands foreach population in which there were two commonbands, 600 bp and 360 bp, in all populations.

The results of this study could be used in a malariacontrol programme to gain a better understanding ofvector transmission and disease epidemiology, andto optimise vector control programmes.

References[1] Ballinger-Crabtree ME, Black WC 4th, Miller

BR. Use of genetic polymorphisms detected by therandom-amplified polymorphic DNA polymerase chainreaction (RAPD-PCR) for differentiation andidentification of Aedes aegypti subspecies andpopulations. Am J Trop Med Hyg, 1992, 47(6):893-901.

Malar

ia

AbstractThis study was undertaken to identify the

determinants of severe malaria morbidity andpredictors of mortality from malaria. All casesadmitted to Hargeisa, Berbera and Boramahospitals were included in the study.Complicated cases accounted for more thanone-third of admitted cases. Diagnostic signsand symptoms of severe malaria were:vomiting, high temperature, anaemia,dehydration, dizziness progressing to coma,oliguria, proteinuria, parasitaemia, icterus, drycough and epistaxis. Determinants ofcomplications in malaria cases included:infection with Plasmodium falciparum,diagnostic and treatment delay, wrong healthbeliefs regarding the disease, suboptimalquality of care, drug resistance, and co-morbidconditions (chronic renal disease, tuberculosis,chronic hepatitis, malnutrition and parasiticinfestations). A negative past history of malariawas strongly associated with encountering asevere malaria attack. Predictors of mortalityfrom severe malaria were cerebral and renalinvolvement.


It was a great accomplishment for Somalilandto carry out such a study while other regions inSomalia were under an emergency situation andcategorized as war zone. The research facedmany constraints attributed mainly to thedeteriorated health infrastructure and low healthstaff motivation.

BackgroundMalaria outbreaks and epidemics in

Somaliland began in 1977 with the flowof refugees from Ethiopia following thewar between the two countries. Thedeterioration of the health infrastructureduring the post-war period interfered withthe achievement of a successful malariacontrol programme in the country.Accordingly, the case fatality rate frommalar ia was increasing, thoughunderestimated due to the lack of a reliableregistry system. Plasmodium falciparumis the predominant protozoa and is mostlytransmitted by the vector Anophelesarabiensis.

This study was undertaken to identifythe determinants of severe malariamorbidity and predictors of malariamortality in the disadvantaged conditionsof one of the least developing countries.

Materials and methods All cases admitted to Hargeisa, Berbera

and Borama hospitals were included inthe study. The items on the questionnairewere assessed for their validity. Thequestionnaire included informationregarding: demographic data, past historyof malaria, reason for admission, detailsof clinical examination and laboratoryinvestigations, as well as medicationsgiven before and after admission. Thefield investigators were asked to write thedetails of follow-up and the outcome oftreatment.Case management Management ofsevere malaria cases was generallyaccording to the recommended WHOguidelines. Treatment approach to thepatients depended upon the generalcondition of the cases. Rapid clinicalassessment was first carried out toevaluate the overall condition of the patientthen drug prescription was performed. Ift he pa t i en t i s coma tosed o rsemicomatosed, Quinine Di-hydrochloride

Assessment ofdeterminants of

severe malaria andmortality due to

malaria

SomaliaHergeisa, Berbera,

Borama

Study period:April 1996–January 1997

Small GrantsScheme

(SGS) 1995 No. 8

Principal InvestigatorDr Abdirahman Abdillah

MohamedMinistry of Health and Labour

Hargeisa, Somalia

Risk factors of developing severe malaria were inaccessibilityto health services, diagnostic and treatment delay, wrong healthbeliefs, suboptimal diagnosis and treatment, and lack of immunityagainst malaria infection.

The information gathered in this study could serve as a basisfor a quality improvement programme through educating healthprofessionals and the community regarding the disease and itsmanagement, ensuring the availability of malaria medications inhealth facilities, and improving patients' access to health services.

The implementation of a system for malaria monitoring andsurveillance in the country is emphasized.

MalariaS o m a l i a


Morbidity and Mortality

64 65

injection was the first drug of choice. In case ofdehydration (vomiting, diarrhoea, high fever) ringerlactate was given, and blood transfusion was givenin case of anaemia (Hb<6mg/dl). Palliative drugssuch as antipyretics, and analgesics were also givenwhenever needed.

Main study findings

Hargeisa General Hospital Over 9 months, 312total malaria patients were admitted, with severecomplicated cases constituting 38.1% of cases (119patients). The case fatality rate was underestimated(3.3%) as most of the patients were managed in theprivate sector. Two-thirds of the admitted patientswere male. This is attributed to the high occupationalexposure of males to the vector in agricultural fields,but might also reflect gender-related inequalities inaccessibility to health services.

Berbera General Hospital The study wasinterrupted 3 months after its onset due to the overalldecline in the number of admitted patients becauseof unfavourable climatic conditions. However, duringthis shortened period, 12 out of 60 admitted malariacases were complicated (20.0%), and no deathswere recorded.

Borama General Hospital Over 7 months (July1996-January 1997), 125 patients with severe malariawere admitted, with the highest percentage in October,and the lowest in January. Severe cases constituted34.3% of all malaria patients (125/364 admitted), witha case fatality rate of 10% of all admitted patientsand 29% of severe cases.

Diagnostic criteria of complicated malariacases Complicated cases accounted for more thanone-third of all admitted malaria patients in the 3hospitals (256/736 admitted). The following signsand symptoms were diagnostic of complicatedmalaria: vomiting, high temperature, anaemia,dehydration, cerebral dizziness to coma, oliguria,proteinuria, parasitaemia, icterus, dry cough andepistaxis.

Determinants of complications in malariacases The severity of malaria resulted from aninterplay of factors which included: infection with P.falciparum, diagnostic and treatment delay, wronghealth beliefs about the disease, suboptimal qualityof care, drug resistance, and co-morbid conditions(chronic renal disease, tuberculosis, chronic hepatitis,malnutrition and parasitic infestations). A negativepast history of malaria was strongly associated withencountering a severe attack of malaria (60% ofsevere cases). Predictors of mortality from severemalaria were cerebral and renal involvement.

Diagnostic and treatment delay Delay indiagnosis and treatment appeared to be a significantdeterminant of severe malaria as more than 80% ofcases were referred from remote areas to the studiedhospitals to receive appropriate care. Reasons fordiagnostic and treatment delay were: living at a fardistance from the health facility (>50 km); lack/difficultyin transportation; rough roads; or due to the use ofnon-prescribed medications at home. In otherinstances, patients sought care late due tosuperstitious beliefs regarding the causes of malariasuch as: chronic constipation, toxins injected bymosquitoes, evil or Satan, and drinking watercontaminated by the eggs of mosquitoes.

Evaluation of malaria management On someoccasions, physicians were not strictly adherent tothe WHO guidelines in patient management. Drugresistance to chloroquine and sulfadoxine/pyrimethamine was recorded.

RecommendationsThe information gathered in this study highlights

the need for educating health professionals on therecent therapeutic guidelines and the communityregarding the disease, ensuring the availability ofmalaria medications in health facilities, and improvingpatients' access to health services.

Malar

ia

AbstractThis study was carried out to evaluate the

effect of community use of bednetsimpregnated with insecticide (permethrin) onthe control of the malaria vectors in Sudan.Two villages, Alakafek and Eredeba, wereselected for the study. Thick blood filmsstained with Giemsa stain were obtained froma randomly selected sample of 160 villagersfrom each village. The parasite density wasrecorded and all positive patients wereweighed and treated with chloroquine (25mg/kg body weight). Individuals were alsointerviewed regarding malaria symptoms.Indoor and outdoor collection of mosquitoesfrom 7 randomly selected houses was carriedout. The community and health authoritieswere educated regarding impregnated bednetusage. The efficacy of the insecticide-impregnated bednets was evaluated on themosquitoes collected from the study andcontrol villages.

Results Anopheles pharoensis was thepredominant species of mosquitoes collectedin the study village, followed by An. arabiensis,with the highest frequency in December.Bioassay results indicated significantly highermortality rates in the experimental groupexposed to insecticide-impregnated bednetscompared to the control group. The prevalenceof positive blood tests during the baseline

survey was 12%, which was significantlyreduced to 2.5% after intervention. There wasa significant difference between both villagesregarding the overall prevalence of positivecases. The prevalence of malaria infectionwas highest among individuals aged 20-29years and the protective effect of insecticide-impregnated bednets was more evidentamong children up to 10 years old. Malariasymptoms were present in 55.6% of casesand 60% of controls, and the maximumparasite density was less in the study group.

Conclusion The results of this study showedthat insecticide-impregnated bednets are anefficacious method for vector control inendemic regions.

BackgroundMalaria is considered one of the major

public health problems in Sudan, withprofound impact on the productivity ofindividuals. As farmers are the mostvulnerable group, a widespread malariainfection among farmers during the rainyseason directly affects agriculture, thecountry's main resource. Insecticides foruse in vector control have been generallyapplied throughout the country, resultingin HCH and DDT resistance. The use ofbiological vector control, such aslarvivorous fish, was not previouslypractised, and the use of bednets wasvery limited and often incorrect. This studywas carried out to evaluate the effect ofcommunity use of bednets impregnatedwith insecticide (permethrin) on the controlof malaria vectors in Sudan.


Study area Two villages, Alakafek andEredeba, were selected for the study.Alakafek village was selected forintervention, while Eredeba was takenas the control. The breeding sites werein low land that retains excess flood andseepage from canals. The populationconsisted of farmers living in huts madeof thatch and in small mud dwellings.

Bednetsimpregnated with

insecticides for thecontrol of the

malaria vectors inSudan

SudanAlakafek and Eredeba

villages, south of Sennartown

Study period:October 1995–November 1997

Small GrantsScheme

(SGS) 1995 No. 14

Principal InvestigatorDr Osman Abd El Nur

Medical Entomology SectionNational Health Laboratory

Khartoum, Sudan

MalariaS u d a n

Vector Control

An. pharoensis was the predominant species of mosquitoescollected in the study area, followed by An. arabiensis.

The prevalence of malaria infection before intervention was 12%,mainly Plasmodium falciparum (11.6%) and rarely P. malariae(0.4%). This was significantly lowered to 2.5% after the use ofinsecticide-impregnated bednets.

There was a significant difference between the study and controlvillages regarding the overall prevalence of positive cases.Furthermore, the impact of impregnated bednets was more apparentin younger age groups (up to 10 years old).

It is recommended to educate communities regarding thebeneficial effects of using insecticide-impregnated bednets andimplementing their use to achieve high control resolution of malariavectors in endemic regions of the world.


66 67

Baseline survey (January-May 1996) Clinicaland parasitological survey: Thick blood films stainedwith Giemsa stain were obtained from a randomlyselected sample of 160 villagers from each village.The parasite density was recorded per 100 fields ofhigh power. All positive cases were weighed andtreated with chloroquine (25 mg/kg body weight).Individuals were also interviewed regarding malariasymptoms.

Vector survey: Mosquitoes were collected at 6:00and 18:00. Seven houses were randomly selectedfor space spraying and knock-down collection. Theparity rate was determined by records of the ovariantracheoles, and salivary glands were crushed forsporozoites.

Intervention phase (June 1996-July 1997)The community and health authorities in Alakafekvillage were educated regarding impregnated bednetusage. Bednets made of opaque cotton fabric,primarily used to protect against sandflies, wereimpregnated over 2 weeks to give a uniform 200mg/m insecticide coating.

Bioassay of the efficacy of insecticide-impregnated bednets The efficacy of theinsecticide-impregnated bed nets (IIBN) was evaluatedon 3-to-5-day-old adult mosquitoes collected fromthe study and control villages. The tests applied werein accordance with the method described by Sohreck,using the WHO plastic cones. Seven tests wereperformed to test the IIBN's efficacy: 3 tests after thefirst impregnation, 3 tests after the secondimpregnation, and the last test after reimpregnationand replacement of lost nets.

Main study findingsA. pharoensis was the predominant species of

mosquitoes collected in the study village (80% and88% of indoor and outdoor catch, respectively),followed by A. arabiensis. The highest frequency ofindoor or outdoor collected mosquitoes was recordedin December for both species in the study village andfor A. pharoensis in the control village. A significantlylower percentage of A. arabiensis was recorded inthe control village compared to the study village.

The prevalence of positive blood tests during thebaseline survey in Alakafek village was 12%, mainlyP. falciparum (11.6%), and rarely P. malariae (0.4%).The intervention phase resulted in a significantreduction in the frequency of positive malaria casesto 2.5%. Furthermore, there was a significantdifference between both villages regarding the overallprevalence of positive cases (2.5% in the study villagecompared to 8.1% in the control village). Theprevalence of malaria infection was highest amongindividuals aged 20-29 years. It is noteworthy thatthe impact of impregnated bednets was more evident

among younger age groups; no children under theage of 5 were diagnosed, and only 0.6% of children5-9 years old were positive for malaria in the studyvillage, compared to 8.4% and 7.1% for these agegroups, respectively, in the control village. Thefrequency of positive cases was slightly higher infemales (M:F ratio of 1:1.45 and 1:1.14 in the studyand control village, respectively). Malaria symptomswere present in 55.6% of cases and in 60% of controls,while the maximum parasite density was 635 in casescompared to 1630 in controls. Similarly, bioassayresults indicated significantly higher mortality ratesof adult mosquitoes in the experimental group exposedto IIBN compared to the control group.

The results of this study showed that insecticide-impregnated bednets is an efficacious method forvector control. It is recommended that impregnatedbednets be used to achieve high control resolutionof malaria vectors in highly endemic regionsthroughout the world. This study also indicates thebeneficial effects of educating communities, regardlessof their educational levels. The role of health educationin malaria vector control is therefore emphasized.

Malar

ia

AbstractThis study was undertaken to evaluate the

impact of several parameters of hill lakes onthe density of the larval population and howthese may affect the biological control of thelarval population using natural predators. Theprospective study was conducted over a 1-year period in north-western Tunisia, where6 lakes were randomly selected and paired.Within each pair, one lake was chosen as astudy lake and the other as a control. Thephysicochemical properties of the water werestudied in the field as well as in the laboratory.Sampling of larvae and vegetation wasperformed twice monthly, and specimenswere transported to laboratories for furtherstudies. Random sampling of Gambusia affinishollbrooki from Lebna Dam was performedand the specimens were transported incontainers to the studied lakes or to thelaboratory. Specific numbers of larvae wereintroduced into an aquarium and theconsumed number of fish and factors affectingpredating efficacy were determined.

ResultsThe physicochemical analyses ofthe water revealed that water quality in the

hill lakes varied considerably and that theywere suitable breeding localities for Anopheleslabranchiae, Culex theileri, Culex pipiens,Aedes caspius and Culiseta longeriolatta. Thefrequency of larvae varied in the differentlakes, with C. theileri as the most frequentand constant species in all types of lakes.The introduction of Gambusia affinis fish inthe 3 studied lakes resulted in a significantdecrease in larval population compared tothe control group.

Conclusion The efficacy of predation wasrelated to the size of the larvivorous fish andthe water temperature while negativelyinfluenced by the presence of vegetation. Itis therefore recommended to introduceherbivorous fish together with larvivorous fishto achieve successful biological vector control.

PublicationsGirab J, Bouattour A. Etude experimentale de

l'efficacité larvivore de Gambusia affinis holbrooki(Girard, 1859) (Poisson-Poecilidae). Arch InstPasteur Tunis, 1999, 76:33-37.

BackgroundA national malaria control programme

was implemented in Tunisia in 1968,resulting in the disease being completelyeradicated. Since then, health authoritieshave followed a preventive policy againstmalaria by controlling the factors favouringthe reintroduction of the disease. Thelakes that were sited at the foot of hillsthroughout the country, primarily as partof irrigation projects, became favourablebreeding sites for the vectors of thedisease. Vector control therefore emergedas a public health concern that neededto be tackled while respecting theenvironment. In fact, the biological controlof mosquitoes has always beenconsidered the most appropriate methodfor vector control. It includes the use ofvertebrate and invertebrate predatorsand enteropathogens such as viruses,bacteria (Bacillus sphaericus and B.thur ingiensis ) , fungi , protozoa,nematodes, and larvivorous fish.

Impact of hill lakeparameters on the

density of theanopheline mosquitoand the control of

larval population bylarvivorous fish

predators

Tunisianorth-west Region, Bejaand Bizerte governorates

Study period:September 1995–1996

Small GrantsScheme

(SGS) 1995 No. 17

Principal InvestigatorDr Ali Bouattour

Institut Pasteur de TunisTunis, Tunisia

MalariaT u n i s i a


Vector Control

Hill lakes were suitable breeding localities for a diversity ofmosquito species, namely: An. labranchiae, C. theileri, C. pipiens,A. caspius and C. longeriolatta. Their frequency varied in thedifferent lakes, with C. theileri as the most frequent and invariablespecies in all types of lakes. The presence of turbidity and waterpollution interfered with the breeding of A. labranchiae, while theseconditions favoured the breeding of C. pipiens and C. longeriolata.A. caspius was rare due to the low salinity of these lakes.

There was a significant decrease in larval population followingthe introduction of Gambusia affinis fish in the 3 studied lakes.

The efficacy of predation was associated with the size of thelarvivorous fish and the water temperature, while it was hinderedby the presence of vegetation. The vegetation provides organicsubstances favouring the growth of micro-organisms necessaryfor the feeding of larvae and form obstacles interfering with thepredating efficacy of the Gambusia fish. It is therefore recommendedthat herbivores be introduced together with larvivorous fish forsuccessful biological vector control.

68 69

Gambusia affinis hollbrooki fish is considered anefficient natural predator of anopheline larvae andwas introduced in Tunisia in 1929 in the La Mannoubaartificial breeding basin. This study was undertakento evaluate the impact of several parameters of hilllakes on the density of larval population and howthese may affect the biological control of the larvalpopulation using natural predators.

Materials and methodsThe prospective study was conducted in north-

western Tunisia (Beja and Bizerte governorates). Sixrepresentative lakes were randomly selected, andmatched for similarities between them and the timingof their construction, thereby forming 3 matched pairsof lakes: recent (1994), intermediate (1990-1992),and old (1984-1987). This subdivision was chosenmainly to study the effect of the development ofvegetation with time as a factor favouring the breedingof the vector. Within each pair, one lake was chosenas a study lake and the other as a control.

The physicochemical properties of the water werestudied in the field as well as in the laboratory.

The field survey consisted of larval sampling bythe dipping method whereby a container of knowncapacity was plunged at randomly selected points inthe lake and the number of larvae per dip wasdetermined. Knowing the volume of water in the lake,the total number of larval population was estimatedusing a special formula. The collected larvae werethen transferred to the laboratories for further studies.The field survey also included the random samplingof vegetation, and these samples were transportedto the laboratories for analysis.

Laboratory analyses The larvae were separatedaccording to their developmental stage. Larvalcounting was performed on mature larvae (stage IV)using binocular lenses. Breeding of immature larvaeand nymphs took place in suitable conditions, tillstage IV (for larvae) or adult stage (for nymphs) wasreached.

Larvae and adults were then identified andpreserved in a suitable container. The relative andseasonal frequencies of species and several indicesproviding information regarding the predominance ofdifferent species in different stations were calculated,and the impact of modification in the biotope on thelarval population was measured.

Biological control Gambusia fish were releasedinto the study lakes (3300 in Sidi M'barek, 3160 inAin Elbabouch, and 1220 in Skhira). Another sampleof fish was transported to the laboratory, kept in anaquarium, and fed on artificial material. A definednumber of larvae was then introduced into theaquarium and the number of larvae consumed bythe fish was determined. The effects of vegetation,larval movement and water temperature on the

predators' efficacy were recorded.

Main study findingsThe physicochemical analyses of the water revealed

that the water in the hill lakes varied from sweet tolow salinity, from moderate to strong alkalinity, andall were well oxygenated. Nitrogenous compoundsvaried according to the month of sampling, and thewaters were rich in organic compounds. These lakeswere suitable breeding localities for mosquitoes,namely: Anopheles labranchiae, Culex theileri, Culexpipiens, Aedes caspius and Culiseta longeriolatta.Their frequency varied in the different lakes, with C.theileri the most frequent and invariable species inall types of lakes. On the other hand, An. labranchiaewas absent from O. Elgraa lake due to its turbidityand water pollution. However, this type of water ismore favourable for the breeding of C. pipiens andC. longeriolata. A. caspius was rare due to the lowsalinity of these lakes. The introduction of Gambusiaaffinis fish in the 3 studied lakes resulted in a significantdecrease in the larval population compared to thecontrol group.

Laboratory studies also indicated a significantassociation between the size of larvivorous fish andthe number of larvae consumed, a lack of preferencefor any particular species, although the fish preferlive rather than dead larvae, and that the efficacy ofpredation was higher with increasing watertemperature. The diversity of mosquito species wasmainly dependent on the heterogeneity of theenvironment; Lake O. Elboul, the richest in vegetation,recorded the highest diversity of species. Thepresence of vegetation was the main determinant ofhigh larval density. This rich vegetation providesorganic substances favouring the growth of themicroorganisms upon which the larvae feed. Thisvegetation constitutes a habitat for these larvae, andwhen dense, it is an obstacle that interferes with thepredating efficacy of the Gambusia fish. It is thereforerecommended that herbivorous fish such asCtenopharyngodon idella or Cyprinus carpio beintroduced along with the larvivorous fish. Biologicalcontrol of mosquitoes using Gambusia affinis andCypinus carpio, the latter also being a protein-richfood source for humans, could be an environmentallysafe and cost-effective alternative to pesticides forachieving successful vector control.

Malar

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AbstractAn intervention study conducted in Al Rahad

area aimed at an assessment of theeffectiveness of impregnating thobs withinsecticide as a vector control method. Twovillages were selected as study and controlvillages.

Results The prevalence of malaria decreasedsignificantly in the study village 1 year afterintervention (from 53.8% to 3.5%) and overthe 4 follow-up surveys. A high protectiveefficacy of 81% was attributed to theimpregnated thobs. All households werecompliant to the use of the impregnated thobs,and kept them folded in their houses. Therewas a high acceptability of this method by thevillagers and negligible side effects wererecorded. The thobs had a significant impacton the mortality rates of the mosquitoes.Anopheles arabiensis and An. rufipis werethe 2 female anopheline mosquitoes identifiedin the 2 villages.

Conclusion Insecticide-impregnated thobsare an effective and safe vector control methodin endemic areas, with high acceptability fromthe community.

Activities conducted in theframework of the project

The community health workers in thevillage health centre, as well as schoolteachers, were motivated regarding

educating the community and studentsconcerning malaria control.

BackgroundThe thob is a wrap, normally made of

cotton, worn by women in Sudan overtheir regular dresses. It is also used asa cover for women and their childrenwhile sleeping. In view of this habit, itwas hypothesized that these thobs, onceimpregnated with insecticides, would bea more acceptable and cost-effectivevector control method compared toimpregnated bednets, particularly sincevillage huts are too small to accommodatebednets. The assessment of theeffectiveness of impregnated thobs as avector control method and the educationof the community on the impregnationmethod were the objectives of this study.

Materials and methodsTwo endemic villages in the Al-Rahad

area were selected: Elregeila and Tendaltias intervention and control villages,respectively.

Parasitological survey Thin andthick blood films were examined byGiemsa stain. A pre-intervention surveywas conducted on a sample of 338 and213 villagers from the study and controlvillages, respectively, followed by 4surveys, 2 during and after thetransmission season. Febrile villagers int he s tudy v i l l age we re a l soparasitologically examined, and positivecases treated.

Thobs distribution and treatmentThe thobs were distributed to all 600villagers in the study village. Eachhousehold was visited and the names ofthe thobs users were recorded. The thobswere made from local cotton fabric(wilaya). The insecticide used in treatingthe thobs was Deltamethrin (K-Othrin EC2.5). A big tree in the centre of the village

Insecticideimpregnation of

material (Sudanesethobs) as a control

method of malaria inan area endemic

with malariatransmission

Sudan Al Rahad area, NorthKordofan, mid-west

Sudan

Study period:March 1999–2000

Small GrantsScheme

(SGS) 1998 No. 19

Principal InvestigatorDr Samia El-Karib

Tropical Medicine ResearchInstitute

National Centre for ResearchKhartoum, Sudan

Insecticide-impregnated thobs are an effective and safe vectorcontrol method in endemic areas, with high acceptability from thecommunity.

The effectiveness of the insecticide-impregnated thobs is higherduring the first 3 months of impregnation and decreased with timeand number of washes. This finding indicated the usefulness ofre-impregnating bednetss at quarterly intervals. However, re-impregnation should only be limited to the transmission seasonto further reduce the cost of this control method.

Supervised, centrally organized thob dipping ensured high re-treatment rates. This method can be successfully applied in malariaendemic areas with a comparable social and behavioural context.

MalariaS u d a n


Vector Control

70 71

was selected as the station for the impregnation.Thenew thobs were rinsed first in a measured amountof water and soaked in a bucket until totally wet.The required amount of insecticide for each thob wascalculated. The thobs were then treated by dippingthem in a mixture of insecticide and water. Each thobwas soaked until the insecticide was completelyabsorbed and then laid flat to dry in the shade.

Bioassay and effectiveness of insecticide-treated thobs The effect of 1, 2, and 3 washes ofthe treated thobs was tested. Adult mosquitoes wereexposed to thob pieces (impregnated, control andwashed) for 3 minutes and a piece of cotton woolsoaked with glucose solution was provided at theend of a holding tube on the nylon gauze during the24-hour holding period. Mortality was recorded 24hours after exposure.

I m p r e g n at e d t h o b s ev a l u a t i o nquestionnaire An evaluation questionnare wasdesigned to assess the acceptance of the communityregarding the thobs and side-effects were recorded.

Night bite collection (NBC) Night bite collectionwas performed monthly in sentinel sites in bothvillages, whereby human-bait collectors performedthe catches in 2 shifts between 18:00 and 06:00. Thecatchers were given chloroquine tablets forchemoprophylaxis. Mosquitoes were collected whilelanding on the host to bite, therefore, the collectorsthemselves acted as bait. A torch was switched onwhen a bite was felt and the mosquitoes were suckedinto an aspirator. The mosquitoes were thentransferred into paper monocups. Anophelinemosquitoes were identified morphologically by thekey method, counted and dissected for sporozoites.

Pyrethrum Space Spraying Collection (PSC)PSC was performed on the morning following nightbite collection in a randomly selected room in eachof the 5 clusters dividing each of the 2 villages. Theselected room was considered a sentinel site. Afterspraying, the mosquitoes were collected and placedin Petri dishes on moist filter-paper (Whatman no.1). Anopheline mosquitoes were identified andcounted, females were classified by stages ofabdominal appearance and then dissected forsporozoites.

Main study findingsThere was a significant difference between the

study and control villages regarding the overallprevalence of malaria (41.1% and 18.8%,respectively). The prevalence of malaria decreasedsignificantly in the study village 1 year after intervention(from 53.8% to 3.5%). Furthermore, there was asignificant difference between the study and controlvillages regarding the prevalence over the 4 follow-up surveys. A high protective efficacy of 81% attributed

to impregnated thobs against malaria infection wasobtained during the transmission season in the studyvillage.

The thobs were re-impregnated twice and allremained in a good condition during the study period.The cost of the impregnated thob per person was LS3650.

The KAP study carried out at the beginning of thestudy indicated that the type of houses in the studyvillage is huts and most people sleep outdoors, thevillagers knew about malaria as a disease, but werenot aware regarding its transmission or controlmeasures. No previous insecticide spraying had beenperformed in the village or its surroundings.

All households were compliant to health educationsessions and to the use of impregnated thobs, andkept them folded in their houses. The evaluationshowed the high acceptability of the method by thevillagers in terms of material quality, adequateness,affordability, and negligible side effects (1.5%). Theyused it for all family members, indoor and outdoor.

The bioassay test showed that 100% knock downoccurred within 3 minutes of exposure of An.arabiensis to the treated thob. All the knock-downmosquitoes died after a holding period of 24 hours.The thobs washed for the first and second time gave100% knock down within 3 minutes and 100%mortality. The bioassay on thobs used for 3 monthsshowed 97.5% mortality after 24 hours and 3 minutes'exposure. For thobs washed once and twice, themortality was 83.3% and 33.3%, respectively. Thebioassay on thobs used for 11 months showed 93.3%mortality after 24 hours and 3 minutes' exposure,and 40% and 26.6% mortality rates for the first andsecond washes, respectively.

Two species of female anopheline mosquitoeswere identified, An. arabiensis and An. rufipis, in the2 villages. There was seasonal fluctuation in the An.arabiensis density in both villages, appearing only inthe wet season (July to October). The indoor restingdensity was very low, and there was no significantdifference between both villages regarding themosquito resting density. This species was found tobe strongly endophagic as about 60% were capturedwhile trying to bite human baits indoors. The earliestperiod of activity was found at 18:00, indoor andoutdoor. Biting continued overnight with a markedincrease of the biting period, both indoor and outdoor,occurring between 20:00 and 02:00, with a peak at22:00-00:00, then gradually declining indoors at02:00-06:00 while disappearing outdoors.

Regarding human-biting rates, An. arabiensis bitingactivity reached its peak in October. An averagevillager was bitten by 2.7 and 3.4 females nightly inthe study and control villages, respectively, duringthe wet season (October-January), and no bites wererecorded in the dry season.

Malar

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AbstractThe aim of this study was to assess the

effectiveness and safety of different pesticidesfor use in areas at risk of malaria. A larvalsurvey was carried out in Fayoum, Egypt todetermine the larval density. Water andsediment analyses were performed onsamples from mosquito breeding sites. Thepotency of different pesticides, includingbiopesticides, insect growth regulators andivermectin, and their mixtures, on larvae wasassessed in the laboratory as well as in thefield. Their efficacy was expressed as themean percentage reduction of larvae in treatedversus control pools.

Results All the pesticides tested exhibitedsignificant larval reduction of the malariavector. Recently introduced ivermectin,tr i f lumuron and neem (alone or incombinations) proved to be equally efficaciousand long-lasting.

Conclusion The study emphasized acombined water management and neemcoating strategy as an environmentally safemethod that could be implemented as a controlmeasure for rice-field mosquitoes. Neem wasrecommended as a measure for achievinghigh control of the malaria vector in endemicregions.


The research team consisted of 8 memberswho worked in close collaboration with theSinnuris and Fayoum Malaria Health Units.Group discussions were held in schools,mosques, and with officials in the Ministry ofIrrigation and Water Resources and theMinistry of Health to increase awareness andto solicit the contribution of individulas andauthorities in the control strategy conductedin their community.

BackgroundFayoum Governorate is considered the

sole focus of malaria in Egypt. P.falciparum is the predominant speciesrecorded, since P. vivax was eradicatedin 1996. The aim of the study was toevaluate the efficacy of new andfrequently used alternative pesticidessuch as Bacillus thuringiensis israelensisand Bacillus sphaericus formulationsunder laboratory and field conditions, asa means for developing an efficaciousand integrated vector control method.


Study areaFayoum is a large agricultural oasis at

an altitude of 20 m below sea level.Selection of the study areas in Sinnurisand Fayoum districts was based on thefrequency of malaria cases and theproximity of breeding sites to residentialareas. The permanent stagnant watersites were randomly selected to representall parts in each district. Informationpertaining to the land characteristics andbreeding places was recorded.

Monthly larval survey A monthlylarval survey was carried out, and siteswith high larval density were chosen asfixed catching stations. Larvae werecollected using the dipping method [1]and transported to the laboratory to becounted and identified. Ten dips were

Comparative efficacystudies usingalternativepesticides

(biopesticides,insect growthregulator andivermectin) on

anopheline-malariamosquitoes in Egypt

EgyptFayoum, south-west of

Cairo

Study period:December1998–November 1999

Small GrantsScheme

(SGS) 1998 No. 47

Principal InvestigatorDr Salwa M Abd-Allah

Central Laboratory of PesticidesAgricultural Research Centre

Alexandria, Egypt

All pesticides tested exhibited significant larval reduction of themalaria vector with long-lasting effect (up to 30 days post-treatment).

A combined water management and neem coating strategycould be implemented as a control measure for rice-field mosquitoes(Anopheles pharoensis), to minimize the hazards of possiblemalarial transmission in the future.

The high frequency of the malaria vector A. sergenti could beattributed to the hydraulic situation of subsoil water, hindering thespraying of swamps with weeds and wild plants, and the suitablemeteorological conditions. Agricultural and other developmentprojects and land excavation have resulted in development of newbreeding sites, which may lead to increased transmission in thefuture. The utmost need for vector control measures with thestudied pesticides, even in low-risk areas, is emphasized.

MalariaE g y p t


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72 73

taken from each location, and the relative density ofmosquitoes as a larval index (number of larvae/dip)was calculated monthly.

Rice-land survey (July-October 1999)Sampling along the rice fields was carried out usingthe standard dipper. Collected anopheline larvaewere counted and identified.

Laboratory tests:

Water samples Water samples were randomlycollected from different locations in the breeding sitesand were categorized according to larval intenstity(high, moderate or negative). The chemical andphysical properties of the water, and the presenceof pesticide residues were determined [2].

Sediment samples Sediment samples werecollected from the upper layer of the pool bottom andanalysed.

Laboratory experiments Laboratory experimentsincluded the assessment of the potency of differentalternative pesticides and their mixtures on the larvae.

Field trials Field trials were conducted to assessthe optimum effective doses of the pesticides in thefield, whereby pre-weighed amounts of differentformulations and their mixtures were sprayed ontothe surface of ponds as treatment measures. Theywere then evaluated for their efficacy as the meanpercentage reduction of larvae in treated versuscontrol pools.

Standard statistical tests were performed. Bioassayswere analysed using probit analysis that computesthe LC50 and LC95 and chi-square goodness of fit forcomparison.

Main study findingsA. sergenti was the most frequent species (79%),

followed by A. multicolor (20.8%), of the collectedlarvae. All tested alternative pesticides exhibitedsignificant larval reduction of the malaria vector (A.sergenti) with long-lasting efficacy (up to 30 dayspost treatment).

There was no significant difference in the percentagelarva reduction between ivermectin, Bacillusthuringiensis (VectoBac 12AS, VectoBac G), lufenuron(Match) and neem 24 hours post treatment, whileBacillus sphaericus (ABG-6490) was the leasteffective. The corncob formulation (VectoBac G)proved to be more efficacious and long-lasting thanthe fluid concentrate (VectoBac 12AS). Recentlyintroduced ivermectin, triflumuron and neem (aloneor in combinations) proved to be equally efficaciousand long-lasting.

The combinations used in this study successfullyreduced the larval population density to less than1.00 larvae/dip (91%-100% larval reduction) up to

30 days post treatment, with LC25 TFM-LC25 DLMcombinations as the most efficacious of all.

A combinated water management and neem-coating strategy is an environmentally safe methodand was acceptable to farmers due to the increasein the yield of cultivated crops. Therefore, it could beimplemented as a control measure for rice-fieldmosquitoes (A. pharoensis) to minimize the hazardsof possible malarial transmission in the future.

Furthermore, owing to its protective effect againstadult mosquito biting, it is recommended to impregnatebednets and clothes with neem in order to achievehigh control resolution of the malaria vector in highlyendemic regions throughout the wor ld .

This study finally suggested that an informationsystem built on the malaria vector control programmecould have a strong impact on communities,incorporating health education, water provision,sanitation and the establishment of primary healthcare.

References[1] Manual on practical entomology in malaria. Part

II: methods and techniques. Geneva, World HealthOrganization, 1975 (available on request from Divisionof Malaria and Other Parasitic Diseases, World HealthOrganization, 1211 Geneva, Switzerland).

[2] Valerio et al. Separation of pesticides, relatedcompounds, polychlorobiphenyls and other pollutantsinto four groups by sil ica gel microcolumchromatography (Application to water surfaceanalysis). Pest Sci, 1991, 31:209-220.

Malar

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AbstractThis project was designed to develop feasible

and acceptable approach(es) for the socialmarketing of insecticide treated nets (ITNs) ina rural population, through primary health careoutlets. Two villages in rural lslamabad weresubjected to context analysis. The socialmarketing strategy was evolved in a series ofmeetings with various stakeholders. Theprimary health care outlets and active supportof community-based health workers andcommunity-based organizations in the projectarea were used to promote, distribute andprovide health education about the ITNs. A 3-phase approach was adopted: A total of 110bednets were sold during 6 months. Data frombuyers and non-buyers were collected.

Results In order to make the price of ITNsaffordable to the community, shifting thestrategy from a single payment to installments,the use of double-bed size ITNs, andminimizing the difference in the price betweeninsecticide-impregnated and non-impregnatedbednets, were the solutions devised.Vaccinators and female health workers werethe most common source of information andpurchase of ITNs. An easy mode of paymentwas considered the most frequent enablingfactor in purchasing bednets, followed by thequality of the bednets and malaria prevention.Almost all users paid installments on time.

Almost two-thirds of non-buyers knew aboutthe availability of lTNs. The main factorshindering non-buyers from purchasing lTNswere the lack of conviction about the utility ofITNs, high cost, and use of other methods.Their main suggestion for promoting ITNs wasincreasing community awareness.

Conclusion The experience of marketingITNs through public-private partnership wasfound to be very useful.

BackgroundThe main emphasis of malaria control

programme nowadays in Pakistan is onsustainable control programmes that canbe implemented in the context of primaryhealth care systems through activecommunity participation. This project wasdesigned to develop potentially feasibleand acceptable approach(es) for the socialmarketing of impregnated bednets (ITNs)in a rural population through primary healthcare outlets.

Materials and methodsTwo villages in rural lslamabad with a

total population of 17 250 were selected.Context analysis was conducted byreviewing the l i terature/records,interviewing key informants andconducting community discussions.

The data collection methods includedthe village profile tool, community dialogueand care-provider dialogue forms. Thesocial marketing strategy was evolved ina series of meetings, interactions, anddiscussions with various stakeholders,using a separately designed tool, i.e. astrategy evolution guideline.

The primary health care outlets and theactive support of community-based healthworkers, i.e. vaccinators and female healthworkers in the project area, were used forpromotion, distribution and healtheducation of the ITNs. Also, the supportof community-based organizations in theproject area was utilized to facilitate theperformance of ITN-related activities by

Evolving a socialmarketing approach

for promoting theuse of insecticide-

impregnatedbednets in rural

Islamabad

Pakistanrural Islamabad

Study period:November 1998–

August 2001

Small GrantsScheme

(SGS) 1998 No. 65

Principal InvestigatorDr Mohamed A. Khan

Association for SocialDevelopment

lslamabad, Pakistan

The role of the primary health care system is very significant inintroducing the ITNs in rural communities and can be furtherenhanced by developing strong linkages and mechanisms withall the stakeholders. The malaria control programme can play akey role in these initiatives by providing strong technical supportfor a systematic approach to establish strong public-privatepartnerships in controlling malaria with new interventions.

The need for readily available low-cost ITNs through local sourceswas identified as a major constraint to ITN access. Interpersonalcommunication with the community-based health workers wasidentified as a enabling factor in promoting ITNs.

There was no statistically significant difference between the costof ITNs and other routine methods used for protection againstmosquitoes. Selling ITNs on installment was found to be a majorenabling factor promoting their purchase and use.

MalariaP a k i s t a n


Vector Control

74 75

primary health care outlets by highlighting specificroles and responsibilities. The bednets were madeavailable for purchase in a single payment orinstallments on a not-for-profit basis.

A 3-phase approach was adopted tooperationalize/pilot the marketing strategy. In theinitial strengthening phase, the baseline supply of10-15 ITNs and information materials was suppliedin advance to the selected primary health care outlets,health workers and members of community-basedorganizations. Training on the proper technique ofmanaging lTNs and their installation was given to theconcerned individuals. The second phase consistedof establishing protocols of the detailed mechanismsfor procurement, initial investment, baseline supply,selling, cash management, record keeping/follow-up, promotion, health education and promotionalmaterials, training and sustainability. The last phaseconsisted of supervising and monitoring theimplementation process by monthly visits to the healthfacilities, health workers and members of thecommunity-based organizations. To keep an updatedinformation system for the management ofcash/installment payments, a customer profile anda monitoring system for the installment tool wasdeveloped. An extensive exercise was designed andconducted to assess the evolved strategy. A multi-method approach was followed including qualitativeand quantitative data collection and analysis tounderstand the providers' and consumers'perspectives of the ITN experience. Focus groupdiscussions were held to share and learn from theexperience of providers involved in implementing thesocial marketing strategy. Data from buyers and non-buyers were collected by using a structuredquestionnaire. A total of 110 bednets were sold during6 months. A total of 56 households who had bought1 or more of these ITNs were interviewed. Informationwas collected on their socio-demographic backgroundand on their knowledge, attitudes and practicesconcerning bednets. For comparison purposes, thissame information was also collected from 56 non-buyer households, from the house to the right of eachbuyer house.

Main study findingsThe price of the bednets was not affordable for

many of the community members. Shifting the strategyfrom a single payment to installments was found tobe effective. Female vaccinators and health workersshowed lower interest compared to males, probablydue to the fact that the difference between the priceof impregnated and non-impregnated bednets neededto be minimized. There was no significant differencebetween buyers and non-buyers regarding socio-demographic variables, their awareness thatmosquitoes are a nuisance and/or a health risk, ortheir history of normal bednet usage. The current

buying practices were not influenced by past historyof use. The majority of individuals claimed that theyhad used some sort of protective measure againstthe mosquito bite, mainly fans. There was nosignificant difference between the 2 groups regardingthe cost of protective measures used prior to theirintroduction to lTNs, the difference in the size ofcatchment population, and female clients havingrelatively limited decision-making regarding ITNs use.It was found that it was easier to demonstrate theuse of ITNs to groups compared to a single buyer.The use of double-bed sized lTNs was found to bemore economic and acceptable. Regarding thedifferent sources of information about lTNs,vaccinators and female health workers (64.3%) werethe most common source of information, followed bymembers of community-based organizations (19.6%),then health facilities posters/pamphlets distributedin the area (each 5.4%). Community-based healthworkers, including vaccinators and Lady HealthWorkers, and members of community-basedorganizations were the main source of ITNs. Theeasy mode of payment was considered the mostfrequent (46.4%) enabling factor in purchasingbednets, followed by the quality of the bednets andmalaria prevention.

The major reported reasons for non-use were thatlTNs were purchased for someone else or that it waslate in the mosquito season. Among the users, 77.5%used the bednets while sleeping outdoors, 10% usedthem only indoors, and 12.5% used the bednets bothindoors and outdoors. The majority said that therewas no constraint to the use of bednets, while otherssaid that installation was a cumbersome process,but no one mentioned hot, windy, or humid weatheras a constraint. Almost all users were convincedabout the efficacy of ITNs and recommended themto their relatives. They also paid installments on time.

Almost two-thirds of non-buyers were informedabout the availability of lTNs through community-based health workers, members of community-basedorganizations and public meetings, but rarely throughhealth facilities or general neighbourhood talk.Purchasing of the ITNs was also through the samesources.

The main factors hindering non-buyers frompurchasing lTNs in spite of their knowledge abouttheir availability were: lack of conviction about theutility of ITNs, high cost, and use of other methods.Their main suggestions for promoting lTNs were:increasing community awareness throughinterpersonal communication, followed by massannouncement through mosques, distributingposters/pamphlets to homes, organizing communitymeetings and holding public demonstrations.

Malar

ia

AbstractThis study was conducted in Fayoum

Governorate to determine the laboratory andfield efficacy of neem oil, Temephos andChlorosol towards anopheline larval density indifferent water bodies, and to study thepreliminary toxicological impact of neem oil onnon-target species. FEBA® detergent wasused to emulsify the neem oil. A village wasdivided into 4 sectors. The water bodies ineach sector were then treated with Temephos,Chlorosol, or neem oil, and 1 sector was leftas a control. To study the field efficacy of neemoil as an alternative larvicide, all water bodiesin one village were treated with neem oil whilethose of another village were treated withChlorosol. Treatment was applied biweekly for7 continuous rounds in the summer. Larvaldensity was monitored for the baseline andregularly thereafter until the 4th week of post-treatment.

Results Neem oil and other tested compoundsshowed variable degrees of toxicity for thenon-target species. The safety of the neem oilwas also proved by testing it on laboratorymice.

Water bodies were free of anopheline larvaefor 4 weeks after a single application ofTemephos or Chlorosol compared to 2 weeksin neem oil-treated water bodies.

No anopheline larvae were detected after 1week of applying the tested larvicides.Moreover, after two weeks. The larval densitywas dramatically reduced with no significantdifference in the anopheline larval densitybetween the 2 treated villages. Residualspraying of neem oil on the walls and ceilingsof the dwellings also showed a significantreduction in the number of anopheline adultsand their positive sites.

Conclusion Neem oil is not recommendedas an alternative larvicide unless justified byacute insecticide resistance.

BackgroundEgypt has been freed from malaria,

except for 2 districts in FayoumGovernorate; Sinnuris and Fayoum.However, there is always the risk ofresumed transmission due to the presenceof the anopheline species in this area andthe long transmission season ofPlasmodium falciparum. This study aimedat evaluating neem as an alternativelarvicide, and comparing it to otherlarvicides in the prevention of resumedtransmission in this area. The neem tree(Azadirachta indica) is a tropical evergreen.Azadirachtin, one of the first activeingredients isolated from neem, has provento be a primary agent for vector control.The simplest neem pesticide is a crudeextract, however, advanced formulationspossess ant i feedant, repel lent ,ovipositional inhibitor and insecticidalproperties.

Materials and methodsTwo villages in Sinnuris District, Fayoum

Governorate, were selected forimplementation of this study: El-Henawyand Abheit El-Hagar villages.

Operationallaboratory and field

usage of neemextract as an

alternative larvicideto control anopheline

vectors and itstoxicological impact

on non-targetspecies in Egypt

EgyptFayoum Governorate


Small GrantsScheme

(SGS) 2000 No. 52

Principal InvestigatorDr Osama M Awad

Department of MedicalEntomology

High Institute of Public HealthAlexandria University

Alexandria, Egypte-mail:

[email protected]

The efficacy of azadirachtin as an insecticide is well documentedin the literature. However, the results of this study cannot supportlarge-scale use of neem oil unless justified by acute insecticideresistance problems. Neem oil is not selective for resistance sinceazadirachtin is a natural mixture of natural substances with variousmodes of action.

It is difficult to judge whether the insecticidal efficacy comesfrom azadirachtin itself or from the detergent added to emulsifythis product. Considering the relatively high amount of detergentadded, it is likely that the larvicide effect resulted more from thephysical impact of the oil than from its insecticidal properties.Although the environmental impact was dramatic, a similar impactis also obtained with a non-insecticidal oil. Therefore, the impactof detergent alone on anopheline larvae, under field conditions,should be investigated in order to draw practical conclusions andmake recommendations.

Neem oil application for wall spraying is not recommendedbecause of its dark green colour, oily nature and the large quantitynecessary for this application.

MalariaE g y p t


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Larvicides tested Neem oil was purchased fromPlasma Power Private Limited, India. Two locallyproduced insecticides were also tested: Temephosand Chlorosol. Temephos consists of 50% Temephos,11% berol, and 39% emulsifiers and white kerosene.Chlorosol consists of 20% chlorpyriphos methyl, 20%fenitrothion, and 60% emulsifiers and solvents.

Laboratory study The ready-made detergent,FEBA, a dishwashing liquid, proved to be the bestemulsifier for neem oil at 20% of neem's concentration.

Laboratory bioassay The susceptibility tests ofanopheline (laboratory and field strains) and Culexpipiens field strains towards neem oil, Temephos andChlorosol were carried out in the laboratory. Moreover,the susceptibility of field strains of anopheline larvaecollected from El-Henawy village against neem oil andanopheline larvae collected from Abheit El-Hagarvillage against Chlorosol were evaluated after 3 monthsof biweekly application of the tested substances.

Laboratory strains of anopheline larvae wereselectively pressured against neem oil for 5 generationsand their susceptibility towards neem oil was recorded.The susceptibility test of the field strains of Daphniamagna and Gambusia affinis against neem oil,Temephos and Chlorosol were carried out, and thetoxicity of laboratory mice against neem oil was studied.

Efficacy of neem oil, Temephos , andChlorosol as anopheline larvicides El-Henawyvillage was divided into 4 sectors, and in each sector,5 different water bodies were chosen and treated withthe above-mentioned substances and 5 water bodieswere left without treatment as the control group. Larvaldensity was determined prior to treatment, and ondays 1, 2, 3, 7, 14 and 28 of post-treatment inaccordance with WHO guidel ines, 1975.

Field efficacy of implementation of neemoil as an alternative larvicide Six selectedwater bodies in El-Henawy village were treated biweeklywith 5% neem oil emulsified in 1% ready-madedishwashing detergent, while those of Abheit El-Hagarvillage were treated according to the schedule of theMinistry of Health and Population of biweekly back-spraying with Chlorosol at 10 ml Chlorosol per 10 litresof water covering 100 square metres of surface area.Seven rounds of biweekly application continued from30 June until 21 September.

Larval densities were monitored prior to applicationand weekly post-application until the end of the aboveschedule and four weeks thereafter (30 June through19 October). Larval density was determined accordingto WHO guidelines, 1975.

Parallel adult density was determined to study theimpact of larviciding on adult density. In each villagea group of 10 houses and another group of 10 animalsheds were randomly selected, and mosquitoes werecollected by the spray-sheet collection method using

pyrethrum solution.

Residual effect of the neem oil Five houseswere randomly selected in El-Henawy village fordetermining the impact of the residual action of neemoil on adult mosquito density. Five percent neem oilplus 1% ready-made dishwashing detergent (500 mlneem oil + 100 ml ready-made dishwashing detergentfor a 10-litre back sprayer) was used to cover 250square meters of indoor walls and ceiling. Adultmosquito density was monitored pre-application andbiweekly for seven rounds (14 July through 21 October).Mosquitoes were collected, identified and their densitywas determined.

Main study findingsReady-made FEBA detergent is better than any

other tested emulsifier for its efficiency, reproducibilityof results, toxicity to the environment and cost. Therewas no significant difference in the susceptibility oflaboratory versus field strains of anopheline larvaetowards crude neem oil. Moreover, the field efficacyof neem oil as an anopheline larvicide is comparableto the other tested traditional larvicides, but it is efficientfor only 2 weeks compared with 4 weeks for the otherlarvicides. Also, neem oil is as efficient as the Chlorosolin reducing the anopheline adult density and thenumber of adult-infested sites.

This study also showed that vegetable oil is almostas toxic as neem oil towards Culex pipiens and Daphniamagna and other tested non-target organisms. Thismight be due to the low concentration of azadirachtinin the crude neem oil (1570 ppm) and the highconcentration of the detergent, and therefore, the mainmode of action of neem oil in this case is through itsphysical properties rather than its azadirachtin contents.Using traditional mineral oils as larvicides might bringthe same results.

The preliminary toxicological impact of crude neemoil on male mice did not prove any significant changesin body weight gain, liver function or blood parameters.

The cost of quantity of the neem oil-detergentemulsion required to control anopheline larvae in acertain area is 2 to 5 times greater than the cost ofChlorosol or Temephos to control the same area.

Conclusions and recommendationsThe results of this study cannot support large-scale

use of neem oil as an insecticide unless justified byacute insecticide resistance problems. Beforerecommending its use as an alternative larvicide, thereis a need to study the impact of long-term fieldapplication and its toxicological impact. The larvicidaleffect of neem oil should also be compared with otherfuel oils.

Malar

ia

AbstractA field study was carried out in South Batinah

Region to evaluate the impact of 3 larviciding-based strategies of vector control. Theevaluation covered 2000 breeding sites in thisregion and continued over 27 weeks.

The first strategy was to use half the amountof Abate (0.5 ppm) weekly, instead of theconventional weekly dose of 1 ppm currentlyused in the National Malaria EradicationProgramme. The second approach was toapply the full dose (1 ppm), but fortnightly.The third method was to search for vectorlarvae and treat only the breeding places ofanopheline larvae. The first method was foundto be as effective as the full dose and thesecond approach was the least effectivelarvicidal method.

Conclusion Compared to the full dose ofAbate currently used in the control programmeof the Ministry of Health, the half dose of Abate(0.5 ppm) was proved to be a more cost-effective and environmentally friendly larvicidal-based vector control method. It is much moreeffective than applying a full dose fortnightlyor treating only larval-positive breeding sites.This vector control method could be adoptedin malaria endemic countries

BackgroundThe National Malaria Eradication

Programme in Oman has achieved

excellent malaria control through casemanagement and vector control, includinglarval control, using chemicals. This studyreports the results of comparing differentlarvicidal strategies for vector control inthe field.

Materials and MethodsOman consists of 10 administrative

regions, each divided into wilayats. At thewilayat level, malaria units are responsiblefor malaria eradication activities. The areaunder each unit is demarcated into daraksfor all operations. Centrally, the Directorateof Environmental Health and MalariaEradication supervises the nationalprogramme.

The study was carried out in 5 coastaldaraks of the malaria-free wilayat of Barkain the South Batinah region.

Geographic reconnaissance wasupdated for the vector breeding locationsby physical verification. Final mapsindicating details of the work were thenprepared. Meteorological data werecollected from Seeb Weather Station. ThepH value in each darak was also recorded.

The intervention The interventionconsisted of using several differentstrategies for applying Temephosinsecticide. Some daraks were treatedusing the existing standard method, butapplying only half the standard weeklydose of Temephos; others were treatedwith the normal 1 ppm dose of Temephos,fortnightly; and in the third group, onlypositive larval breeding sites were treated.One darak was left without interventionto observe the natural conditions and asa control for comparison to the above-mentioned strategies.

Supervisory teams closely directed thesprayers and regularly delivered the dailyprogrammes for spraying. All daraks werevisited weekly to assess the impact of thetreatment strategy on larva control.

Malaria vectorcontrol through threedifferent larviciding

strategies

Oman Wilayat Barka, South

Batinah Region

Study period:May 2001–January 2002

Small GrantsScheme

(SGS) 2000 No. 72

Principal InvestigatorDr Salahuddin Parvez

Department of EnvironmentalHealth

and Malaria EradicationMinistry of Health

Muscat, Sultanate of Oman

MalariaO m a n


Vector Control

A half dose of Abate (0.5 ppm) is more cost-effective comparedto a full dose, with less harmful effect to the environment. Hencethis method could be applied on a wide scale in endemic countries.

In countries targeting elimination, it is recommended to stratifythe country according to receptivity and vulnerability to malaria,and apply the tested larvicide wherever there is a risk of resumingmalaria.

The frequency of positive breeding sites for the vector in thisregion ranged from 4% to 6.6% in June and increased during themonths of August to December.

Anopheles culicifacies and An. stephensi are the only speciesidentified in this region.

78 79

Vector susceptibility to Temephos A pre-intervention baseline evaluation of the vectorsusceptibility to Temephos was performed usingWHO standard methods for susceptibility testing.Larval density tests were also conducted.

Main study findings

Baseline pre-intervention survey: June 2001There was no significant difference between the tanksregarding the presence of the vector during this period(ranging from 4 to 6.6%). The only species identifiedwere An. culicifacies and An. stephensi, and theformer was much more prevalent (vector density: 10-25/100 dips for An. culicifacies compared to 0-7/100dips for An. stephensi). Vector susceptibility tests toTemephos were also conducted in each darak.

Intervention period: 30 June-December2001

Control daraks Vector breeding in this areamarkedly increased during the period August-December, reaching 20-50 larvae/dip withoutapplication of any vector control measure.

Weekly half-dose treatment with TemephosIn this intervention, there was a significant decreasein vector density in the treated daraks, with vectornumbers declining from an initial 30 larvae/dip tonone after 4 cycles, and this was maintained till theend of the study.

Fortnightly normal dose of 1 ppm Althoughthere was a significant decrease in vector densitywith this intervention, it was not 100% effective.

Weekly breeding checking and treating ofpositive sites While this intervention was moreeffective in reducing vector density compared to thefortnightly normal dose of 1 ppm, it was less effectivecompared to the weekly half-dose treatment withTemephos.

Vector breeding sites Twenty percent of potentialbreeding sites were positive for the vector during thestudy period. However, there was a significantdifference between sites regarding the presence ofthe vector. Interestingly, repeated identification oflarvae was reported up to 10 times, indicating atendency of the vector to use the same breeding siteor "habitat" in this situation.

Larvicidal strategy Multivariate logistic regressionanalysis showed that weekly half-dose treatment withTemephos provided the most protective effect againstpositivity of breeding sites. This was adjusted for theeffect of temperature and pH value of the daraks.

Vector susceptibility tests There was nosignificant difference before and after interventionregarding vector susceptibility to Temephos.

Cost-benefit analysis of different strategiesThe cost for larvicide was reduced by 50% usingweekly half-dose applications of Temephos, whichwas proved to be as effective as using the full dose,and was the most effective tested strategy. Thefortnightly cycle was the most economic method, butwas the least effective.

Conclusions and recommendationsA half dose of Abate is a cost-effective larvicidal-

based vector control that could be applied on a widerscale in endemic countries. However, in countriestargeting elimination, it is recommended to stratifythe country according to receptivity and vulnerabilityto malaria, and apply the tested larvicide where thereis a risk of resuming malaria. Testing the half doseof Abate for a two-week cycle is also recommendedto provide a further reduction in cost and harm to theenvironment.

Malar

ia

AbstractThis study was conducted in an

onchocerciasis endemic village southernDarfour sate. Male individuals aged 15-60years were interviewed regarding itching andmusculo-skeletal pains. Individuals with skinlesions were examined for dermatologicalsigns of onchocerciasis. The degree of severityand morbidity of onchodermatitis skin lesionswere assessed and graded. Ninety patientswith moderate or severe onchodermatitis wereselected and randomized into one of thefollowing ivermectin treatment schedules 3,6, or 12 monthly treatment. Ivermectin wasadministered in single oral dose of 150 µg/kg. Ivermectin was distributed to the rest of thevillagers as part of the first ivermectin massdistribution programme conducted in this area. The three study groups were followed every3 months and compared regarding the timeneeded for disappearance, improvement orrecurrence of symptoms, or changing in theseverity of the skin lesions.

Results Itching whether troubling or severewas the commonest symptom reported by allpat ients wi th moderate or severe

onchodermatitis. Both quarterly and biannualivermectin treatment lead to significantreduction in prevalence of itching while 12months treatment resulted in partial declinein its intensity. Moreover, quarterly andbiannual schedules resulted in significantreduction in the prevalence and intensity ofAcute and Chronic Papular Onchodermatitis,and Lichenified Onchodermatitis.

Muskuloskeletal complaints was theprevalent symptom in onchodermatitis. Boththe joint, bone, and backache showed goodresponse to ivermectin treatment irrespectiveof the treatment schedules.

Onchocercal depigmentation was the onlyskin lesion not responding to ivermectintreatment, however, ivermectin prevented thedevelopment of new skin lesions.

Conclusion There was no significantdifference between biannual and quarterlyivermectin treatment schedules regarding theimprovement of signs and symptoms ofonchodermatitis.

The 6 monthly ivermectin treatmentschedule proved to be a cost-effectiveschedule for controlling moderate or severeonchodermati t is in endemic areas.

BackgroundOnchocerciasis has long been

associated with a high incidence ofdetrimental effects on socioeconomicdevelopment and public health in endemicareas. Ocular compl icat ions ofonchocerciasis and the response totreatment with ivermectin, both have beenextensively invest igated. Othermanifestations related to infection withO. volvulus such as onchodermatitis andmuskuloskeletal pains have received lessattention. Only recently the psycho-socialimportance of skin disease becameevident. Symptoms such as itching canlead to sleeplessness, fatigue, weaknessand less capacity to work.

There is enough evidence that skinlesions and itching are reduced withivermectin. Yet, the optimum scheduleof treatment is not worked out especially.Muskuloskeletal symptoms due to

The prevalence of onchocerciasis in a village in Southern Darfoursate, accounted to 35% using the method of Rapid EpidemiologicalMapping of Onchocerciasis (REMO). These highly endemic areasshould be therefore carefully subjected to mass drug administrationof ivermectin to control the detrimental effects of the disease onthe inflicted populations.

There is no significant difference between biannual and quarterlyivermectin treatment schedules regarding the improvement ofsigns and symptoms of onchodermatitis.

Quarterly and biannual schedules result in significant reductionin the prevalence and intensity of itching, Acute and ChronicPapular Onchodermatitis, Lichenified Onchodermatitis, andmuskuloskeletal complaints.

Onchocercal depigmentation is the only skin lesion not respondingto ivermectin treatment, however, ivermectin prevented thedevelopment of new skin lesions.

The 6 monthly ivermectin treatment schedule proved to be acost-effective schedule for controlling moderate or severeonchodermatitis in endemic areas.

OnchocerciasisS u d a n


Drug StudiesThe response ofonchodermatitis

and itsmusculoskeletal

complaints todifferent treatment

schedules withIvermectin

SudanRadom focus, Mirarie,Southern Darfour Sate

Study period:January 1997–October 1998

Small GrantsScheme

(SGS) 1996 No. 10

Principal InvestigatorDr Omer Zayed BarakaDepartment of Medicine

Faculty of MedicineUniversity of Khartoum

Khartoum, Sudane-mail :

[email protected]

80 81

onchocerciasis could have a major socioeconomicimpact especially in farmer communities, where itwas found to be the major cause of morbidity andloss of working days. However, little is known aboutthe response of these complaints to ivermectin. Anearlier observation by the research team followingmass drug administration of ivermectin indicated thatthis complaint improved at the follow-up visit. Thisstudy was conducted aiming at determining theoptimum treatment interval with ivermectin forcontrol l ing onchocerciasis skin disease.

Materials and methods A village in Radomfocus, Mirarie, southern Darfour sate, where ivermectinhas not been previously distributed, has beenselected. The prevalence of onchocerciasis was 35%using the method of Rapid Epidemiological Mappingof Onchocerciasis (REMO). The village census wasperformed and all male individuals aged 15-60 yearswere enrolled in the study.

The format developed for WHO Multicountry studyof the economic impact and social cost of non-ocularonchocerciasis was used. Symptoms of itching andmusculo-skeletal pains were recorded and gradedas (0/1/2/3) according to the ability of the patient tocarry out the respective activity such as to work orsleep. The dermatological signs of the disease weredetermined. Individuals with skin lesions were carefullyexamined for dermatological signs of onchocerciasis.The degree of severity and morbidity ofonchodermatitis skin lesions were carefully assessedand graded into four grades. Ninety patients withmoderate or severe onchodermatitis were selectedand randomized using stratified random samplinginto three groups of 30 patients each. Stratificationwas based on different grades of skin lesion severity.The lesions were also mapped and photographed.Each of the three study groups were randomlyallocated to one of the following ivermectin treatmentschedules 3, 6, or 12 monthly treatment. administeredin a single oral dose of 150 µg/kg. Ivermectin wasdistributed to the rest of the villagers as part of thefirst ivermectin mass distribution programmeconducted in this area.

The three study groups were followed every 3months using the same initial methodology.

Data analysis consisted of determination of timeneeded for disappearance, improvement or recurrenceof symptoms, or changing in severity of the skinlesions.

Main study findingsSixty-three out of the 90 patients had completed

the study. There was no statistically significantdifference between the three study groups regardingtheir response to the three treatment schedules.

Itching was the commonest complaint manifestedby all the study population (63 patients). The intensity

of itching varied between troubling (55%) or severe(40%). The best response of itching to ivermectintreatment was obtained 6 months following the initialdose. Annual treatment resulted in reduction of thepatient suffering causing a decline in the severity ofitching but did not lead to significant decline in theprevalence of this symptom. Following treatment,both 3 and 6 month ivermectin schedules resulted in90 % reduction of the prevalence of itching 12 monthspost treatment, with no significant difference betweenboth schedules regarding prevalence or intensity ofthis symptom.

Muskuloskeletal complaints such as joint, bone orbackache were reported in 84% and 92% of the studypopulation, respectively. There was significantreduction in the prevalence and intensity of thesecomplaints following treatment, with no significantdifference in the efficacy of the three treatmentschedules.

Acute Papular Onchodermatitis (APOD) wasreported in 79% of the study population. The biannualand quarterly ivermectin treatment schedules resultedin significant decline in the prevalence of APOD from61% and 90% respectively to 15% each, at 12 monthspost treatment. Up to 6 months post treatmentschedules had comparable results. However,significant changes were evident in both the biannualand quarterly treatment schedules during subsequentfollow-up visits.

Regarding Chronic Papular Onchodermatistis(CPOD), it accounted for 68, 81, and 85% in annual,biannual and quarterly ivermectin treatmentschedules. The annual treatment schedule did notlead to any significant reduction in the prevalence ofthe skin lesions. The quarterly and biannual ivermectintreatment schedules had effectively reduced theprevalence of the lesion, while the three scheduleshad reduced the severity of the lesion. This wasmanifested by disappearance of excoriation with orwithout super infection.

Lichenified Onchodermatitis (LOD) was reportedfrom 70-73% of patients in the three groups. Therewas no significant difference between the threeschedules regarding the prevalence or intensity ofinfection. However, the biannual and quarterlyschedules recorded comparable results at 9 and 12month follow-up.

It is worth mentioning that depigmentary skinchanges were the most disfiguring and stigmatizinglesion of onchodermatitis. This was reported in 40to 54% of the studied groups and did not record asignificant improvement with treatment. Few patientswho presented with very early brown pigmentationreverted to normal. Patients initially presenting withlate brown pigmentation gradually progressed toleopard skin which was irreversible.

Onch

ocerc

iasis

AbstractIn a previous study carried out by the author

between 1988 and 1991, a schistosomiasiscontrol programme was undertaken in Mariout,west Alexandria, an area reclaimed from thedesert . The basel ine s i tuat ion ofschistosomiasis was established in a sampleof 3 villages with a total population of 6577individuals. The prevalence of Schistosomamansoni in the 3 villages was 22%, 28% and40%. A survey of the water channels revealedthat 60%-80% of them dried up betweenirrigation rounds. Biomphalaria alexandrinawas detected in channels permanentlycontaining water. The proportion of infectedsnails was unexpectedly high (7.5%, 15.8%and 17.3%). In 1989, all positive cases werecalled for treatment with a single dose ofpraziquantel. This was followed by an abruptdrop in the disease prevalence of infection to18.5%, 14.9% and 15.4%. In 1990, snail controlusing 70 ppm of the dry form of the plantmolluscicide Ambrosia maritima (damsissa)was applied. This led to a 90% drop in thesnail population and in the number of infectedsnails. Schistosomiasis prevalence showed afurther slight decline, with disease prevalencerates becoming 17%, 14% and 12%. No controlactivities were undertaken thereafter. Theobjectives of the present project were todetermine the level of schistosomiasis in thearea, 3 years after the cessation of control

measures, to find an explanation for casesconverting to positive, and for cases revertingto negative.

Results Revision of the census data in 1993demonstrated an overall net increase of thepopulation (including new settlers) by 14.6%as compared to the census of 1988. Theprevalence of S. mansoni was still on thedecline and the rates had become 13.6%,14.4% and 9.0%. A survey of the waterchannels did not reveal any significant changesin the total length of the water channels or ofthe infected ones. The water network had notbeen modified. The snail population hadincreased compared to the baseline findings,but the proportion infected was very low (0%,0.5% and 0.9%). A study of the conversionindicated that the yearly incidence was 11%,whereas it was 14% before control. It wasfound that 20% of converting cases reportedworking outside the area, 21.5% were newsettlers and the remaining 60% were probablyinfected locally. A study of reversion revealedthat only 7% of positive cases had been treatedin the previous 3 years. It was found that 30%of cases were missed by the Kato-Katztechnique (2 slides).The remaining 63% couldbe considered self-cured during the 3-yearperiod. This finding was explained by the factthat the mean life span of the S. mansoni adultworm is 3-4 years. Accordingly, in the absenceof re-infection, 20-30% of the infectedindividuals lose their infection every year.

Conclusion An integrated control programme,applied once, could control schistosmiasis inboth the human and snail hosts for a periodof 4 years . However, the increase in snailpopulation points to the possibility of reachingthe original level of infection. These findingsgenerally call for application of integrated controlmeasures every 3 years.

PublicationsFarag H. Evaluation of an integrated approach

to schistosomiasis control in a resettlement areawest of Alexandria. EMHJ, 1996, 2(3):558-559.

BackgroundMariout, 40 km to the west of Alexandria,

consists of land reclaimed from the desert,and irrigated by El-Nubarieh canal. An

Evaluation of anintegrated approachof schistosomiasis

control(chemotherapy andsnail control) in aresettlement area,west of Alexandria

EgyptMariout area, western

Alexandria Governorate

Study period:December 1993–1994

Small GrantsScheme

(SGS) 1993 No. 6

Principal InvestigatorDr Hoda Farag

Department of ParasitologyMedical Research Institute


Integrated control measures for schistosomiasis could besuccessfully achieved by a single dose of praziquantel (40 mg/kgbody weight) and snail control using 70 ppm of the dry form of theplant molluscicide Ambrosia maritima (damsissa).

The Kato-Katz technique (2 slides) records a considerablepercentage of false-negative cases (30%) when used in lowinfection intensity (fewer than 100 epg).

The mean life span of the S. mansoni adult worm is 3-4 years.Accordingly, in the absence of re-infection, 20%-30% of the infectedindividuals lose their infection every year. However, the increasein snail population points to the possibility of reaching the originallevel of infection with population movement from areas where nocontrol measures were implemented. The application of integratedcontrol measures every 3 years is therefore recommended.

SchistosomiasisE g y p t


Control

82 83

integrated schistosomiasis control programme wasimplemented in this region during the period of 1988-1991. In the baseline survey, the prevalence ofSchistosoma mansoni in 3 villages in this area was22%, 28% and 40%. The proportion of infected snailswas unexpectedly high (7.5%, 15.8% and 17.3%).Positive cases were treated with a single oral dose ofpraziquantel. This was followed by an abrupt fall inthe disease prevalence of infection to 18.5%, 14.9%and 15.4%. Snail control was performed by applyingthe plant molluscicide Ambrosia maritima (Damsissa)to all infested water channels. One year later, thecontrol programme was evaluated and was consideredsuccessful. The present study was conducted by thesame research team, aiming at determining theprevalence of schistosomiasis in this area, 3 yearsafter the cessation of control measures.

Materials and methodsFollowing the same methodology as the initial study,

3 previously surveyed villages (Orabi, El-Gazayer,and Palestine), out of a total of 26 in the area, with atotal population of 6577 individuals, were selected.Maps of the houses and water channels around thevillages were prepared and a census of the populationwas recorded. A stool survey of all villagers wasperformed using the Kato-Katz (2 slides) methods.Snails were collected, counted, crushed and examinedunder the dissecting microscope for trematodeinfections. Villagers were interviewed according to aquestionnaire including information regarding populationmovements, such as having an occupation outsidethe village or being a new settler in this area, andhistory of anti-schistosomal treatment during the initialstudy. Stool specimens were obtained from 191individuals with a low intensity of infection. Eachspecimen was examined by Kato-Katz smear (2 slides),formol ether concentration and simple sedimentation.

Main study findingsThe revised census indicated a 14.6% increase in

the population. It was observed that a proportion ofthe population moved to urban areas, while new settlersadded 26% to the original population. Theenvironmental conditions in this area were unfavourablefor the snail habitats; the soil is sandy with limitedwater supply, and accordingly, the majority of waterchannels becomes completely dry between irrigationrounds. However, B. alexandrina was identified in thefew canals and drains containing water all year roundor that dried for only short periods. The snails werecollected from the 3 villages throughout the year, withtheir numbers peaking in April. El-Gazayer villagerecorded the highest frequency of snails, followed byOrabi and Palestine. Compared to the baseline survey,there was a 2-fold increase in the number of snails inEl-Gazayer and Orabi villages, and a significantreduction (50% reduction) in the number of snails

collected from Palestine village. In spite of the overallincrease in the snail population, the proportion infectedwas very low (0%, 0.5% and 0.9%). The study of thewater network revealed that in Orabi, the stretch ofthe canal carrying water increased together with theinfested channels. In El-Gazayer, there was a reductionin the length of canals and the number of harbouringand infected snails. In Palestine, in spite of the increasein the amount of water, the length of infested channelshad not changed and no canals were infected.

The prevalence and intensity of infection in the 3villages were still declining one year after the applicationof the control measures in Orabi and Palestine, therebyindicating a decrease in the transmission potential inthese 2 villages; the prevalence dropped to 13.6%and 9.0%, respectively, and the intensity from 52 to34 epg, and from 49 to 32 epg, respectively. On theother hand, there was a decrease in the mean eggcount of positive cases in El-Gazayer while theprevalence was maintained at 14.4%. Infection inchildren younger than 5 years of age was significantlyreduced to 1.2% compared to 10% in 1988 and 2.7%in 1991.

Study of conversion (negative to positive)This was mainly related to working outside the areaand to new settlers.

Study of reversion (positive to negative) Onehundred forty five positive cases in 1991 were negativein the present study, 7% reported treatment duringthe previous 3 years, and 30% were missed, therefore,the remaining 63% could be considered self-curedduring the 3-year period. This finding was explainedby the fact that the mean life span of the S. mansoniadult worm is 3-4 years. Accordingly, in the absenceof re-infection, 20%-30% of the infected individualslose their infection every year.

It was also found that 30% of cases were missedby the Kato-Katz smear technique (2 slides).

Conclusions and recommendationsAn integrated control programme, applied once,

could control schistosomiasis in both the human andsnail hosts for a period of 4 years if the same conditionsare maintained (no re-infection). However, the increasein the snail population points to the possibility ofreaching the original level of infection, especiallyconsidering the significant population movement.These findings generally call for the application ofintegrated control measures every 3 years.

Schis

tosom

iasis

AbstractThe prevalence of schistosomiasis in Ma'rib

Province, and the distribution and infectionrate with Schistosoma cercariae of the snailintermediate hosts in Ma'rib Dam wereinvestigated. A total of 2650 urine and 2023stool samples were examined in the centralhospital of Ma'rib Province, Republic of Yemen.

Results The overall prevalence ofSchistosoma mansoni was 35.1% and of S.haematobium 18.3%, while 1.2% of the infectedsubjects had mixed infections. The infectionrate for both urinary and intestinalschistosomiasis was higher in males than infemales. The highest prevalence rates wereobserved in males and females aged 16 to 20years. All infected persons were treated witha single dose of 40 mg/kg praziquantel.Biomphalaria arabica, the intermediate hostof S. mansoni, was found in all 3 surveyedlocations, while Bulinus truncatus, theintermediate host of S. haematobium, wasfound in 2 locations only. The overall rate ofinfections of Biomphalaria arabica was 1.65%while the infection rate of Bulinus truncatuswas 1.3%. Snail control with Bayluscide wasperformed throughout the canals, especially

those with a higher probability of water contact.

Publications1. Nagi MA, et al. Epidemiological, clinical and

haematological profile of schistosomiasis inYemen. EMHJ , 1999, 5 (1) :177-181.

2. Extension of the National SchistosomiasisControl programme to all Governorates. El-Thawra Newspaper, Yemen, September 1995.

BackgroundSchis tosomias is due to both

Schistosoma haematobium and S.mansoni is endemic in Yemen. Accordingto a rough estimation made in 1971 andconfirmed by further studies done through1982, more than one million people ofYemen have one or both types of infection.In view of the lack of information regardingschistosomiasis conditions in Ma'ribProvince, where the largest dam in thecountry is located, the present study wasconducted to assess the prevalence ofurinary and intestinal schistosomiasis inthe inhabitants of Ma'rib Province, thedistribution of the snail intermediate hostin 3 localities around the Ma'rib dam andtheir infection rate with Schistosomacercariae.

Materials and methodsA total of 2650 urine and 2023 stool

samples were collected from patients withvarious medical problems who had beenadmitted to the general laboratory in thecentral hospital of Ma'rib Province, Yemen.Urine samples were examined by theNucleopore technique, and stool sampleswere examined by the Kato-Katztechnique. All urine and stool sampleswere collected between 11:00 and 14:00.All infected persons were treated with asingle dose of 40 mg/kg praziquantel.

Ma'rib Dam is located near Ma'rib town,120 km from Sana'a. The distancebetween the dam site and the old city is11 km. It is located 3 km from the site ofthe historic Ma'rib Dam. The reservoir ofthe dam and its main canal were opened

Schistosomiasiscontrol in Ma'rib

governorate

Republic of YemenMa'rib governorate


Small GrantsScheme

(SGS) 1993 No. 48

Principal InvestigatorDr Musaid Ahmed NagiNational Schistosomiasis

Control ProgrammeMinistry of Health

Sana’a, Republic of Yemen

The prevalence of S. mansoni and S. haematobium infection inMa'rib governorate was 35.1% and 18.3%, respectively, and 1.2%of the infected individuals had mixed infection.

The highest infection rates occurred in the age group of 16 to20 years, with higher rates in males, which was attributed to theirincreased exposure during work in the field, swimming, bathing,or fishing in infested water.

The Biomphalaria arabica snail was more widespread thanBulinus truncatus and the rate of infection of Biomphalaria withSchistosoma cercariae was higher than that of Bulinus, indicatingthat the types of snail breeding habitats are the main determinantsof the species of parasite infecting inhabitants of different areas.

Schistosomiasis control was performed by praziquanteladministration to all infected cases and by snail control usingBayluscide. Destruction of all infested locations around the dam,proper disposal of sewage to decrease human exposure to infectedsnails, mass treatment and educational programmes to increaseawareness of the problem were recommended.

SchistosomiasisR e p u b l i c o f Y e m e n


Control

84 85

in December 1986. Three localities (the main gateof the dam, Diversion A and Diversion B) wereinvestigated for the presence of snails. Each site wasinspected twice each season for 2 years. Snails weresearched through examination of submerged andemergent plants. A minimum of 1 hour was spent ateach site during each visit. Snails found were placedin wide-mouthed screw-cap containers and broughtalive to the laboratory for identification. The methodof Teesdale et al was used to examine the infectedsnails. Snail control with Bayluscide was performedthroughout the canals, especially those with a higherprobability of water contact.

Evaluation of the control measures was notperformed by the research team.

Main study findingsThe prevalence of S. mansoni and S. haematobium

infection accounted for 35.1% and 18.3%,respectively. In both urinary and intestinalschistosomiasis, the highest rates of infection occurredin the age group of 16 to 20 years, and it was higherin males than in females, though this difference wasnot significant. Fourteen subjects (1.2% of the infectedindividuals) had mixed infection. Bulinus truncatus,the intermediate host of S. haematobium, was foundin 2 habitats (the main gate of the dam and DiversionB) out of the 3, while Biomphalaria arabica, theintermediate host of S. mansoni, was found in all the3 inspected habitats.

The rate of infection of Biomphalaria arabica withS. mansoni cercariae was 1.65% while the rate ofinfection of Bulinus truncatus with cercaria of S.haematobium observed during the survey was 1.3%.This was consistent with reports from Taiz province,that Biomphalaria arabica was more widespread thanBulinus truncatus and the rate of infection ofBiomphalaria with Schistosoma cercariae was veryhigh in comparison to that of Bulinus [1].

It seems that the density of both Biomphalariaarabica and Bulinus truncatus is in continuousincrement. In 1985, a team from World HealthOrganization (WHO) made an extensive study toassess the potential impact of Ma'rib dam on thehealth conditions in Ma'rib province, but they did notfind the snail hosts for schistosomes [2]. In 1990,Arfaa [3] found both snails in two ponds immediatelydown stream of the main canal of the dam. In 1991,Nagi [4] found that canals of irrigation systems ofMa'rib dam were heavily infested with snails, and hestated that, in some places, shells covered waterplants like salt. In view of what is mentioned above,the high rate of infection recorded among theinhabitants of Ma'rib province is not surprising.

Conclusions and recommendationsThe differences in the prevalence rates of the 2

Schistosoma species may be attributed to the relativeprevalence of the species of snail intermediate hoststransmitting them. The higher infection rate amongmales was attributed to their increased exposureduring work in the field, swimming and bathing, orfishing in infested water. Furthermore, the types ofsnail breeding places and their suitability for thebreeding of Bulinus or Biomphalaria snails are themain determinants of the species of parasite infectinginhabitants of different areas.

It was recommended to discover and destroy allinfested locations around the dam. Proper disposalof sewage to decrease human exposure to infectedsnails, mass treatment and educational programmesto increase awareness of the problem to control thedisease, were also recommended.

References[1] Hazza YA, Arffa F, Haggar M. Studies on

schistosomiasis in Taiz Province, Yemen ArabRepublic. Am J Trop Med Hyg, 1983, 32:1023-1028.

[2] Chen K, Delfini LF, Ahmed NY. Health aspectsof the Marib Dam and irrigation project, YARASS.Report WHO EM/VBC/84.

[3] Arffa F. Schistosomiasis in the Republic ofYemen. WHO Assignment report , 1990,YES/PDP/001.

[4] Nagi MA. Report of the Ministry of Health, 1991.

Schis

tosom

iasis

AbstractFour water courses in Abis, rural Alexandria,

with the same conditions and snail densitywere chosen for this study. All methods wereapplied simultaneously in May. Snails werecol lected monthly to evaluate themalacological effect. A single application of100 ppm dried Ambrosia maritima decreasedthe snail density significantly within one month.The snail density was maintained at a verylow level until the end of the year. Fresh Azollapinnata applied at a dose of 280 ppm loweredsnail density to a level intermediate betweenAmbrosia maritima and Bayluscide. The snailpopulation began to build 5 months later.Bayluscide applied once at a dose of 1 ppmsignificantly lowered the snail density. Thislowering was maintained for 4 months afterwhich the snail density began to build up andremained high until the end of the study period.Clearing of vegetation was followed by anabrupt lowering of snail density, and this wasmaintained for a period longer than thatfollowing Bayluscide and Azolla.

PublicationsAllam A.F. Evaluation of different means of

control of snail intermediate host of Schistosomamansoni. Journal of the Egyptian Society ofParasitology, 2000, 30(2):441-450.

BackgroundT h e s u c c e s s f u l c o n t r o l o f

schistosomiasis should be based on anintegrated approach which includes thecontrol of the intermediate snail host. Forthis reason, there is a continuous needfor the development and evaluation ofnew, inexpensive, but highly effectivemolluscicides. Many developing countriesare reluctant to embark on chemical snailcontrol programmes, using costlysynthetic compounds from industrializednations. National and internationalinstitutions are currently giving increasingattent ion to the study of plantmolluscicides in the hope that they mayprove to be cheaper and more readilyavailable for snail control compared tosynthetic chemicals.

In Egypt, the annual herb, Damsissa(Ambrosia maritima), is a biologicalmoluscicide, and could be a part of theecosystem. It was reported that this planthas lethal effects on snails and their eggsdue to its active moluscicide components,main ly Ambros in and Damsin.

The Azolla plant is a symbiotic algalassociation that grows on the watersurface. It is widespread in the westernhemisphere as well as in Egypt. Thisplant plays an important role in theprocess of nitrogen fixing in the soil andas food for domestic animals; it is also adesirable organic fertilizer in rice fields.This plant lowers the survival, growth andfecundity of B. alexandrina snails.Bayluscide is still recognized as the mosteffective chemical molluscicide, and it ispredominantly used in snail controlprogrammes. However, the cost ofchemical molluscicides is soaring to suchan extent that governments are reluctantto include their purchase in budgets.Clearing vegetation is one of the importantphysical methods of snail control. Thepresent study aimed at evaluating

Control of snailintermediate host of

schistosomiasisusing differentmolluscicides

Egyptrural Alexandria

Study period:January 1997–

March 1998

Small GrantsScheme

(SGS) 1996 No. 49

Principal InvestigatorDr Amal Farahat Allam



A single application of Bayluscide at a dose of 1 ppm succeededin lowering the snail density for a short period of time, not exceeding3 months. On the other hand, this method is costly and was provedto be toxic for aquatic organisms such as fish.

A single application of A. maritima at a dose of 100 ppmconsiderably reduced the number of snails. The snail populationremained low for at least 7 months.

The snail density decreased after a single application of Azollapinnata (280 ppm) on Biomphalaria alexandrina snails. The numberof snails was maintained at a low level for 4 months, then increased.

Clearing of vegetation was followed by the abrupt lowering ofthe snail density, and this low level was maintained for 8 months.

It was concluded that the best results for control of B. alexandrinacould be obtained by clearing vegetation together with theapplication of Damsissa.



Control

86 87

different methods of B. alexandrina snail control,namely chemical (Bayluscide), biological (plantmolluscicides, Ambrosia maritima and Azolla pinnata),and physical (clearing of vegetation) means.

Materials and methodsThis study was conducted in Abis 10 (Esbet El-

Matar), 10 km west of Alexandria. Water courseschosen for this study were based upon: the presenceof a significant number of B. alexandrina snails, theirsimilarity in size and conditions, being equally distantfrom the village and with comparable human activities,water volume, pH, salinity, and presence or absenceof vegetation. Twenty equidistant sampling stationswere determined for each water body. The snailswere collected by a wire net with 3 adjacent dipsevery 25 metres.

Molluscicides were applied during May 1997 asfollows:

Chemical moluscicide: Bayluscide was appliedat a dose of 1 ppm in the chosen drain using a dripfeed technique with a drain dispenser that deliversa constant flow of the molluscicide.

Plant molluscicides: A. maritima was obtainedin its dry form from Aswan and applied at a dose of100 ppm. The dry Damsissa plant was applied inweighed nylon mesh bags, each contained 1/2 kg.The bags were distributed in alternation along bothbank sides. A. pinnata was collected in its fresh formfrom different water courses outside the area. Thefresh plant was applied by dispersal in alternationalong both banks. The concentration of the plant was280 ppm W/V. The land owner was asked to removethe vegetation from the fourth drain by the usualmanual method. Snail collection was repeated usingthe same standard technique, 2 weeks afterapplication of the control method, and then monthlyuntil the end of the study.

Main study findingsBayluscide application at a dose of 1 ppm led to a

rapid decrease in snail density that was maintainedfor 3 months. This was followed by a rapid increasein the snail population, and the snail density remainedhigh until the end of the study. Thus, a singleapplication succeeded in lowering the snail densityfor a short period of time; then, it is likely that thechemical was washed away with the flowing water,and the environment became suitable again for thesnails. Other main drawbacks appear to be its highprice and its adverse effects on non-target organisms,particularly fish, at the concentrations used to controlthe snails.

The malacological data indicated that control of B.alexandrina was successful in the water courseswhich were treated with A. maritima. A single

application of 100 ppm of the plant considerablyreduced the number of snails. The snail populationremained low for the rest of the year. This prolongedaction may be due to the fact that the dried plant wasfixed in situ and was not washed away with the flowingwater. Furthermore, the plant was not toxic whenused at the molluscicidal concentration.

This study revealed that the snail density decreasedafter a single application of Azolla pinnata (280 ppm)on the B. alexandrina snails. The number of snailswas maintained at a low level for 4 months, then theirnumbers increased. Compared to Bayluscide, thetotal number of snails collected was lower after Azollaapplication, while compared to Damsissa it washigher. The clearing of vegetation was followed byan abrupt lowering of the snail density that continuedfor 8 months. The decrease in snail density wasmaintained for a longer period, longer than that whichfollowed the application of Baylluscide or Azolla. Thismay have occurred because the snails were deprivedof their food and shelter, and were not subsequentlyable to repopulate the water course in short periodof time.

It was concluded that the best results for control ofB. alexandrina would be obtained by clearingvegetation together with the application of Damsissa.

Schis

tosom

iasis

AbstractAn intervention study was conducted in

Khemir, northern Sana'a, whereby integratedcontrol of urinary schistosmiasis was performedby means of chemotherapy and healtheducation. The study also aimed atinvestigating the validity of reagent strips asa rapid diagnostic tool, and their cost-effectiveness together with visible haematuriafor rapid screening of Schistosomahaematobium infection. Houses and schoolsin the study area were randomly selected. Abaseline survey was performed using aquestionnaire and urine analysis. A total of8540 individuals were treated with praziquantel40mg/kg body weight, single dose. Weeklyhealth education classes were held in schoolswhere health education posters weredistributed. Other health education sessionswere also held in schools for non-enrolledchildren and their parents. A post-interventionsurvey was also performed by questionnaireand urine analysis

Results There was a significant decrease inthe prevalence of infection 14 months post-intervention. The overall prevalence of S.haematobium infection dropped from 58.9%to 5.8%. Furthermore, there was a significantdecrease in the frequency of heavy infectionsfrom 40% to less than 20% of infections. Therewas no significant difference between malesand females, nor between literate and illiterateindividuals regarding the prevalence or intensityof infection. Health education sessions resulted

in a significant decrease in the frequency ofcontact with water sources and moreadherence to preventive measures.

Conclusion Reagent strips and visiblehaematuria could be cost-effective screeningmethods for the disease only if applied inremote areas with limited accessibility to healthservices. However, reagent strips are far lessreliable diagnostic tools compared tomicroscopic urine examination.

BackgroundSchistosomiasis has been a public

health problem in Yemen for the last 2-3 decades. Its endemicity together withother intestinal parasites has beenfrequently reported. However, there havebeen no previous reports regarding thesituation in Khemir or integrated methodsdeveloped for schistosomiasis control inthe whole country. The present study wastherefore conducted to evaluate the impactof integrated surveillance and controlmethods on the burden of schistosomiasisin this area, as well as studying the validityof using reagent strips as a rapiddiagnostic tool, and its cost-effectiveness,together with visible haematuria, for rapidscreening of the S. haematobium infection.


Setting Al- Khemir, located 90 km northof Sana'a, is an agricultural areadepending on rain and ground water forirrigation and domestic use.

Design This was an intervention studytargeting community and schoolchildren.

Baseline community survey A listof the houses in the study area (1000)was prepared and a random sample of100 houses (863 individuals) wasselected. Each individual was interviewedusing a questionnaire and was subjectedto urine examination.

Baseline school survey A sampleof 20% of schoolchi ldren (287

Evaluation of anextended school-based programmefor the control of

Schistosomahaematobium in an

endemic area inYemen

Republic of YemenWadiaa District, Al-

Khemir, northern Sana'a,Yemen

Study period:May 1999–April 2000

Small GrantsScheme

(SGS) 1999 No. 6

Principal InvestigatorDr Musaid Ahmed El-Nagi

National SchistosomiasisControl ProgrammeMinistry of Health

Sana'a, Republic of Yemen



Control

An integrated school-based programme consisting ofchemotherapy and health education proved to be effective incontrolling urinary schistosomiasis in endemic areas.

The integrated programme had a significant impact on prevalence,lowering the relative contribution of heavy infections among positiveindividuals, as well as changing the behaviour of the community,manifested in the adoption of preventive measures against thedisease.

Reagent strips and visible haematuria could be cost-effectivescreening methods for S. haematobium infection in remote areaswith limited accessibility to health services.

schoolchildren), randomly selected from the 14 schoolsof the area was included in the study. These childrenwere also interviewed using a questionnaire and weresubjected to urine examination.

Intervention The integrated methods ofschistosomiasis control consisted of chemotherapyfor S. haematobium infection and health education.A total of 8540 individuals were treated withpraziquantel 40mg/kg, single dose. These wereschoolchildren, non-enrolled school-age children, andpreschool children and their parents. School teachersfrom all 14 schools in the district participated in a 2-day seminar. Weekly health education classes wereheld in schools where health education posters weredistributed. Other health education sessions werealso held in schools for non-enrolled children andtheir parents. They were educated about the diseaseand briefed about the study. They were also givendetailed information regarding methods of healtheducation for the different groups, chemotherapy forschistosomiasis and its dosage, and record-keeping.One teacher from each school was appointed as thecoordinator for the control activities in his/her school.

Community evaluation survey (14 months postintervention) The houses in the study area (1000)were listed and a study sample of 100 houses (913individuals) was randomly selected. Evaluation wasperformed via questionnaire and urine analysis.

School evaluation survey (14 months postintervention) Three hundred and twenty threeschoolchildren, consisting of 20% of all schoolchildrenin the area, were subjected to post-interventionevaluation via questionnaire and urine analysis.

Validation of reagent strips The reagent stripswere compared to microscopic urine examination todetermine their validity as rapid diagnostic tools forS. haematobium.

Main study findingsThe overall prevalence of S. haematobium infection

was 58.9%. The age-specific prevalence was 48.3%,61.3% and 69.3% among preschool, schoolchildren,and non-enrolled school-age children, respectively.The prevalence of infection was 55.2% among 16-40years old individuals, compared to 42.9% in olderindividuals.

Regarding the intensity of infection, the majority ofinfections (60%) were light (50 eggs/10 ml urine) and40% of infections were heavy. The frequency of lightinfections in children (15 years old) was significantlyhigher than heavy infections. By contrast, the frequencyof heavy infections was significantly higher than lightinfections in the older age groups. There was nosignificant difference between males and females,nor between literate and illiterate individuals regarding

the prevalence or intensity of infection. There was asignificant decrease in the prevalence of infection 14months post-intervention. The overall prevalencedropped to 5.8%, ranging from 4.9% among 6-15year-old children to 7.8% among individuals aged 16-40 years. Furthermore, the majority of infections werelight infections (81.1%), regardless of age or gender.

Impact of health education The frequency ofcontact with water sources significantly decreasedfrom 95% and 98% to 9% and 3.6% in the communityand school surveys, respectively. Similarly, adherenceto preventive measures rose from almost none to97% and 88% in the community and school surveys,respectively. Almost all the interviewed populationswere treated and attended health education sessions(up to 97% chemotherapy and 99% for healtheducation). They reported that they knew about theprogramme from the mobile team of the programme(90%), from the head of the community (88.1%), anda limited number (15.3%) reported school sessionsas being their source of information. There was mutualsatisfaction with the programme from those whoimplemented the programme as well as from itsrecipients. In fact, health education sessions wererated as very good or excellent by 85% of the studiedpopulation, and almost all community leaders andschool teachers reported the smooth implementationof the programme.

Validation of reagent strips Compared to theparasitological examination of urine, the reagent stripsrecorded low sensitivity (56.5% and 56.95% incommunity and school surveys, respectively), lowspecificity (57.3% and 52.9%, respectively), limitedpositive predictive value (72.2% and 68.3%,respectively), and low negative predictive values ofaround 40% in both surveys. There was badconcordance between these reagent strips andparasitological examination of urine (Kappa = 0.1 inboth surveys).

Cost-effectiveness of screening by thereagent strips and visible haematuria Thescreening methods greatly reduced the cost of thecontrol programme in the study area (Wadiaa), whichis a remote area with limited facilities.

Conclusions and recommendationsAn integrated school-based programme of

chemotherapy and health education is an effectivemeans to control S. haematobium in endemic areas.Reagent strips and visible haematuria could be usedfor screening the disease in remote areas with limitedaccessibility to health services, however, reagentstrips are far less reliable diagnostic tools comparedto microscopic urine examination.

88 89

Schis

tosom

iasis

AbstractSchistosoma circulating antigens were used

to indicate the infection intensity and to assesscure. An immunoglobulin G2a (IgG2a) mousemonoclonal antibody was used in a fast dot-enzyme-linked immunosorbent assay (ELISA;FDA) for rapid and simple diagnosis ofschistosomiasis in the field. Seven hundredEgyptians were examined for Schistosomamansoni and other parasitic infections. A rectalbiopsy was done as a "gold standard" forindividuals showing no S. mansoni eggs intheir faeces. Egg counts were obtained by theKato-Katz smear method. Specific anti-schistosome IgG antibodies were evaluatedin sera by ELISA. Urine samples from the 700individuals were tested by FDA for detectionof the circulating antigen. The assay showeda sensitivity of 93% among 433 infectedindividuals and a specificity of 89% among 267noninfected individuals. FDA showed thehighest efficiency of antigen detection (91%)compared with the efficiency of antibodydetection by ELISA (75%) and stool analysis(60%). In addition, FDA detected infectedpatients with 20 eggs/g of faeces. Also, thesensitivity of FDA ranged from 90% to 94%among samples from patients with differentclinical stages of schistosomiasis. All the assay

steps could be completed within 30 minutesat room temperature for 96 urine samples. Themonoclonal antibody identified a 74-kDaantigen in different antigenic extracts of S.mansoni and S. haematobium and in the urineof infected individuals. In addition, a 30-kDadegradation product was identified only in theurine samples. On the basis of these results,FDA could be used as a rapid tool for thesensitive and specific diagnosis of Schistosomainfection.

Publications1- El-Masry SA et al. Detection of circulating

antigens in individuals infected withschistosomiasis using monoclonal antibody andfast dot-ELISA. EMHJ, 1996, 2(2):340.

2- Attallah AM et al. Immunochemicalcharacterization and diagnostic potential of a 63-kilodalton Schistosoma antigen. Am J Trop MedHyg, 1999, 60(3):493-497.

3- Attallah AM et al.Rapid detection of aSchistosoma mansoni circulating antigen excretedin urine of infected individuals by using amonoclonal antibody. J Clin Microbiol, 1999,37(2):354-357.

4- Attallah AM et al. Fast-Dot ELISA usingurine, a rapid and dependable field assay fordiagnosis of schistosomiasis. J Egypt SocParasitol, 1997, 27(1):279-289.

BackgroundThe dot enzyme-linked immunosorbent

assay (ELISA) type of immunodiagnostictest has been reported for use in thedetection of schistosomiasis. In the presentstudy the sensitivity and specificity ofcirculating antigen detection in urine wasevaluated by a newly developed fast dot-ELISA assay (FDA) and compared withthose of standard traditional techniquesfor the rapid and simple diagnosis ofhuman schistosomiasis in the field.

Materials and methodsSeven hundred individuals were

subjected to full clinical examinations,stool, urine and blood examinations. Arectal biopsy was done as a "gold

Detection ofcirculating antigens

in individualsinfected with

schistosomiasisusing monoclonal

antibodies and fastdot-ELISA

EgyptMansoura Governorate

Study period:June 1994–1995

Small GrantsScheme

(SGS) 1993 No. 28

Principal InvestigatorDr Samir El-Masry

Gastroenterology CentreMansoura University

Mansoura, Egypt

The fast dot-ELISA assay (FDA) for the detection of Schistosomacirculating antigen excreted in urine had a higher sensitivity (93%)than microscopic examination of eggs in stool (35%) and a higherspecificity (89%) than anti-schistosomal antibody detection inserum by ELISA (56%).

FDA had sensitivities ranging from 90% to 94% for the differentclinical stages of schistosomiasis and could detect the schistosomeantigen in urine samples from patients with light infections of 20epg of faeces.

The developed FDA proved to be a simple, rapid, sensitive, andspecific enzyme immunoassay. The urine samples are usedwithout any treatment, 96 samples could be run in about 30minutes, and all assay steps are done at room temperature withno need to sophisticated equipment. Therefore it could be usedin the field as part of a mass screening programme forschistosomiasis.



Diagnosis

90 91

standard" for individuals showing no S. mansoni eggsin their faeces.

Schistosoma antigenic extract preparationSoluble worm antigen preparation (SWAP), cercarialantigen preparation (CAP), and soluble egg antigen(SEA) were prepared, and the protein contentmeasured, then the specimens were aliquoted andstored at 70 °C.

BRL4 MAb An anti-Schistosoma monoclonal antibodyMAb (BRL4 MAb) had been generated by theestabl ishment of mouse hybridomas [1].

Immunoblotting Polyacrylamide gel electrophoresiswas carried out in thick, 10% vertical slab gels (Bio-Rad) under reducing conditions. After separation bysodium dodecyl sulfate-polyacrylamide gelelectrophoresis, the gel was electroblotted onto anitrocellulose (NC) filter at 60 V for 2 hours. Afterblocking with 5% on fat milk, the NC filter was incubatedovernight with the BRL4 MAb diluted 1:50. Goat anti-mouse immunoglobulin G (IgG) alkaline phosphatasewas used at a dilution of 1:500 for 2 hours at roomtemperature and 5-Bromo-4-chloro-3-indolylphosphate-Nitro Blue Tetrazolium (BCIP-NBT)premixed substrate solution (Sigma) was used tovisualize the reaction. The reaction was stopped withdistilled water. Then the filter was dried and kept inthe dark.

Indirect ELISA A polystyrene flat-bottom microtitreplate was coated with 2.5 µg of SWAP per ml incarbonate-bicarbonate buffer (pH 9.6). After blocking,50 µl (per well) of 1:200-diluted human serum samplesin 0.05% (v/v) phosphate-buffered saline (PBS) Tween20 (PBS-T20) was added and the plate was incubatedat 37 °C for 2 hours. After washing, 50 µl (per well) ofanti-human IgG alkaline phosphatase conjugate (wholemolecule; Sigma) diluted 1:500 in 0.2% (w/v) non-fatmilk in PBS-T20 was added and the plate wasincubated at 37 °C for 1 hour. One mg of para-nitrophenyl phosphate (Sigma) per millilitre was usedas a substrate, and the absorbance was read at 405nm with an EL311 microplate autoreader (Bio-TekInstruments).

Fast dot-ELISA assay FDA was carried out witha Hybri-Dot Manifold (Bethesda Research Laboratories,Richmond, Calif.) to detect the Schistosoma circulatingantigen in urine. The NC filter (Sigma) was washed ina bath of distilled water and soaked in a bath of PBS(pH 7.2), each for 1 minute. Then, the NC filter wasoverlaid on a filter-paper presoaked in PBS and heldin the manifold. Fifty µl of a urine sample was addedto each well, and after suction, the NC filter was air-dried and then washed in 0.3% PBS-T20 for 1 minuteon a shaker. Blocking of nonspecific binding sites onthe filter was done with 2% (w/v) nonfat dry milk inPBS-T20 for 15 minutes on a shaker. After removal

of the blocking solution, the BRL4 MAb was added ina dilution of 1:500 in PBS-T20 for 5 minutes withcontinuous shaking. After washing, anti-mouse IgGalkaline phosphatase conjugate (Sigma) was addedat a dilution of 1:500 in blocking buffer for 5 minuteswith continuous shaking. The filter was washed withPBS for 3 minutes. An insoluble purple product wasdeveloped after the addition of the BCIP-NBT premixedsubstrate solution (Sigma) for about 3 minutes. Thereaction was stopped with distilled water, and the filterwas dried and kept in the dark. Positive controls forthe assay were the affinity-purified antigen either fromSWAP or from the urine of infected individuals andneat urine samples from infected individuals. Negativecontrols were urine samples from noninfectedindividuals. FDA allows a semiquantitative reading ofthe resulting coloured spot in case of antigen detection(i.e. a positive test result). The purple colour that wasproduced varied in its intensity from weak (1+ or 2+)to strong (3+ or 4+). Positive controls with thesedifferent colour intensities were used. A colourlessspot was produced in the case of a negative test result.The resulting colour for the tested sample was thencompared and related to the colour of one of thepositive and negative controls with the naked eye.

Main study findingsFDA had a higher sensitivity (93%) than microscopic

examination of eggs in stool (35%) and a higherspecificity (89%) than anti-schistosomal antibodydetection in serum by ELISA (56%). Moreover, FDAhad sensitivities ranging from 90% to 94% for thedifferent clinical stages of schistosomiasis. Also, itcould detect the schistosome antigen in urine samplesfrom patients with light infections of 20 epg of faeces.The circulating antigens were detected in individualswith low egg counts [2].

We can conclude that the FDA developed on thebasis of an IgG2a MAb for the detection of Schistosomacirculating antigen excreted in urine proved to be asimple, rapid, sensitive, and specific enzymeimmunoassay. The urine sample was used withoutany treatment, the assay needs no sophisticatedequipment, and 96 urine samples could be run inabout 30 minutes. In addition, all assay steps weredone at room temperature. The assay could thereforebe used in the field as part of a mass screeningprogramme.

References[1] Attallah AM et al. Immunochemical purification

and characterization of a 74.0 kDa Schistosomamansoni antigen. J Parasitol, 1998, 84:301-306.

[2] De Jonge N et al. Presence of the schistosomecirculating anodic antigen (CAA) in urine of patientswith S. mansoni or S. haematobium infections. AmJ Trop Med Hyg, 1989, 41:563-569.

Schis

tosom

iasis

AbstractThe aim of this study was to examine the

efficacy and safety of praziquantel (PZQ) inthe treatment of the Schistosoma mansoniinfection, studying the predictors of treatmentfailure and the performance of the IgG avidityELISA tests in differentiating between acuteand chronic schistosomiasis. All villagers livingin Abis 4 village, rural Alexandria, Egypt, weresubjected to stool examination, and the eggcounts per gram stools (epg) were calculated.Subjects were then interviewed and examined.A single dose of PZQ was administered to allpositive stool patients. Twenty four hours aftertreatment, all treated subjects were visitedand interviewed concerning side effects. Stoolanalysis was performed 2 months aftertreatment and a second dose of PZQ wasgiven to individuals who remained positivefor S. mansoni infections. Cure rates andintensity reduction rates were calculated.Immunoglobulin levels (specific ELISA IgM,IgG antibodies and IgG avidity) inschistosomiasis patients were studied beforeand after PZQ administration.

Results PZQ is an efficacious anti-schistosomal drug as the current studyrevealed a cure rate of 91.1% afteradministration of the first dose and an overallcure rate of 98.7% after the second dose. Alonger duration of water canal exposure wasfound to be a significant risk factor for PZQincomplete cure (positive after first PZQ dose).PZQ was well tolerated with nausea, diarrhoeaand dizziness as the only significant side-effects. Stool examination proved to be morereliable to detect cases with incomplete curethan were the studied immunologicalparameters.

Conclusion PZQ is a safe and efficaciousdrug. The IgG avidity test could not distinguishbetween acute and chronic stages ofschistosomiasis. The sharp rise of IgM inchildren after PZQ treatment can be attributedto their higher rates of re-infection comparedto adults. Educating villagers regarding thedisease and the harmful hazards of watercanal exposure, as well as mass PZQadministration for children are recommended.

Publications1. Abou Basha LM et al. Performance of IgG

Avidity in an endemic area for schistosomiasisin Egypt. EMHJ 2002:8(1) In press.

2. Abou Basha LM et al. A field study onresistance of Schistosoma mansoni strain topraziquantel: an answer for a question. EMHJ2002:8(3) In press.

BackgroundPraziquantel (PZQ) is currently the drug

of choice for treatment of S. mansoniinfection as it is highly efficacious andwell tolerated in a single dose, it is alsowidely used for morbidity control throughpopulation-based mass treatment.Variations in the cure rates between areascan be explained by differences in pre-treatment egg counts, in rapidness andintensity of re-infection. Re-infection oftenoccurs rapidly, prepatent infections (2-5weeks) do not respond well to treatmentand may mature to productivity weeksafter treatment. The cured patients maycontinue to excrete dead eggs for up to

Resistance ofSchistosoma

mansoni Egyptianstrain to

praziquantel: a fieldstudy

EgyptAbis district, rural

Alexandria governorate

Study period:1998-1999

Small GrantsScheme

(SGS) 1998 No. 12

Principal InvestigatorDr Laila M Abou-BashaParasitology Department

Medical Research InstituteAlexandria Univerisity

Alexandria, Egypt

The prevalence of S. mansoni in rural Alexandria accounted for20.5%, with the highest frequency in the age group 15-30 years,but with a low grade of intensity of infection. Males recorded higherprevalence and intensity of infection compared to females.

PZQ is an efficacious anti-schistosomal drug with an overallcure rate of 98.7% after the second dose, while the first dose left7.6% of treated patients partially cured.

The only significant predictor of partial cure after the first dosewas the duration of water canal exposure.

PZQ was well tolerated with mild and self-limited side-effects.Nausea, diarrhoea and dizziness were the only significantcomplaints.

The IgG avidity ELISA test cannot be used to distinguish betweenacute and chronic stages of schistosomiasis in endemic areasbecause there is a continuous state of re-infection that hindersthe sharp distinction between acute and chronic infections.

The high rate of re-infection among children resulted in a sharprise in IgM levels after treatment. Mass semi-annual PZQadministration and the organization of a health educationprogramme to this vulnerable group are therefore recommended.



Drug Studies

92 93

2 months which cannot be distinguished from viableeggs by the widely applied Kato-Katz method,therefore re-infection cannot be determined exceptafter 6-12 weeks.

Recently, an assay measuring the antigen bindingavidity of the IgG antibody was developed to separatelow-avidity antibodies produced at an early stage ofinfection from those with higher avidity that reflectspast infection. This test may help in the assessmentof cure; low avidity was found after successfultreatment and high avidity in resistance to treatment.

The aim of this study was to examine the resistanceto PZQ and the risk factors for non-response totreatment. The performance of the IgG avidity ELISAtest in differentiating between acute and chronicinfections was also evaluated.

Materials and methodsA map of Abis villages in rural Alexandria was

prepared and Abis 4 village was randomly selectedas the study area. All households (n = 2577) wereasked to provide morning stool samples, and thenumber of eggs per gram faeces was determined byaveraging egg counts on 3 modified Kato-Katz thicksmears. All subjects were interviewed using a pre-tested questionnaire. The collected informationincluded: sociodemographic, lifestyle, history of anti-schistosomal treatment and medical past history.Clinical examination of a representative sample wascarried out in the field.

Blood samples ELISA IgM, IgG and IgG aviditywas performed on 111 consenting patients, and acomplete blood count was performed to detectoesinophilia.

PZQ treatment A single dose of PZQ (40mg/kgbody weight) was administered to all positive stoolpatients on December 1998 and January 1999 (non-transmission season). Pregnant and lactating womenwere excluded from the study. Twenty four hoursafter treatment, all treated subjects were visited andinterviewed concerning side effects. Patients whosuffered from vomiting were given a replacementtherapy. Two months after treatment (February-March1999) stools were collected and examined and epgstools were calculated. Blood samples were collectedfrom 28 consenting patient who remained positivefor S. mansoni to determine specific ELISA IgM, IgGantibodies and IgG avidity.

A second dose of PZQ was given to 40 individualswho remained positive for S. mansoni infections.

Cure rates and the intensity reduction rates werecalculated. A matched control group equal in numberto the cases was given a placebo to study thetolerance and safety of the drug.

Main study findingsS. mansoni is still endemic in this area with an

overall prevalence of 20.5%. Prevalence increasedwith age to reach a plateau at 15-30 years, then wasnearly stable thereafter. The intensity of infectionwas low according to the WHO staging system(geometric mean less than 100 epg). Males recordedhigher prevalence and intensity of infection comparedto females. PZQ is an efficacious anti-schistosomaldrug as the current study revealed a cure rate of91.1% after the first dose and an overall cure rate of98.7% after the second dose. Therefore, only 7.6%of treated patients were partially cured after the firstdose and the recorded response failure of PZQ was1.3%. However, the second dose yielded 100% curein children and an egg intensity reduction rate of69.9% in adults who remained positive after thesecond dose. A longer duration of water canalexposure was found to be a significant risk factor forPZQ partial cure. This is explained by the fact thatcontinuous exposure to water infested with cercariaeallows the presence of both mature and immatureforms. PZQ will kill only the mature worms, leavingthe immature forms to mature and excrete eggs instools weeks af ter PZQ administ rat ion.

PZQ was well tolerated with mild and self-limitedside-effects. Nausea, diarrhoea and dizziness werethe only significant complaints as compared to amatched control group of equal number that wasgiven a placebo.

This study also showed that stool examinationproved to be more reliable to detect cases with partialcure than immunological parameters that failed todistinguish untreated from partially cured individuals.

Therefore, the IgG avidity test cannot be used todistinguish between acute and chronic stages ofschistosomiasis in endemic areas because there isa continuous state of re-infection that hinders thesharp distinction between acute and chronic infections.

There was a sharp rise of IgM in children afterPZQ treatment which can be attributed to re-infection.The high rate of re-infection in children demonstratesthe importance of hygienic behaviors as well asensuring access to chemotherapy. Mass semi-annualPZQ administration to children together with a healtheducation programme are therefore recommended.

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AbstractEcological studies on Bulinus truncatus and

Planorbarius metidjensis were carried out inAgadir province, Morocco. B. truncatus wasfound in perennial water bodies, P. metidjensisin temporary water courses, and the speciesin rare habitats. Two annual generations wereobserved for B. truncatus, with the first fromJuly to November and the second fromNovember to June. P. metidjensis had 3 annualgenerations (May-November, October-February, and January-May). Newborn snailswere exposed individually or in pairs of mixedspecies to miracidia of Schistosomahaematobium before being killed and examinedhistologically 24 hours after exposure.

Results Miracidial penetration was greaterin P. metidjensis than in the other species.Most of these larvae penetrated through themantle in B. truncatus and P. metidjensis.Numerous live sprorocysts were found in P.metidjensis whereas a rather low number wasfound in B. truncatus. Sporocyst migration inthe snail body left sequellae consisting oftunnels. The maximum density of thesporocysts was in the foot and mantle.

Conclusion It was hypothesized that theplanorbid infection by S. haematobium occursduring the first days of snail life.

Publications1. Moukrim A, Zekhnini A, Rondelaud D.

Observations écologiques sur les hûtesintermédiaires de Schistosoma haematobiumBilhariz dans la province d'Agadir (Maroc). Bulletinde la Société Française de Parasitologie, 1993,11(2):223-230.

2. Zekhnini A, Mansir A, Moukrim A. Nouvellesdonnées sur les hôtes intermédiaires deSchistosoma haematobium dans la provinced'Agadir (Maroc). La pénétration du miracidumet son devenir chez trois pulmones. Bulletin dela Société Française de Parasitologie,1993,11(1):85-94.

3. Yacoubi B et al. Schistosoma haematobium:comparative studies on the characteristics ofinfection in three populations of Planorbariusmetidjensis from the Agadir province in southMorocco. Parasitol Res, 1999, 85(3):239-242.

4. Zekhnini A et al. Schistosoma haematobium:comparative studies on prevalence and cercarialshedding according to the shell diameter ofPlanorbarius metidjensis at miracidial exposure.Paras i to l Res , 1997, 83(3) :303-305.

5. Moukrim A, Zekhnini A, Rondelaud D.Schistosoma haematobium: influence of thenumber of miracidia on several characteristicsof infection in newborn Planorbarius metidjensis.Paras i to l Res , 1996, 82(3) :267-269.

BackgroundIn Moroccco, Agadir is the region mostly

affected by schistosomiasis due toSchistosoma haematobium. Two snailswere identified by the Public HealthServices as intermediate hosts for theparasite: Bulinus truncatus Audouin, aclassic intermediate host for S.haematobium , and Planorbar iusmetidjensis Forbes. This latter specieswas proved to be an intermediate host forthe parasite in southern Portugal, but itsrole has never been confirmed in northernAfrica and several contradicting resultshave been reported. A preliminary studywas undertaken by the same researchteam in 1987 and proved the presence ofthe 2 snails in the region. The presentstudy was therefore conducted in orderto study the ecology of the region withspecial reference to the distribution of

Contribution to thestudy of molluscschistosomiasis

hosts in the regionof Agadir

Moroccoregion of Agadir, southern

Morocco


Small GrantsScheme

(SGS) 1993 No. 43

Principal InvestigatorDr Abdullatif Moukrim

Biology DepartmentFaculty of Science

Ibnou Zohr UniversityAgadir, Morocco

This study provided information regarding the distribution of B.truncatus and P. metidjensis in Agadir region.

B. truncatus snails were detected in perennial water bodies, P.metidjensis in temporary water courses, and the 2 species in rarehabitats.

P. metidjensis has 3 annual generations. Miracidial penetrationis greater in P. metidjensis than in B. truncatus. Most of the larvaepenetrate through the mantle in both species. Numerous livesprorocysts were found in P. metidjensis whereas a rather lownumber was found in B. truncatus.

The planorbid infection by S. haematobium occurs during thefirst days of snail life.

The results of this study provided strong and definitive evidenceabout the role of P. metidjensis as an intermediate host for S.haematobium.

SchistosomiasisM o r o c c o


Ecology

94 95

Schistosoma intermediate hosts, to study the biologyof the snails, and to determine the role of each snailin disease transmission.

Materials and methodsStudied habitats The ecological studies wereperformed in the region of Agadir, whereschistosmiasis cases have been detected since 1980.The water collections and irrigation canals weresurveyed at regular intervals (1-3 months) to identifythe species of snails in this region.Laboratory investigations The physical andchemical characteristics of water samples obtainedfrom the stations were studied. Water analysis wasperformed once every 2 months. Every month, 200snails randomly selected from the 3 habitats weretransported to the laboratory, 100 snails weresubjected to a test of cercarial emission, while therest was fixed and crushed for detection of the parasitein its state of evolution. Furthermore, some newbornsnails were exposed to the miracidum of S.haematobium. A histological control was used tofollow the infestation and the development of thesnail in the host. The same experiment was repeatedunder different conditions (snail size, number ofmiracidia, temperature and the amount of lightavailable for the experiment).

Main study findingsEight sites of Schistosoma snails were detected in

Agadir region. The ecological study revealed thefollowing distribution of snail habitats:

1. B. truncatus, the classical host of S.haematobium, was present in the perennial waterbodies. It was detected in 5 habitats: irrigation zonesof Massa and Ouled Taima, rivers and the lake ofTanal, bridges of Youssef ben Tachfin and AbdelMoumen.

2. P. metidjensis was detected in sources andtemporary water courses. It was found in 4 habitatsin Assaka, Immozzer, Sidi Belkacem.

3. The 2 snails coexisted in 2 habitats: Ait Ouadrimand Ida Ougnidif.

The biology of the 2 snails was studied in Massa,Ouled Taima, Sidi Belkacem and Assaka sites Twoannual generations were observed for B. truncatus,with the first from July to November, and the secondfrom November to June. The life cycle of P.metidjensis presented 3 annual generations (May-November, October-February and January-May).These results indicate that the newborns of theschistosomiasis snail were present all year round.

It was noticed that newborn B. truncatus and P.metijensis, when exposed individually or in pairs ofmixed species to one miracidium of S. haematobium,miracidial penetration was greater in P. metidjensis.Most of these larvae penetrated through the mantle

in the 2 snail species. Numerous live sporocysts werefound in P. metidjensis whereas a rather low numberwas found in B. truncatus. The results of this studyindicate that the miracidia are capable of penetratingthe body of the snail and that no sporocysts can beformed during the first 24 hours. This is indisagreement with reports stating that the sporocystswere killed by the amoebicidal reaction of the snail.However, the penetration of miracidia detected inthis study could be explained by the fact that newbornshave immature immune systems allowing thedevelopment of sporocysts without being killed bythe amoebicidal reactions. According to thishypothesis, snail infection should take place withinthe first few days of the snails' lives in order to reachmaturation. At 24 hours, the sporocysts were detectedmainly in the foot, and in the kidney and to a lesserextent in other sites. The sporocysts are in a stateof continuous movement during the first days untilthey finally settle in the salivary glands of the snail.The other characteristics of the sporocysts(physiology, length, number of germinating cells)were less studied due to the small number ofsporocysts.

ConclusionsThis study provided information regarding the

distribution of B. truncatus and P. metidjensis in theAgadir region. The detection of the 3 generations ofthe P. metidjensis all year round in this region is afinding of public health importance as the presenceof newborns indicate the later development into larvalstage.

Furthermore, these results provided strong anddefinitive evidence on the role of P. metidjensis asan intermediate host for S. haematobium in Agadirregion.

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AbstractUrinary schistosomiasis transmission in Seil

Al-Hasa, Jordan, was reported due to thepresence of the intermediate host, Bulinustruncatus snails, favourable environmentalconditions and foreign cases passingschistosomes. This work was thus undertakento study the ecology of Seil Al-Hasa and itsrelation to the presence of B. truncatus alongthe Al-Hasa stream. Eight stations wereselected to monitor the presence of snailsalong the Al-Hasa stream. The physical andchemical parameters of the stream water wereanalysed. Snails were collected from all sites,transported to the laboratory for identificationand B. truncatus snails were exposed weeklyto light to stimulate the shedding of cercariaefor a period of 3 months.

Results Six species of snails were collectedfrom Seil Al-Hasa. B. truncatus snails wererecorded from 2 sites in different months.They were found attached to stones in thewater bed or to aquatic plants. None of the200 collected snails shed cercariae after 3months of laboratory observation. There wasno significant difference between the sitesregarding the physical and chemicalparameters of the water. However, infectedsites recorded higher water temperatures andlower velocities compared to the others,indicating that B. truncatus snails were ableto survive by attaching themselves to stones.

Conclusion This study emphasized the role

of water temperature and velocity inschistosmiasis transmission and the need toconduct more studies on the ecology of B.truncatus.

BackgroundB. truncatus snails were recorded in

Jordan during the period 1975-1985 fromdifferent sites: springs, irrigation poolsand dams [1]. The presence of foreignworkers, mainly Egyptians, infected withurinary schistosomiasis increased theprobability of local transmission of thedisease as the Jordanian B. truncatuswas proved to be an intermediate hostfor Schistosoma haematobium [2].Furthermore, autochthonous cases werealso reported from Seil Al-Hasa, southernJordan, where the Al-Hasa stream isused for land irrigation. The hightemperature of the springs' water thatfeeds this stream might explain the rapidproliferation of B. truncatus snails in thisarea in spite of Bayluscide use by theMinistry of Health. This research wasthus undertaken to study the ecology ofSeil Al-Hasa and its relation to thepresence of B. truncatus in the stream.

Materials and methodsEight stations were chosen as sampling

points to monitor the presence of snailsalong the stream. The stations werevisited biweekly, and snails were collectedand transported to the laboratory foridentification. At each visit, the followingparameters were determined in eachstation: water temperature, dissolvedoxygen, pH, electric conductivity, andwater speed. In addition, bimonthly watersamples were subjected to water analysisfor: calcium, magnesium, sodium,potassium, chlorides, carbonates,bicarbonates, sulfates, nitrates, iron,manganese and zinc. Water analysis wasperformed in the Water Research Centre,

Ecology of Bulinustruncatus in Seil Al-

Hasa, KarakGovernorate, Jordan

JordanKarak Governorate

Study period:August 1994–July 1995

Small GrantsScheme

(SGS) 1993 No. 47

Principal InvestigatorDr Elias K Saliba

Department of BiologicalSciences

Faculty of ScienceUniversity of Jordan

Amman, Jordan

Six species of snails were collected from Seil Al-Hasa: Theodoxusmacri, Melanopsis praemorsa, Melanoides tuberculata, Gyrauluspiscinarum, Lymnaea truncatula and Bulinus truncatus.

The sites infected with B. truncatus recorded higher watertemperature and lower velocity compared to other sites free fromthe snails, suggesting the role of these 2 factors in schistosomiasistransmission.

The results of this study indicate the need to conduct moreecological studies not only in endemic areas, but also in non-endemic areas to prevent the transmission of the disease in areaswith favourable environmental conditions

SchistosomiasisJ o r d a n


Ecology

96 97

University of Jordan, according to the standardmethods [3]. In addition, B. truncatus snails collectedfrom the sites were placed in the laboratory and wereexposed weekly to light to stimulate the shedding ofcercariae for a period of 3 months.

Main study findingsSix species of snails were collected from Seil Al-

Hasa. These were: Theodoxus macri, Melanopsispraemorsa, Melanoides tuberculata, Gyrauluspiscinarum, Lymnaea truncatula and Bulinustruncatus. The latter was recorded from 2 sites duringdifferent months: from the first site during April, May,August, and September, and from the second siteduring May, June, and October. Hundreds of snailsof this species were found attached to stones in thewater bed or to aquatic plants. None of the 200collected snails shed cercariae after 3 months oflaboratory observation. There was no significantdifference between the sites regarding the physicaland chemical parameters of the water. However,infected sites recorded higher water temperaturesand lower velocities compared to others. Watertemperatures at the infected sites were 6-10 °C higherthan at the other sites, and the water velocities rangedfrom 0.5 m/sec to 0.9 m/sec in the infected sitescompared to 0.65 m/sec to 3 m/sec in the other sites.

These findings suggest that B. truncatus snailswere able to survive in the 2 sites by attachingthemselves to stones, while the water velocity in theother sites was too strong such that the snails, ifpresent, would be washed away and would not survivethe water currents. These results are consistent withthose obtained from a similar study conducted inZarqa River, where B. truncatus snails were foundattached to stones in the River where a water velocitydid not exceed 0.78 m/sec [4].

The identification of snails at these sites wasfollowed by notification of the Ministry of Health,whereby snail control activities were initiated withmolluscicides.

RecommendationsThe results of this study indicate the need to conduct

more ecological studies, not only in endemic areas,but also in non-endemic areas, to prevent thetransmission of the disease in areas with favourableenvironmental conditions. The study emphasized therole of water temperature and velocity inschistosmiasis transmission and endemicity and thenecessity to perform in-depth analysis of these 2factors. The disappearance of snails from the studiedregion for several months after snail control activitieshighlighted the importance of continuous monitoringof the situation and the application of molluscicidesto limit the spread of the disease.

References[1] Saliba EK, Abdul-Hafaz S, Tawfik MR.

Schistosomiasis in Jordan: An unwelcomed guest.Parasitology Today, 1986, 2:91-93.

[2] Ayed RE, Saliba EK. Further studies on therelationship between Bulinus truncatus from Jordanand Schistosoma haematobium from Egypt. JapaneseJ Parasitol, 1985, 34:155-163.

[3] Greenberg AE, et al. Standard methods for theexamination of water and waste water, 16th ed.Washington, DC, APHA, AWWA and WPCFPublication offices, 1985.

[4] Burch J (ed). Schistosomiasis and other snailmediated diseases in Jordan. University of Michigan,1986:224.

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AbstractArtificial intelligence-based approaches has

been used in the fields of computer scienceand medical diagnosis, but relatively little inepidemiological studies. This study reportsthe results of the application of "state-of-the-art" neural networks methodology (NNs) (aspecial category of artificial intelligence (AI))to the study of schistosomiasis. A NNsstructure was designed that would processand fit epidemiological data collected from251 schoolchildren, aged 6-15 years, usingfirst-year data to predict second-year andthird-year infection rates. Data collected over3 years included: age, sex, water canal andfield exposure, medical history, and the resultsof stool and urine analyses. The Schistosomamansoni infection rate was 50% for thebaseline. Modelling was based on thestandard backpropagation algorithm in whicha suitable configuration of the model is builtusing the first year's data to optimize itsperformance.

Results The performance of the NNs modelin the first year compared favourably withlogist ic regression, but with bettergeneralization of predicting criteria forschistosoma infection over time. The NNsmodel was as sentitive and specific as logisticregression, and with lower false-positive andfalse-negative rates. Furthermore, the relativecontribution of clinical morbidity to theprediction of infection was more evident usingthe NNs model.

Conclusion NNs-based models could beused as a tool in widescale controlprogrammes, utilizing data based on unstableegg excretion and insensitive laboratorytechniques, and would solve the issue of theimpracticability of obtaining more than onestool or urine specimen.

PublicationsHammad TA, et al. Comparative evaluation

of the use of artificial neural networks formodelling the epidemiology of schistosomiasismansoni. Trans R Soc Trop Med Hyg, 1996,90(4):372-376.

BackgroundNeural networks (NNs) methodology is

an artificial intelligence-based approachand appears to provide a highly promisingcomputer-based methodology for patternrecognition and analysis of underlyingdisease dynamics. This methodology hasbeen recently accepted by the scientificcommunity to deal with diagnostic medicalproblems [1]. The objectives of this studywere to design and test the feasibility ofapplying NNs methodology in a real-timemanagement system of schistosomiasiscontrol as a long-term goal, and to testthe ability of this tool to predict infectionunder different conditions over time. Theperformance of such alternative non-statistical modelling was also comparedwith that of the conventional statisticalmodels.

Materials and methodsData were obtained from a previous

Feasibility of real-time assessment of

schistosomiasiscontrol programmes:

optimization viapattern recognition,computer simulation

and cost-effectiveness

EgyptMinia Governorate

Study period:August 1994–July 1995

Small GrantsScheme

(SGS) 1993 No. 7

Principal InvestigatorDr Tarek A Hammad

Department of ParasitologyFaculty of Medicine

Minia UniversityMinia, Egypt



Epidemiology

Both the statistical and the neural networks (NNs) models hadcomparable sensitivity and specificity, with higher sensitivity andlower specificity of the logistic regression model, while the NNsmodel maintained the same pattern over years, with fewer false-positive and false-negative rates. Furthermore, clinical morbidityhad more relative contribution in the prediction of infection withthe NNs model than with the logistic regression model.

Potential of neural networks methodology in schistosomiasiscontrol

1. Identification of risk factors of schistosoma infection by utilizing data from different areas under different circumstances, and could be also incorporated in the Geographic Information Systems (GIS).

2. This methodology could be a cost-effective tool for monitoring infected populations if used in a serial screeningmanner with laboratory techniques.

3. It can be used in management of control programmes byprocessing real-time field data about the disease assembled at different locations on a real-time schedule,and producing an output comparable to the outcome in the real population.

study conducted in Sobtas, an Egyptian village in theNile Delta during 1978-1980. The study populationconsisted of primary schoolchildren up to the sixthgrade (n = 251). The collected data included: age,sex, canal water exposure, field exposure, medicalhistory, and results of stool and urine analysis. Alogistic regression model was fitted using infectionas the dependent variable, with age, sex, swimmingin the water canal, field work, hepatomegaly, firmliver, splenomegaly and blood in stools as independentvariables. A NNs structure was designed to processand fit the collected epidemiological data using thefirst year's data to predict second and third yearinfection rates.

Steps of neural network analysis [2]1. The system (i.e. a community endemic for

Schistosoma mansoni) was observed and datacollected to make a training set consisting of inputvariables for each individual: age, sex, swimming,etc.

2. The network representing the connectionsbetween the input and output vectors was built usingthe NeuroWindows computer package.

3. The training was accomplished by applying aninput vector, computing the predicted output andcomparing it to the observed one, and adjusting theNNs weights to minimize the difference. Each inputvector is applied in turn and the network is partiallytrained. After a large number of applications of theinput vectors, the network "converges" to a solutionthat minimizes the difference between the desiredand measured system ouputs.

4. Cross-validation was finally performed byobserving how the network acted as a test data set(20% of the data).

Performance assessment Models were builtusing the first year's data. A training subset of data(80%) was used for modelling and a test subset(20%) was used for cross-validation. Both NNs andlogistic models were applied to the second and thirdyear's data of the same population to predict infection.Predictions of the 2 models were compared to theactual infection rates.

Main study findingsAccording to the statistical model, 1-year-old children

were found to be 1.2 (1.06-1.39) times more likely tobe infected than younger ones, and males 2.8 (1.6-4.96) times compared to females. Apart fromsplenomegaly, other independent variables werepositively associated with infection but not statisticallysignificant. The 2 models had comparable sensitivityand specificity, with higher sensitivity and lowerspecificity of the logistic regression in the last 2 years,while the NNs model had the same pattern over the3 years with lower false-positive and false-negative

rates Furthermore, clinical morbidity had more relativecontribution in the prediction of infection with the NNsmodel than did the logistic regression model.

This methodology is able to utilize data generatedfrom studies about snail infectivity in water canalsfrom different areas under different circumstances.This can be accomplished by training the NNs networkto predict highly infective foci. Examples of the typeof data that might be useful in building the trainingset for the model are: temperature, pH, depth ofwater, water current velocity, types and density ofvegetations, snails information (density, size, infectedproportion, etc.), estimated number of infectivecercariae at a given water site based on cercariometry,etc. Furthermore, this methodology could be usedwith GIS software to characterize the environmentalconditions that increase the risk of schistosomainfection, thus identifying high risk areas.

This methodology might also be applied to theepidemiological data to identify subtle populationpatterns associated with infection. This would increasethe effectiveness of the follow-up process in assessinginfection rates in the community if used in a serialscreening manner with laboratory techniques.Furthermore, it might be used to study the possibilityto reduce the training sample size with a reliablepattern of disease recognition, or to find the minimalsubset of variables allowing accurate prediction.These approaches would reduce resources for datamanagement and monitoring of exposed populations.

Real-time field data about the disease could alsobe provided based on data collected at differentlocations on a real-time schedule. Furthermore, theoutput is made to match the outcome of the realpopulation, therefore, it could be utilized in managingthe control strategies by providing a quick, accurateand low cost predictive tool.

ConclusionsNNs modelling showed reasonable performance

when applied on population data with changingpatterns over time. The potential of NNs-based modelsas an aid to widescale control programmes ispromising.

References[1] Baxt WG. Use of an artificial neural network for

the diagnosis of myocardial infarction. Ann InternMed, 1991, 115(11):843-848.

[2] Cheng B, Titterington DM. Neural networks: Areview from a statistical perspective. StatisticalScience, 1994, 9(1):2-54.

98 99

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AbstractDuring the period 1 April to 31 August 1994,

a case-control study was conducted in MaitamValley. The aim of this study was to investigatethe health impact resulting from thecontamination of the water course of the valleyby the products of a malfunctioning sewerageproject.

Results The schistosomiasis prevalencewas found to be 14.8% in the contaminatedreg ion compared to 4 .6% in thenoncontaminated upper valley. The odds ratiowas 3.6 for schistosomiasis, 2.1 for trichuriasis,1.9 for ascariasis, 1.0 for amoebiasis, 2.1 forgiardiasis and 0.6 for hymenolopiasis(Hymenolepis nana). These findings confirmthe adverse impact of uncompleted projectson human health.

Conclusion The results suggest an urgentneed for the chemical treatment of the waterfrom the Ibb sewerage project, by usingchlorine, insecticides and filtration to destroythe parasites, snails, various insects andactive bacteria, under the supervision of theGeneral Directorate of Public Health in IbbGovernorate.

Publications1- Al-Haddad AM, Assabri AM. Health impact

of uncompleted sewerage project in MaitamValley, IBB city-Republic of Yemen. YemenMedical Journal (YMJ), 1998, 2(2):68-76.

2- Al-Haddad AM. Research Abstract. Newfoci of schistosomiasis transmission in Yemen.Eastern Mediterranean Health Journal, 1995,1(2):294-295.

BackgroundThe water course in Maitam Valley runs

from the southern border of Ibb city upto 20 km. The valley is characterized bygood weather all year round and atemperature range of 10-30 °C. The watercourse flows all year and intensifies inrainy seasons (spring and summer). Thepopulation of the valley is approximately20 000 individuals, and farming is theirmain occupation. Their daily activity islinked to the water canal in washing,swimming, and irrigation. In 1990, a watersupply and sewerage project wasestablished for Ibb city. The treatmentplant, using the activated sludge process,was built at the head of the valley, about5 km south of the city. The projectcollected different wastes that weretreated mechanically, chemically andbiologically. A few years after itsimplementation, the chemical treatmentwas no longer applied. As a result,untreated water was used by the villagersfor domestic purposes. Furthermore, thesolid disposal remaining from waterseparation was collected by machinesand distributed to the farms to be usedas untreated manure thereby creating anadditional source of pollution, especiallywhen rain washes the parasites' eggsand bacteria into the water course.

Owing to the hazards that themalfunctioning of this project has broughtto the environment, the present studywas conducted aiming at evaluating thecont r ibut ion o f the pro jec t toschistosomiasis epidemiology in thevalley.

New foci ofschistosomiasistransmission in

Yemen

Republic of YemenMaitam Valley, Ibb

Governorate

Study period:April–September 1994

Small GrantsScheme

(SGS) 1993 No. 24

Principal InvestigatorDr Ahmed Al-Haddad

Department of CommunityMedicine

Faculty of MedicineSana'a University

Sana'a,Republic of Yemen

The prevalence of Schistosoma mansoni in the lower region ofthe Maitam Valley, where water was contaminated by the Ibbsewerage project, was significantly higher than that of a controlgroup living in the upper part of the valley.

The contaminated lower region of the valley recorded significantlyhigher prevalence of trichuriasis, ascariasis, giardiasis andhymenolopiasis, and 80% of villagers living in the contaminatedregion suffered from allergic dermatitis which was mainly attributedto the bacteria used in the biological processing of the manure.

The study recommendations were: the urgent application ofchemical treatment for the water of the Ibb sewerage project, toincrease the number of manure drying basins to allow for thecompletetion of the drying process thereby decreasingenvironmental hazards, to educate the public regarding the disease,and to establish a health centre for the delivery of diagnostic andcurative services.



Epidemiology

100 101

Materials and methodsA case-control study was conducted in Maitam

Valley, Ibb Governorate during the period 1 April to31 August 1994. The census of the valley wasconducted and the study population included villagersresiding in the valley's villages. A representativesample of villagers were subjected to medicalexamination, laboratory investigations (stool analysisusing the Kato-Katz technique, urine and bloodanalysis), free treatment of positive patients withpraziquantel (40 mg/kg body weight), as well as freetreatment of any incidental disease or emergency.Sociodemographic data were also collected duringthe survey.

Daily health education sessions regardingschistosomiasis and its snail intermediate host werealso conducted in the villages, including measuringsnail intensity in the water course. These sessionswere held in mosques, weekly market gathering andother venues. Health education aids were postersand visual aids.

Main study findingsThe census conducted in the present study revealed

a total population of 20 334, indicating an annualincrease of 3.3% in comparison with the previous1986 census. A total of 1994 villagers were surveyed,and 1290 of them were residing in the upper part ofthe valley that was not affected by the project'scontamination. These were taken as a control group.The identified snail intermediate host in the regionwas Biomphalaria pfeifferi, with an average densityof 16.6%. The prevalence of S. mansoni in thecontaminated region of the valley was 14.8%, with ahigher frequency among males, accounting for 17.7%compared to 11.9% in females. On the other hand,the prevalence of the disease in the control regionwas 4.6% (8% in males and 1.4% females).

Similarily, there was a significant increase in theprevalence of other intestinal parasites in thecontaminated region as compared to the controlregion: trichuriasis (29.2% compared to 16.2%),ascariasis (56.1% compared to 40.3%) and giardiasis(23.4% compared to 12.4%). However, there was nosignificant difference between the 2 regions regardingthe prevalence of ameobiasis (67% compared to66%). Taenia species also recorded a significantincrease among residents of the contaminated region(9.3% versus 6.3%). Interestingly, 80% of villagersliving in the contaminated region reported variablegrades of allergic dermatitis up to skin ulceration, acondition which was not seen in the control group.This dermatitis was mainly attributed to the bacteriaused in the biological processing of the manure.

Conclusions and recommendationsIt was concluded that urgent chemical treatment

for the water of the Ibb sewerage project wasmandatory in order to kill active bacteria, helminthsand parasite eggs. It was recommended to increasethe number of manure drying basins to at least 6,because the 2 available basins did not allow for thecompletion of the drying process, thereby increasingenvironmental hazards. It was also recommended tocommunicate the hazards of water canal exposureto the villagers through continuous health educationsessions. Finally, this study emphasized the need toestablish a health centre for the delivery of diagnosticand curative services.

Schis

tosom

iasis

AbstractThe prevalence, intensity and incidence of

schistosomiasis and soil-transmittedhelminthiases among schoolchildren in anignored area in Yemen were determined. Thestudy aimed to investigate the impact of singledoses of praziquantel or albendazole or both,and how they relate to sanitary, socioeconomicand behavioural practices on the prevalenceand intensity of infections. Out of a totalnumber of 897 pupils, 453 were randomlyselected from Al-Mahweet town and 444 fromrural surrounding areas. Millipore filtration,modified Kato-Katz and precipitationtechniques were applied for urine and stoolanalysis.

Results Prevalence rates were 28% forschistosomiasis, 61% for ascariasis, 21% fortrichuriasis, 2% for fascioliasis, 0.3% forenterobiasis, 0.7% for hook worm infectionand 0.2% for strongyloidiasis. Factors foundconfounding the relationship betweenschistosomiasis and residence, under logisticregression analysis, were sex and frequencyof water contact. The probability of infectionwith schistosomiasis for boys who reside inrural Al-Mahweet and visit the water sourceis 0.52, compared to 0.30 for those who residein Al-Mahweet town. Odds ratio estimatesaccounted for were 2.5 for residence, 1.7 for

water contact and 3.2 for boys. With regardsto other helminthic infections, availability oflatrines remained the only significantdeterminant of infection.Treatment reducedthe infection rate of Schistosoma mansoni by62.5%, Trichuris trichiura by 48% and Ascarislumbricoides by 24%.

Conclusion Annual campaigns of treatmentcan reduce the burden of Schistosomiasismansoni, trichuriasis and ascariasis. On theother hand, for Schistosoma haematobium,the appropriate time interval for interventionshould be shortened according to the findingsof a properly designed intervention studybefore being used as a single control measure.Since 77% of the children were infected withother helminths, mass treatment should beextended to cover all children. For those boysin rural Al-Mahweet who are continouslyexposed to the water source, mass treatmentfor schistosomiasis was recommended sincethe prediction of infection rate reached 52%.

PublicationsRaja'a YA et al. Some aspects in the control

of schistosomosis and soil-transmittedhelminthosis in Yemeni children. Saudi Med J,2001, 22(5):428-432

TrainingA mobile team of researchers and

technicians was trained on the methods ofconducting epidemiological and field surveys.

BackgroundBoth Schistosoma haematobium and

S. mansoni are endemic in Yemen, witha prevalence ranging from 20%-40%.However, there is a lack of informationregarding the disease in some regionsof the country such as Al-MahweetGovernorate. This study was conductedin order to estimate the prevalence ofschistosomiasis and other soil-transmittedhelminthiases in schoolchildren in Al-Mahweet Governorate.

Materials and methodsAl-Mahweet city, 113 km from Sana'a,

Schistosomiasisamong

schoolchildren in Al-Mahweet

Governorate, north-west Yemen

Republic of YemenAl-Mahweet Governorate,

North-west Yemen


Small GrantsScheme

(SGS) 1996 No. 6

Principal InvestigatorDr Yahia A Raja'a

Faculty of Medicine and HealthSciences

Sana'a UniversitySana'a, Republic of Yemen

email: [email protected]

Prevalence rates of helminthic infections among schoolchildrenwere: 22% for Schistosoma mansoni infection, 7% for Schistosomahaematobium infection, 61% for ascariasis, 21% for trichuriasis,2% for fascioliasis, 0.3% for enterobiasis, 0.7% for hook worminfection and 0.2% for strongyloidiasis.

Availability and use of latrines were two factors implicated in thetransmission of soil-transmitted helminths. Use of latrines, sex,water contact and age were the determinants of Schistosomainfection.

There was significant lowering in the prevalence and intensityof infection after treatment in S. mansoni, A. lumbricoides, and T.trichiura, but not in S. haematobium or other soil-transmittedhelminths.

Mass chemotherapy, health education and snail control measureswere the main treatment recommendations.



Epidemiology

102 103

lies about 2000 m above sea level, with extensiveagriculture that depends on rains and groundwater.There are about 430 schools in the entire governoratewith a total of 57 000 students enrolled. A total of498 students (186 girls and 312 boys) were randomlyselected from the mixed primary schools in Al-Mahweet town. Four hundred and fifty three additionalstudents were randomly selected from the rural areasof Al-Mahweet Governorate. Therefore, a total of 951children were enrolled in the study.

Detailed personal and behavioural information werecollected. Midday urine and stool samples wereobtained in containers from each individual andexamined using the Millipore filtration technique andthe modified Kato-Katz technique for urine and stoolanalysis, respectively. Other ova, cysts or larvaewere also recorded. Single doses of praziquanteland/or albendazole were administered whenever aninfection was confirmed.

Follow-up was conducted one year later by urineand stool collection. Children were re-examined andthose with a positive infection were treated again,while other children with soil-transmitted helminthiasiswere referred to the hospital.

Main study findingsA total of 948 students participated in the study

and fulfilled a complete set of investigations. Morethan half of these students (51%) were living in urbanareas and the remaining children were from thesurrounding villages. Almost half of the children hadaccess to latrines, but there was a significantdifference between those living in urban and ruralareas regarding the use of latrines, with 78.3% usinglatrines in urban compared to 17.4% for those livingin rural areas.

Prevalence rates were 22% for Schistosomamansoni infection, 7% for S. haematobium infection,61% for ascariasis, 21% for trichuriasis, 2% forfascioliasis, 0.3% for enterobiasis, 0.7% for hookworm infection and 0.2% for strongyloidiasis. Factorsfound confounding the relationship betweenschistosomiasis and residence, under logisticregression analysis, were sex and frequency of watercontact. The probabil i ty of infection withschistosomiasis for boys who reside in rural Al-Mahweet and visit the water source is 0.52, comparedto 0.30 for those who reside in Al-Mahweet town.Odds ratio estimates accounted for via residencewas 2.5, via water contact 1.7 and via boys 3.2. Withregard to other helminthic infections, the availabilityof latrines remained the only significant determinantof infection.

There was significant lowering in the intensity ofinfection (geometric mean) after treatment of casesof S. mansoni infection but no difference in cases ofS. haematobium infection. Similarily, the prevalence

of infection was significantly reduced in cases of S.mansoni, A. lumbricoides, and T. trichiura. On theother hand, there was no significant lowering in theprevalence of S. haematobium infections or othersoil-transmitted helminthiases after treatment.

Conclusions and recommendationsThis study revealed that helminthic infections were

affecting a considerable proportion of schoolchildrenin this area. Annual campaigns for examination andtreatment were recommended in order to reduce theprevalence and morbidity of schistosomiasis in thispopulation. A mass chemotherapy campaign for theother helminths could be beneficial in the control ofthese infections.

The availability and use of latrines were two factorsimplicated in the transmission of soil-transmittedhelminthiases. Use of latrines, sex, water contactand age groups were the determinants of Schistosomainfection.

Health education of the community to encouragethe construction and use of latrines would producea significant impact on the control of helminthicinfections. It was finally recommended to conductsurveys on the snail population in the different waterbodies and to plan a snail control strategy for thearea.

Schis

tosom

iasis

AbstractThe introduction of irrigation schemes to the

Sinai through the El-Salaam canal to reclaimnew areas is an ongoing project. Althoughthese irrigation channels provide substantialbenefits in the form of employmentopportunities and decreased dependence onfood imports, it may create a new environmentallowing for the transmission of schistosomiasisto the local population. The current study wastherefore conducted to determine theprevalence and risk factors of schistosomiasisin both bedouin and immigrant populationsand to evaluate their knowledge, beliefs andbehaviour regarding the disease. Moreover,a follow-up study was conducted to evaluatethe response 6 months after praziquanteltreatment of infected persons.

A cross-sectional study was conductedwhereby individuals were interviewed andsubjected to clinical examination and laboratoryinvestigations. Risk factors for schistosomiasisin cases of infection were studied bycomparison with non-infected controls. At thebeginning of the second phase, all positivecases were treated with praziquantel andevaluated 6 months later.

Results The overall prevalence ofschistosomiasis was 6.2%. It varied amongdifferent groups and areas: it was 5% inbedouins, 7.6% in immigrants, 10% in relativelyold reclaimed areas, and 5.1% in recentlyreclaimed areas. Immigration from the NileValley, residency in relatively old reclaimedareas, male gender and illiteracy were themain risk factors for schistosomiasis in theSinai. There were significant differences inknowledge and beliefs between bedouins andimmigrants. The behaviour in both groupstowards the disease was nearly similar.Furthermore, there was no significant differencebetween both groups regarding the 6-monthresponse to single praziquantel administrationas 80% of cases remained negative, indicatinga slow rate of reinfection in the area.

Conclusion The present study emphasizedthe need to optimize future strategies in orderto prevent the establishment of schistosomiasisin the newly reclaimed areas.

BackgroundEmerging infectious diseases can be

defined as infections that have newlyappeared in a population or arethreatening to increase in the near future.Specific factors precipitating diseaseemergence can be identified includingecological, technological, biological ordemographic factors that place people atincreased risk of contracting infection.Therefore, in the case of a dramaticchange in the environment, such as thatwhich accompanies new irrigation projects,there is a need for effective surveillanceand control of emerging diseases. InEgypt, 2 major irrigation schemes for landreclamation have been recent lyimplemented: the El-Salaam irrigationproject in the northern Sinai peninsula,and the Toshka project in southern Egypt.This study was conducted to determinethe prevalence of schistosomiasis in thenewly reclaimed areas of the northernSinai peninsula, to identify the risk factorsof schistosomiasis infection, and toevaluate the knowledge, beliefs and

Epidemiology ofschistosomiasis inbedouins of Sinai,Egypt, after the

introduction of Nilewater

Egyptnewly reclaimed areas in

the Sinai

Study period:January 2000–April 2001

Small GrantsScheme

(SGS) 1999 No. 24

Principal InvestigatorDr Mohamed Salah KhedrInternal Medicine Department,

Clinical Epidemiology UnitFaculty of Medicine, Suez Canal

UniversityIsmailia, Egypt

The only type of schistosomiasis present in the newly reclaimedareas in northern Sinai was Schistosoma mansoni at a prevalenceof 6.2%. These results highlighted the need for active surveillancealong the El-Salaam canal in order to prevent the establishmentof the disease in this area.

Male sex, farming occupation, living in established rather thannew agricultural areas, immigration from the Nile Valley, and lowersocioeconomic status were the significant risk factors for thedisease.

There was a significant difference between migrants and bedouinsregarding their knowledge and beliefs of schistosomiasis. Thereaction to health education and its interpretation, as well as thebehaviour towards the disease, were comparable in the 2 groups.

There is a low rate of reinfection in this area with 80% of casesremaining negative after 6 months of treatment.

An integrated approach consisting of chemotherapy and targetedhealth education was recommended in order to limit reinfectionand to control the disease.



Epidemiology

104 105

behaviour of the studied communities and theirresponse to a single dose of praziquantel, 6 monthsafter treatment.

Materials and methodsThe study population included all individuals aged

6-60 years. Data on knowledge and beliefs relatedto water contact and schistosomiasis were collectedusing a semi-structured questionnaire from the familyheads and health workers, while the behaviour andpractice of the inhabitants were observed. A cross-sectional survey of the study area was undertakenaccording to a list of a representative sample ofhouseholds. The collected information includeddemographic data and place of origin. All inhabitantsincluded in the survey were subjected to: clinicalexamination for schistosomiasis or its complications,stool examination, urine analysis, abdominalultrasonography, and serological investigation byindirect haemagglutination test.

The evaluation of the knowledge, beliefs andbehaviour was performed by interviewing arepresentative sample of bedouins (530 individuals)and immigrants (450 individuals).

A nested case-control study design was used tostudy the risk factors of schistosomiasis amonginfected cases as compared to non-infected controls.

All positively diagnosed individuals treated withpraziquantel (40 mg/kg body weight) during the initialcross-sectional survey were evaluated 6 months laterin a follow-up study.

Main study findingsThe prevalence of Schistosoma mansoni in the

newly reclaimed areas in northern Sinai was 6.2%,and no cases of S. haematobium were diagnosed.There was a significantly higher prevalence ofschistosomiasis in areas with established agriculturecompared to areas with recently implementedagricultural activities related to water contact. Bycontrast to infection in the Nile Valley, the prevalenceof infection was higher in older individuals; this couldbe explained by the fact that most infections wereacquired during visits to the Nile Valley, which ismostly an adult activity. Male sex and the farmingoccupation were the significant risk factors for thedisease mainly due to the greater liability of thesegroups to exposure to water canal. Measures ofsocioeconomic status such as educational level werealso implicated with contracting infection. Furthermore,immigrants were at a significantly higher risk forschistosomiasis compared to bedouins.

There was a significant difference betweenimmigrants and bedouins regarding the knowledgeand beliefs of schistosomiasis. The reaction to healtheducation and its interpretation, as well as the

behaviour towards the disease were comparable inthe 2 groups.

After 6 months of praziquantel administration, 80%of infected bedouins and immigrants remainednegative, indicating that the reinfection rate wasrelatively limited during a 6-month period.

Conclusions and recommendationsThe emergence of schistosomiasis in the newly

reclaimed land in North Sinai highlighted the needfor active surveillance along the El-Salaam canal inorder to prevent the establishment of the disease inthis area. An integrated approach consisting ofchemotherapy and targeted health education wasrecommended in order to limit reinfection and tocontrol the disease.

Schis

tosom

iasis

AbstractThis study aimed at determining the current

status of schistosomiasis infections in theGezira-Managil scheme and the impact of anew irrigation system on transmission.Schoolchildren from 15 randomly selectedschools were subjected to parasitologicalexamination of stool and urine. All children ³10years old were interviewed regarding risk factorsof disease transmission, and a sample ofchildren was clinically examined. Thetransmission of schistosomiasis was studiedin Agando village, Gezira area and in 2 camps,by parasitological survey of the residents aswell as by studying the effect of drying andcleaning canals on snail density and infectionwith schistosomes.

Results The prevalence of infection among3170 schoolchildren was 15.8% and 1.8% forSchistosoma mansoni and S. haematobium,respectively, being significantly higher in theManagil than in the Gezira area, with nosignificant difference between the 2 areasregarding the intensity of infection. Thesignificant risk factors in the 1648 interviewedchildren were: farming as the paternaloccupation, living in houses built with material

other than red bricks, proximity to a water canal,unavailability of water supply and latrines, andpast history of infection and treatment. In thecamps, the prevalence of S. mansoni infectionwas significantly higher than that in the villages.The drying and cleaning of canals reduced thesnail density and the snail intermediate hosts.

Conclusion S. mansoni infection is thepredominant type of schistosomisis in the area.An integrated control programme particularllytargeting the camps in the neighbourhood ofthe villages was highly recommended.

PublicationsHilali AH et al. Infection and transmission pattern

of Schistosoma mansoni in the Managil irrigationscheme, Sudan. Ann Trop Med Parasitol, 1995,89(3):279-286.

BackgroundThe prevalence of S. mansoni in the

Gezira-Managil scheme is high while thatof S. haematobium is generally low andvariable from one village to another. Acomprehensive control strategy consistingof chemotherapy, focused mollusciciding,health education and improvement of watersupply and sanitation was adopted andimplemented by the Blue Nile HealthProject (BNHP) during the period 1980-1990. In April 1999, the GeziraAdministration Board adopted a newsystem for improving irrigation, consistingof drying and cleaning canals before theirrigation season. This was expected tolower the snail density in the scheme. Thisstudy was conducted to determine thecurrent status of schistosomiasis infectionsand the impact of the adopted canalmaintenance programme on i tstransmission. The study also aimed atproviding information related to the patternof water supply and sanitation in the areaas well as assessing the knowledge,attitudes and practices of schoolchildrentowards the disease.

The presentsituation of humanschistosomiasis inthe Gezira-Managil

scheme, Sudan:prevalence and

transmission pattern

Sudan Gezira-Managil scheme,

Sudan

Study period:August 1999-2000

Small GrantsScheme

(SGS) 1999 No. 51

Principal InvestigatorDr Abdelmoneim HilaliParasitology Department

National Health LaboratoryKhartoum, Sudan

S. mansoni infection is the predominant type of schistosomiasisin the Gezira-Managil scheme. Both types of schistosomiasis weremore prevalent in the Managil compared to the Gezira area, wherethe Blue Nile Health Project had been previously implemented.This highlights the effectiveness and the long-lasting impacts ofintegrated control programmes in highly endemic areas.

The main determinants of schistosomal infection are: farming,water supply, sanitation, housing conditions, and past history ofinfection and treatment.

Camps in the neighbourhood of villages significantly contributeto the transmission of the disease in the Gezira-Managil schemeand should be targeted for intervention in future control programmesin the area.

Cleaning and drying of canals conducted by the new irrigationsystem had a significant impact on lowering the snail density inthe area. The tendency of snails to colonize the tails of canals,where stagnant water enhances the growth of vegetation, indicatesthe importance of proper maintenance of new irrigation systems.

SchistosomiasisS u d a n


Epidemiology

106 107

Materials and methodsThe prevalence of schistosomiasis was studied in

15 randomly selected schools from the 45 schools inthe area where the Blue Nile Health Project wasimplemented. Parasitological examination of stool andurine samples was performed. Stool specimens wereexamined using the locally modified Kato-Katz methodand urine specimens were examined using the simplesedimentation technique.

All children ³10 years old were interviewed using aquestionnaire. The information collected wassociodemographic data, housing conditions such aswater supply and sanitation, water contact activities,knowledge about schistosomiasis, and history ofschistosomiasis infection and treatment. Children from4 schools (2 from each area) were subjected to clinicalexamination.

The transmission of schistosomiasis was studied inAgando village, Gezira area, which includes the villageand 2 labour camps, as well as 2 neighbouring labourcamps with residents coming from western Sudan.These camps are highly dependent on the water canaland lack services such as water supply, health services,and schools.

A census of the 2 camps was performed and stooland urine samples were collected and examined fromall the residents.

The effect of drying and cleaning the canals fromvegetation and silt accumulation on the snail populationand transmission of schistosomiasis was studied inthe canals around Angado village in the Gezira area.This activity was performed before the irrigation season,and snails were collected monthly from different sitesat the head, middle, and tails of minor canals. Snailswere also collected from human water contact sitesin the village area and from the neighbouring campsin order to determine the seasonal changes in snaildensities and infection with schistosomes. Snails werecollected using scoops (10 scoops from each site),and subsequently counted and sorted by species ina field laboratory. Biomphalaria pfiefferi and Bulinustruncatus were examined for schistosome infection.

Infected schoolchildren and camp residents weretreated with praziquantel (40mg/kg body weight).

Main study findingsA total of 3170 schoolchildren were subjected to

stool and urine examination. The prevalence andintensity of infection were 15.8% and 112.7 eggs/g forS. mansoni, compared to a prevalence and intensityof 1.8% and 7.3 eggs/10 ml, respectively, for S.haematobium. The prevalence of both species wassignificantly higher in the Managil area than in Gezira(24.2% and 9.5%, respectively) for S. mansoni, and3.8% and 0.3%, respectively, for S. haematobium. Onthe other hand, there was no significant difference inthe 2 areas regarding the intensity of infection.

The prevalence was significantly higher in males

than in females and increased with age, being higheramong older children. For S. mansoni, the prevalencewas 20% among males and 6.2% in females, comparedto 2.6% and 0%, respectively, for S. haematobium.

Regarding the clinical examination, there was nosignificant association between the results ofparasitological examination and certain signs andsymptoms such as abdominal pain, blood in the stool,diarrhoea and fever. On the other hand, there was asignificant association with visible haematuria andsplenomegaly. No hepatomegaly was reported amongthe schoolchildren examined.

A total of 1648 children were interviewed regardingthe risk factors for schistosomal infection. The mostsignificant identified risk factors were: farming as apaternal occupation, poor housing conditions such asliving in houses built with material other than red brickssuch as mud or straw, and proximity to the water canal.The risk of infection was significantly associated withthe unavailability of water supply and latrines. Housetap water was reported to exist for 97% and 56% ofschoolchildren interviewed in Gezira and Managil,respectively. Furthermore, past history of infection andtreatment was a significant risk factor for infection.Half the interviewed children did not know about thehazards of water canal contact, and 27.2% did nothave any information about the mode of transmissionof the disease, however, knowledge and awarenessabout the disease did not have a positive impact ondisease transmission.

The prevalence of infection in the labour camps wassignificantly higher than that in the villages, amountingto 39.9% and 0.8% for intestinal and urinaryschistosomiasis, respectively. The prevalence ofinfection was higher in males than in females, but thiswas not statistically significant. The age-specificprevalence was 45.4% among children younger than10-years old, reached a peak in the age group 10-19years (67.3%), then decl ined thereafter.

The effect of drying and cleaning canals was reflectedin the snail density, which decreased, particularly inthe Gad Elain minor canal. The snail intermediatehosts were absent around Angado village, but werefound in low densities in the camps' (Gad Elain, Tobaand Elku) minor canals, mainly in the tail parts.

Conclusions and recommendations S. mansoni infection is the predominant type of

schistosomiasis in the area. The lower prevalence ofinfection in the Gezira compared to the Managil areahighlights the long-lasting impact of the Blue NileHealth Project for schistosomiasis control. Provisionof a safe water supply, better housing conditions, andmaintenance of the new irrigation system in the schemewere the main study recommendations. There wasalso a major emphasis on the need to implement acontrol programme targeting the camps in theneighbourhood of the villages.

Schis

tosom

iasis

AbstractThe long-term effect of single-dose

praziquantel on morbidity and mortality fromSchistosoma mansoni was investigated insurveys in 1987 and 1994 in central Sudan.Prevalence of infection dropped from 53% to34%, and intensity of infection (> or = 400eggs/g of faeces) from 31% to 18%. Therewas a reduction in hepatomegaly andhepatosplenomegaly, although splenomegalyalone was unchanged. Prevalence ofperiportal fibrosis decreased from 14% to10%. Endoscopic investigation of patientswith fibrosis showed a reduction inoesophageal varices from 47% to 30%.Mortality due to bleeding varices was high(community-wide, up to 11/100 infectedpatients with bleeding). Thus praziquantelmass treatment can be spaced to a muchlonger period, reducing the expense oftreatment, delivery and distribution.

PublicationsKheir MM et al. Effect of single-dose

praziquantel on morbidity and mortality resultingfrom intestinal schistosomiasis. EMHJ, 2000,6:926-931.

BackgroundAlthough much research has been

performed in the epidemiology, treatmentand immunology of schistosomiasis, thereis a paucity of information regarding themorbidity and mortality of this disease.Many of the studies were hospital basedand limited in the selection of patients.In a survey conducted during the period1985-1987, the prevalence of Symmers'fibrosis in the population was 13.6%, of

whom 54% reported oesophageal varices[1]. In another retrospective study, annualtreatment of villagers produced regressionin the signs of mild fibrosis and protectedagainst the occurrence of periportalfibrosis [2]. However, the protective effectof a single dose of praziquantel (PZQ)was not studied, nor the frequency of itsadministration required to produce sucha protective effect. The objectives of thisstudy were: to examine the effect of singlepraziquantel therapy in the long-termreduction of hepatosplenomegaly andSymmer's periportal fibrosis by comparingthe results of the current survey,conducted in 1994, with those of an initialsurvey conducted in 1987 in the samesetting and using the same methodology;to evaluate the natural history of fibrosisand/or oesophageal varices in patientstreated with praziquantel, 7 years earlier,during the initial 1987 survey.

Materials and methodsThe records of the earlier studies

conducted in the eighties were reviewedin order to identify the villages previouslysurveyed and Abu Jin village, Gezirastate, was selected as the study area.The village is located between the 2 Nilerivers in Sudan, 150 km south-west ofKhartoum. A complete census of thevillage was performed followed by tracingthe causes of deaths through the localdispensaries and El-Ribie Hospital (localdistrict hospital). The prevalence ofinfection and its intensity were determinedby stool examination using the Kato-Katzmethod. A random sample of 240 villagerswas interviewed for the following: historyof intestinal and urinary schistosmiasis,jaundice, haematemesis, past history ofhaematemesis , and h is tory o fantischistosomal treatment. The villagerswere also subjected to cl inicalexamination of the liver, spleen, ascites,anaemia, and s igns o f por ta lhypertension.

Long-term effect ofsingle dose mass

praziquantelchemotherapy on

morbidity andmortality of S.

mansoni in Gezira,Sudan

SudanGezira district


Small GrantsScheme

(SGS) 1993 No. 23

Principal InvestigatorDr Musa M Kheir

Department of MedicineFaculty of Medicine

Khartoum UniversityKhartoum, Sudan

SchistosomiasisS u d a n


Morbidity

A single-dose of praziquantel had a significant impact on thedisease prevalence, intensity of infection, burden of chronic liverdisease and mortality due to schistosomiasis mansoni.

Mass treatment can be spaced to a much longer period, reducingthe expenses of treatment, delivery and distribution.

The villagers were finally referred for ultrasoundexamination, and those with Symmer's fibrosis werere-assessed and carefully questioned about previousattacks of haematemesis and loss of blood.

In 1987, 32 patients were randomly selected amongthose with Symmer's fibrosis, and their degree ofperiportal fibrosis (PPF) was recorded. They weresubjected to endoscopic examination for evidenceof oesophageal varices, and the degree of variceswas graded. The follow-up of this cohort of patientsin 1994 revealed that 2 patients had died fromhaematemesis, and the remaining 30 patientsaccepted participation in the study. None of themhad a history of haematemesis, jaundice or abdominaldistension. They were subjected to: stool analysis,clinical examination, ultrasonography and endoscopy.

Main study findingsDuring the period 1987-1994, the village population

was rather stable with only few immigrants or newsettlers (1190 compared to 1080 villagers in 1994and 1987 censuses, respectively). There was nochange in the social habits or farming-affecting watercontact, nor in the sanitary system; no additional pit-latrines or tap water supplies were introduced apartfrom the old common tap. The prevalence andintensity of infection were significantly reduced in1994 compared to the initial study in 1987 by thesingle dose of praziquantel administered 7 yearsearlier; the prevalence of schistosomiasis droppedfrom 52.5% to 33.9%, the frequency of the peakintensity in 10-19 year-olds was also reduced from73.3% to 51%, and the frequency of egg counts>400/g was reduced from 31% to 17.7%. On theother hand, there was no significant differencebetween the 2 surveys regarding the frequency oforganomegaly (hepatomegaly and/or splenomegaly).

Considering the cohort of 32 patients previouslydiagnosed and treated in 1987, the single dose ofpraziquantel administered in 1987 resulted in asignificant drop in the frequency of infection, egg loadand intensity of infection, and a significant reductionin the frequency of organomegaly, exceptsplenomegaly, which could be attributed to concurrentinfections, e.g. malaria. Interestingly, a single doseof praziquantel resulted in the complete regressionof fibrosis in 3 patients with grade 1 PPF, and 11patients remained grade 1 PPF over this 7-yearperiod without developing haematemesis or melaena.Similarly, regarding the endoscopy results, patientsregressed to a less severe grade of oesophagealvarices during that period. The overall frequency ofoesophageal varices in patients previously diagnosedas having Symmer's fibrosis in 1987 was significantlyreduced from 47% to 30% over these 7 years.

Conclusions and recommendationsIn spite of frequent schistosomal reinfection, a

single dose of praziquantel administration resultedin a significant decrease in the prevalence of infectionand the regression of early signs of schistosomalmorbidity. The results of this study also proved theeffectiveness of administering the drug at widerintervals (up to 7 years), instead of the usual annualor biannual praziquantel administration, which couldbe a cost-effective approach for schistosomal controlin communities with limited resources.

References[1] Homeida Ma, Ahmed S, Dafalla AA, et al.

Morbidity associated with Schistosoma mansoniinfection as determined by ultrasound: a study inGezira, Sudan. Am J Trop Med Hyg, 1988, 39: 196-201.

[2] Homeida Ma, Dafalla AA, Nash TE. Effect ofantischistosomal chemotherapy on prevalence ofSymmer's periportal fibrosis in Sudanese villages.Lancet, 1988, ii, 437-440.

108 109

Schis

tosom

iasis

AbstractHepatosplenic schistosomiasis and human

fascioliasis coexist in the Abis area of ruralAlexandria. This study was conducted in orderto assess morbidity in patients with single orcombined infections. Stool samples werecollected twice from 3658 villagers in Abis Ivillage and examined by the Kato-Katztechnique. A random sample of positive cases(n = 281) was subjected to a detailed morbiditystudy. All cases were hospitalized andsubjected to c l in ical examinat ion,ultrasonography, liver function tests and testsfor connective tissue markers. A liver biopsywas performed in 37 patients. Results werecompared with healthy controls matched forsex and socioeconomic status.

Results Single and combined infections withSchistosoma mansoni affected 29.5% of therural population (21.5% schistosomiasis and8% fascioliasis). S. mansoni infection wasmore prevalent among young adults (15-35years of age), while fascioliasis and mixedinfections were more prevalent among

children. Males were more infected withschistosomasis and mixed infectionscompared to females, while females weremore infected with fascioliasis. The majorityof affected cases, whether in schistosomiasisor fascioliasis, recorded low intensity ofinfection. The highest procollagen III peptidelevels were observed in cases with mixedinfections. The serum fibronectin level waslower in cases compared to controls, and inschistosomiasis cases compared to Fasciolacases. Younger age was a significant predictorfor elevated procollagen level. In cases withschistosomiasis, serum fibronectin waspositively correlated with serum albumin, andprocollagen III peptide was positivelycorrelated with serum alkaline phosphatase(SAP). Egg count was directly correlated withSAP in Fasciola cases. In mixed infections,the Schistosoma egg count was positivelycorrelated with SGOT and SGPT, serumfibronectin was directly correlated withprothrombin activity, and procollagen IIIpeptide with SAP.

Conclusion Single and combined infectionswith S. mansoni and Fasciola proved to bea relevant public health problem in ruralAlexandria, Egypt. Procollagen III peptidecould be used as a marker for early diagnosisand management of liver affection, therebycausing regression of the signs of fibrosis.

PublicationsAbu Basha et al. Hepatic fibrosis due to

fascioliasis and/or schistosomiasis in Abis 1village, Egypt. EMHJ, 2000, 6:870-878.

BackgroundHepatosplenic schistosomiasis is the

most important form of morbidity causedby S. mansoni. Human fascioliasis isanother additional hazard to the liver.These 2 conditions coexist in the Abisarea, rural Alexandria, Egypt. Thepurpose of this study was to assessmorbidity in patients with schistosomiasis,fascioliasis and combined infections.

Materials and methodsThe selection of Abis I village was

Morbidity due tosingle and combined

hepatosplenicschistosomiasis andfascioliasis in the

vicinity ofAlexandria, Egypt

Egypt Abis, rural Alexandria

Study period:January 1994–1995

Small GrantsScheme

(SGS) 1993 No. 37

Principal InvestigatorDr Laila Abou Basha



Single or combined infections with Schistosoma mansoni (21.5%)and Fasciola (8%) proved to be a relevant public health problemin Abis I village, as 29.5% of the surveyed population had eitheror both infections.

Procollagen III peptide levels were elevated in cases with mixedinfections, followed by Schistosoma mansoni, indicating that mixedinfection has stimulated fibroblastic proliferation and procollagensynthesis.

The serum fibronectin level was lowest in schistosomiasis cases,intermediate in mixed, and highest in Fasciola cases, indicatingmore fibrogenesis induced by Schistosoma infection.

Younger age is a significant predictor for elevated procollagenlevel due to age-related variability in fibroblastic stimulation triggeredby these parasitic infections.

Connective tissue markers can identify active ongoing fibrosis,while ultrasonography refers to old ones. Procollagen III peptidecould be used for early diagnosis and management, and thereforeregression of the signs of fibrosis. The survey emphasized theimportance of prophylactic strategies directed to children living inthis area.



Morbidity

110 111

based on a previous survey that confirmed theendemicity of schistosomiasis and fascioliasis in thisvillage. A map was prepared and the houses, totalling589, were enumerated. Random selection of 403houses was performed. Stool samples were collectedtwice from 3658 villagers and examined by the Kato-Katz technique. A random sample of positive cases(n = 281) was subjected to detailed morbidity study.Exclusion criteria were: history of jaundice, viralhepatitis, history of intake of anti-rheumatics,analgesics, drugs for schistosomiasis or fascioliasisduring the last 6 months.

All cases were hospitalized and subjected to clinicalexamination, ultrasonography, and blood sampleswere collected for complete blood picture, liver functiontests, hepatitis B surface antigen, connective tissuemarkers, fibronectin and procollagen III peptide. Liverbiopsy was performed in 37 patients (14 withschistosomiasis, 14 with fascioliasis, and 9 with mixedinfections). Results were compared with healthycontrols matched for sex and socioeconomic statusas cases.

Main study findingsThe prevalence of S. mansoni was 21.5% (19%

single infection and 2.5% mixed infection withFasciola). The prevalence of Fasciola infection was8% (5.5% single and 2.5% mixed with S. mansoni).S. mansoni infection was more prevalent among theage group 15-35 years (25.4%), followed by children(19.6%), while fascioliasis and mixed infections weremore prevalent among children (9% and 4%,respectively), followed by adults (4% in both). Maleswere more infected than females with schistosomiasis(30.6% versus 13.4%), while females were moreinfected with fascioliasis (10% versus 7%). Mixedinfections were more frequently encountered in males(2.8%) compared to females (2.3%). Regarding theintensity of infection, 83%, 15% and 2% sufferedfrom low, moderate and heavy intensity ofSchistosoma infection, respectively. In fascioliasis,91.3% suffered from low intensity of infectioncompared to 8% and 0.7% of moderate and heavyintensity of infection, respectively.

Detailed morbidity study The highest procollagenIII peptide levels were observed in cases with mixedinfections, followed by S. mansoni, indicating thatmixed infection has stimulated fibroblastic proliferationand procollagen synthesis. The serum fibronectinlevel was lower in cases compared to controls, andin schistosomiasis cases compared to Fasciola cases,indicating that the fibrogenesis induced bySchistosoma infection is more than that induced byFasciola. In mixed infection, the serum fibronectinlevel was of intermediate level, which could beattributed to the immunoregulatory mechanismguarding against dramatic decrease in serum

fibronectin level in concurrent infection. The decreasein fibronectin level in liver diseases was attributed todisorders in coagulation.

On the other hand, procollagen peptide III levelwas increased in cases, and was significantly higherin children compared to adults. After adjusting forconfounding variables, age remained a significantpredictor for elevated procollagen level. This couldbe explained by age-related variability in fibroblasticstimulation triggered by such parasitic infections.There was no association between connective tissuematrix markers and the grade of liver fibrosisdiagnosed by ultrasonography. This was explainedby the fact that the tissue markers can identify activeongoing fibrosis, while ultrasonography refers to oldones.

There were significantly higher levels of SGOT andserum alkaline phosphatase in patients affected withsingle or mixed parasitic infections compared tocontrols. In cases with schistosomiasis, serumfibronectin was positively correlated with serumalbumin such as the situation in alcoholic cirrhosis.Furthermore, procollagen III peptide was positivelycorrelated with SAP, indicating active hepatic fibrosis.

In cases with fascioliasis, the egg count was directlycorrelated with SAP, which could be explained bythe fact that the number of eggs reflect the adultFasciola worms that may lead to biliary stasis andobstruction.

In cases with mixed infection, the egg count waspositively correlated with SGOT and SGPT forSchistosoma eggs but not for Fasciola's. In this groupof individuals, the decrease in serum fibronectin wasdirectly correlated with a corresponding decrease inprothrombin activity, and procollagen III peptide wasalso correlated with SAP.

Conclusions and recommendationsSingle and combined infections with S. mansoni

and Fasciola proved to be a relevant public healthproblem in rural Alexandria, Egypt. The surveyhighlighted the importance of prophylactic strategiesdirected to children living in this area. The increaseof procollagen III peptide indicated active fibrosis,and it was therefore recommended to use thisconnective tissue marker for early diagnosis andmanagement, and therefore the regression of thesigns of fibrosis.

Schis

tosom

iasis

AbstractThis study was conducted to estimate the

impact of schistosomiasis on thesocioeconomic level and on the quality of life(QOL) and productivity of workers. All workersin Misr Textile company, Kafr-El-Dawar,Beheira Governorate, suffering from active orchronic schistosomiasis were included in thestudy. An equal number of healthy controlsmatched for job similarities to the cases werealso included. Data collection forms includedinformation regarding sociodemographic,occupation, housing conditions, clinical data,and knowledge of and attitudes towardsschistosomal infection. The WHOQOLinstrument for measuring quality of life wasused. Data were also collected from the medicalrecords of workers in the outpatient andinpatient settings as well as from the personalrecords of the workers available in thecompany. The latter included informationregarding workers' productivity, sickness, injury,and days lost. Disability data were obtainedfrom the social insurance department andcross-checked with the labour office of thetown. Schistosomiasis stages ranged fromactive infection to urinary cancer forSchistosoma haematobium or haematemesisfor S. mansoni.

Results Most patients suffered from S.mansoni, the most frequent symptoms beingdysuria, frequent micturition and dysentery.

Hepatomegaly and splenomegaly weredetected in 29% and 39%, respectively. Fewpatients had ascites, ankle oedema orhaematemesis. A minority reported bladderulcers, ureteric strictures, cor pulmonale orheart failure. Patients had significantly betterknowledge, higher rates of utilization of medicalservices, and greater economic burden anddisability rates compared to the controls.Furthermore, the mean productivity score ofpatients was lower than that of controls. Patientsrecorded significantly lower levels of quality oflife, and this was strongly associated with thestage of the disease. QOL domains, additionalworking hours, total incentives, and the totalcost of illness could be used as discriminatorsfor the schistosomiasis.

Conclusion Schistosomiasis has a negativeimpact on the quality of life and productivity ofthe affected individuals.

PublicationsKamel MI et al. Impact of type and stage of

schistosomiasis on quality of life and productivityof infected workers. J Egypt Soc Parasitol, 2001,31(1): 153-167.


Training of data collectors and fieldsupervisors included discussion of theobjectives and importance of the study,orientation about the questionnaire, methodof data collection and editing. The practicaltraining of company physicians was carriedout in the company hospital.

BackgroundAn effective disease intervention is one

that produces a net improvement in thebeneficiaries' quality of life and/or increaseslife expectancy. Unlike developedcountries, there is a scarcity of informationon the measures of the ultimate output ofhealth care in developing countries. Qualityof life (QOL) is measured as physical andsocial functioning and as perceivedphysical and mental well-being. Health-related quality of life (HRQL) includesaspects of physical, psychological, andsocial well-being issues for people.

Impact ofschistosomiasis onthe quality of life and

productivity ofworkers

EgyptKafr-El-Dawar, Beheira

Governorate

Study period:October 1998–

March 2000

Small GrantsScheme

(SGS) 1998 No. 7

Principal InvestigatorDr Mohamed I Kamel

Community MedicineDepartment

Faculty of Medicine Alexandria University

Alexandria, Egypte-mail:

[email protected]

Patients recorded better knowledge, higher rates of utilizationof medical services, and considerably higher degrees of economicburden and disability rates compared to the control group.Productivity of patients was lower than that of controls.

Patients recorded a significantly lower quality of life in thefollowing domains: physical, independence, psychological, spiritualand social. But the environmental domain was lower in the controlgroup. A better quality of life was invariably linked with less severityof the disease.

QOL domains, additional working hours, total incentives, andtotal cost of illness could be used as discriminators for the disease.

The results of this study emphasize the need for planning ahealth education programme as well as pre-employment andperiodic clinical examinations of workers at high risk of contractingschistosomiasis.



Morbidity

112 113

Although schistosomiasis is endemic in many tropicalregions of the world, studies have only dealt with thediagnostic and clinical aspects of the disease. Mostof the studies dealing with the impact of schistosomiasisused mortality and morbidity as the main indicators.However, morbidity studies seldom included the impactof the disease on the physical and socioeconomicdevelopment of an affected individual and on theeconomic and social consequences affectingcommunity development. Therefore, this study wasconducted to estimate the impact of schistosomiasison the socioeconomic level as well as on the qualityof life and productivity of workers.

Materials and methodsAll workers in Misr Textile company, Kafr-El-Dawar,

Beheira Governorate, suffering from active or chronicschistosomiasis were included in the study. An equalnumber of healthy controls matched for job equivalenceto the patients were also included.

Study tools Four types of questionnaire were usedfor data collection. The first included informationregarding sociodemographic, occupational and homeenvironment, and clinical data of the disease. Datarelated to knowledge and attitudes towardsschistosomal infection were also included in this firstquestionnaire. The WHOQOL instrument for measuringQOL was used in a second questionnaire. Theinstrument consists of 26 questions, each of five-pointscale. The scale consists of five domains: domain 1is concerned with the physical and independencequality, domain 2 with the psychological and spiritualaspect, domain 3 with the social aspects, and domain4 with the quality of the environment.

A third form was designed to collect data from themedical records of workers in the outpatient andinpatient settings. A fourth form compiled data fromthe workers' records in the company such as themeasures of productivity (e.g. production scores,penalties, incentives, discipline and salary), as wellas data related to sickness, injury and days lost.Disability data were obtained from the social insurancedepartment and cross-checked with the labour office.

Data collection was performed by staff members ofthe Community Medicine Department, Faculty ofMedicine and Faculty of Nursing as well as thecompany physicians.

QOL score The score of each domain was calculatedso that the positive and negative questions would addup in the same direction. The total score was thentransformed onto 0-100 score.

Production score According to the company criteria,each worker was given a score between 0 and 100each month. The total score was calculated by additionof the monthly scores. Absence frequency, severityrates and proportional sickness disability rates were

also calculated.

Scoring of schistosomiasis stages This scoreranged from active infection (score 0) to urinary cancerfrom S. haematobium or haematemesis from S.mansoni (score 5).

Calculation of cost The receipts of hospitals,investigations performed, drugs prescribed and anymedical procedure were included in the calculation ofcosts.

Main study findingsSchistosomiasis workers and controls were

comparable regarding age, gender, job category andalmost all were married. The control group and theirspouses had significantly higher educational levels,tended to live in urban areas, recorded a higher meancrowding index and income, and also lived in betterhousing conditions (including sewage and refusedisposal, electricity, etc.) compared to the patients.

Most of the patients (78.8%) suffered from S. mansoniand only 7.6% from S. haematobium, while the restsuffered from mixed infections. The most frequentsymptoms were dysuria, frequent micturition, anddysentery. Hepatomegaly and splenomegaly weredetected in 29% and 39% of infected workers,respectively. Few patients had ascites, ankle oedemaor haematemesis. A minority reported bladder ulcers,ureteric strictures, cor pulmonale or heart failure.

Patients had significantly better knowledgeconcerning all aspects of the disease. They alsorecorded higher rates of utilization of medical services,and their mean costs for these services was significantlyhigher than for the controls. Disability rates were alsohigher among patients, but there was no significantdifference between both groups regarding sicknessabsence rates, and the mean productivity score ofpatients was lower than that for controls.

Regarding QOL, sufferers recorded significantlylower general domain, physical, independence,psychological, spiritual and social domains, but theenvironmental domain was lower in the control group.This was strongly associated with the stage of thedisease; a better QOL was always associated withearlier stages of the disease. QOL domains, additionalworking hours, total incentives, and total cost of illnesscould be used as discriminators for the disease, asrevealed by multiple logistic regression analysis.

RecommendationsThe results of this study highlighted the need for

planning and implementation of a health educationprogramme for workers. Pre-employment and periodicclinical examinations of this organized sector of thecommunity is also emphasized.

Schis

tosom

iasis

AbstractThis study was conducted with the aim of

identifying the frequency and risk factors ofsevere schistosomal morbidity in ruralAlexandria, and evaluating the role ofprocollagen III peptide, a connective tissuematrix marker, in predicting schistosomalmorbidity. Abis 4 village was randomly selectedas the study area. All 2577 households wereasked to provide morning stool samples, andthe intensity of infection was calculated. Anestimated sample size of 1082 subjects wasrandomly selected, interviewed and examined.Adults with clinically detected organomegalyand an equal number of healthy villagers werereferred for detailed investigations. A 5-itemportal hypertension score was developedconsisting of portal vein diameter, spleenlength, collaterals, direction of portal bloodflow and degree of periportal fibrosis. Subjectsrecording 2 or more abnormal findings of theseitems were considered cases that developedsevere morbidity, while the remaining groupwas taken as a control. A randomly selectedsample of children was also referred to the

hospital for detailed investigations. Childrenwith an enlarged left lobe of the liver and withperiportal fibrosis more than 3 mm wereconsidered cases that developed earlymorbidity, and the remaining group of childrenwere considered controls.

Results The overall prevalence Schistosomamansoni accounted for 20.5%, but with a lowintensity of infection. Seropositivity of HCVantibodies was found to be strikingly high(45.9%) in adults >35 years old with clinicallydetected organomegaly. Older age and heavywater canal exposure were the only significantrisk factors for developing clinically detectableorganomegaly. Previous viral infection (anti-HCV and/or anti-HbsAg) was the onlysignificant risk factor for developing highprocollagen levels, portal hypertension score2, and AST/ALT ratios greater than 1 (i.e.severe morbidity). High procollagen levelswere also predicting severe portal hypertensionand high AST/ALT ratios. Regarding children,older age and positive stool analysis were theonly determinants of developing highprocollagen levels, while schistosomal infectionproved to be the only significant risk factor fordeveloping early liver morbidity.

Conclusion This study revealed that S.mansoni and viral hepatitis infections are themain causes of chronic liver diseases in Egypt.The risk of concomitant viral infection shouldtherefore be emphasized in planning preventiveprogrammes.

PublicationsZaki A et al. Morbidity of schistosomiasis

mansoni in rural Alexandria, Egypt. J Egypt SocParasitol, accepted for publication on the 3rd ofFebruary, 2003.

BackgroundSchistosomal morbidity appears to vary

from one geographical area to another,being related to the strain of parasite, thehost and the environment. It is widelyaccepted that most persons with S.mansoni in fect ion in the sameschistosomal endemic area, living insimilar environmental conditions, andprobably with comparable frequency of

Identification ofgroups at high risk

of severeschistosomalmorbidity: a

suggested plan forcontrol

EgyptAbis district, rural

Alexandria UniversityAlexandria governorate


Small GrantsScheme

(SGS) 1998 No. 36

Principal InvestigatorDr Adel Zaki

Department of Medical StatisticsMedical Research Institue


The prevalence of S. mansoni in rural Alexandria, Egypt, is20.5%, with a low intensity of infection.

Clinical examination proved to be a nonreliable method for thedetection of liver morbidity. The assessment of liver morbidityshould include ultrasonography, hepatitis virus investigations andliver function tests. Connective tissue matrix markers were primarilyassociated with viral infections in adults and S. mansoni infectionin children. Since they escalate the costs of morbidity assessmentwithout adding to the information requested, they are notrecommended for the routine assessment of liver morbidity.

The combined risk of schistosomiasis and hepatitis virusesshould be taken into consideration in planning control strategiesin endemic areas.

Infection with S. mansoni proved to be a risk factor for developinghigh procollagen levels and early liver morbidity in children. Healtheducation programmes and semi-annual chemotherapy targetingthis vulnerable age group of the rural community will result in theregression of early liver morbidity, thereby protecting children fromprogressing to the incurable morbidity stages.



Morbidity

114 115

water canal exposure, show different degrees ofmorbidity. Several factors may contribute to itspathogenesis including the intensity and duration ofinfection, genetic predisposition, blood group, andtiming of treatment. Schistosomal morbidity can beassessed by physical examination, laboratoryinvestigations and ultrasonography. Recently,connective tissue matrix markers were used aspredictors of the degree of fibrosis. Periportal hepaticfibrosis (PPF) is a main sequel of schistosomalinfection, however, the degree of fibrosis cannot beexactly foretold. Some patients may progress to thelate stages of shrunken liver, while others may stopat an intermediate stage of hepatic fibrosis.

This study was conducted aiming at the identificationof the frequency and risk factors of severeschistosomal morbidity in rural Alexandria, in additionto the evaluation of the role of procollagen III peptide,a connective tissue matrix marker, in predictingschistosomal morbidity.

Materials and methodsA map of the Abis villages in rural Alexandria was

prepared, and Abis 4 village was randomly selectedas the study area. All 2577 households were askedto provide morning stool samples and the number ofeggs per gram of faeces (epg) was determined byaveraging egg counts on 3 modified Kato thicksmears. An estimated sample size of 1082 subjectswas selected by a systematic random samplingmethod. Subjects were interviewed using a pre-testedquestionnaire. The information collected included:sociodemographic, lifestyle, history of antischistosomaltreatment and medical past history. Clinicalexamination was carried out in the field. A scoringsystem for water canal exposure was developedtaking into consideration the nature, frequency andduration of exposure.

Morbidity study in adults Adults with clinicallydetected organomegaly and an equal number ofhealthy villagers were referred for liver function tests,procollagen type III peptide, HCV-antibodies, HbsAg,and abdominal Doppler ultrasonography. A 5-itemportal hypertension score was developed consistingof: portal diameter >13mm, spleen length >120mm,presence of collaterals, hepatofugal direction of portalblood flow and periportal fibrosis ³ grade 2. Subjectsrecording 2 or more of these items (portal hypertensionscore ³2) were considered cases that developedsevere morbidity, while the remaining group wastaken as a control.

Morbidity study in children Randomly selectedchildren were referred to perform the sameinvestigations previously described for adults. Childrenwith an enlarged left lobe liver and periportal fibrosismore than grade 1 (>3mm) were considered cases

that developed early morbidity, and the remaininggroup of children were considered controls.

Main study findingsS. mansoni is still endemic in this area, with an

overall prevalence of 20.5%. Its prevalence increasedwith age to reach to a plateau at 15-30 years of age,then was nearly stable thereafter even in olderindividuals. The intensity of infection was lowaccording to the WHO staging system (geometricmean less than 100 epg). Males recorded higherprevalence and intensity of infection compared tofemales.

Factors potentially affecting schistosomal morbiditywere studied and these included: age, sex, intensityof infection, water canal exposure habits, education,antischistosomal treatment, past history of comorbidliver conditions and risk factors for liver morbidity.

This study revealed that S. mansoni and viralhepatitis infections are the main causes of chronicliver diseases in Egypt. The seropositivity of HCVantibodies was found to be strikingly high in adults>35 years old; in fact, age was the only significantdeterminant of HCV seropositivity in this study. Clinicalexamination proved to be a nonreliable method withlow sensitivity, low negative predictive value, moderatespecificity and high positive predictive value fordetection of morbidity. In spite of these limitations,older age and heavy water canal exposure were theonly significant risk factors for developing clinicallydetectable organomegaly. Previous viral infection(anti-HCV and/or anti-HbsAg) was the only significantrisk factor for developing high procollagen levels,portal hypertension score ³2, and AST/ALT ratio ofmore than 1 (i.e. severe morbidity). High procollagenlevels were also predicting severe portal hypertensionand high AST/ALT ratios.

Regarding children, age (older than 10 years) andpositive stool analysis were the only significant riskfactors for developing high procollagen levels. Positivestool analysis proved to be the only significant riskfactor for developing early morbidity (enlarged leftlobe liver and PPF >3mm).

The preventive strategies against the historicalpublic health challenge of schistosomiasis in Egyptianvillagers is expected to change dramatically withincreasing awareness about the risk of concomitantviral infections.

Schis

tosom

iasis

AbstractThis project aimed at studying the effect of

the involvement of members of the IraqiWomen's Federation (IWF) on the outcomeof the strategy of directly observed treatmentshort course (DOTS) in the treatment oftuberculosis patients. A total of 172 newlydiagnosed cases in Saddam City, Baghdad,were systematically randomized into 2 groups,one using the conventional method ofadministering DOTS in primary health carecentres, and the other with involvement ofmembers of the IWF supervising DOTS.

Results Involvement of the IWF had asignificant impact on the outcome of the DOTSstrategy for tuberculosis treatment. The curerate in the intervention group was 83.7%,compared to 68.6% in the control group. Allpatients in the intervention group werecompliant, compared to 86% in the controlgroup. The cure rate for patients was foundto be significantly negatively associated withthe number of doses missed, and the cut-offpoint was found to be 8 doses. Smearconversion rates were significantly higher inthe intervention group compared to the controlgroup. On the other hand, neither the defaulterrate nor mortality was affected by thisintervention.

Conclusion This study provides evidencethat involvement of nongovernmentalorganizations would significantly improve thetreatment outcome of tuberculosis patientsunder the DOTS strategy. The introductionof this intervention in the National TuberculosisControl Programme is highly recommended.

BackgroundThere have been several reports

regard ing the invo lvement o fnongovernmental organizations (NGOs)in home visiting of tuberculosis patientsin order to supervise the directly observedchemotherapy short course, DOTS.Moreover, NGOs have collaborated withnational tuberculosis programmes inDOTS implementation, health education,contact tracing, defaulter retrieving,advocacy and financial support totuberculosis patients. This study aimedat evaluating the impact of theinvolvement of the Iraqi Women'sFederation (IWF) in improving the DOTSoutcome compared to the conventionalmethod of DOTS administration.

Material and methodsAn intervention study was conducted

from February to December 2001 in thePrimary Health Care Centres of SaddamCity, Baghdad. A total of 172 newlydiagnosed cases were systematicallyrandomized into intervention or controlgroups (86 in each group). Diagnosistook place in the Tuberculosis Institute,Baghdad, based on 3 consecutivepositive sputum smears by Ziehl-NeelsenStain.

Intervention Twenty members of theIWF were nominated and selected fromthe local branch of the IWF in SaddamCity and trained for 2 days on the problemof tuberculosis and on the direct dailyclose supervision of DOTS. Trainingincluded how to approach patients dailyand how to administer the antituberculosisdrugs directly from their hands during the2 months of intense treatment of DOTS.They were trained on the adherence tothe visit, the schedule of treatment andthe provision of direct daily hand-to-mouth

Effect of theinvolvement of

members of the IraqiWomen's Federation

(IWF) on theoutcome of DOTS

strategy forpulmonary

tuberculosis patientsin Iraq: an

intervention study

IraqBaghdad

Study period:February2001–December 2001

Small GrantsScheme

(SGS) 2000 No. 8

Principal InvestigatorDr Amjad Daoud Niazi

Department of CommunityMedicine

Saddam College of MedicineKadmiya, Iraq


This study reported the beneficial impact of involvingnongovernmnental organizations on the outcome of tuberculosispatients managed under the DOTS strategy.

There is a need to implement this successful intervention on awider scale in the country and in other countries where tuberculosisis a public health problem.

A minimum of 8 missed doses of anti-tuberculous drugs couldsignificantly affect the cure of patients.

TuberculosisI r a q


Control

116 117

therapy for patients assigned to them; thereafter,they were responsible for their patients' monthlyfollow-up. They were also taught to educate thepatient and his family on the disease and itstransmission, and trained in the completion of thequestionnaires. These individuals were strictlyobserved by the tuberculosis coordinator. After theperiod of intensive treatment using the same regimen(rifampicin, isoniazid, pyrazinamide, ethambutol orstreptomycin), the patients were referred to thetuberculosis coordinator to complete their treatmentof daily rifampicin and isoniazid administered by thenearest primary health care centre.

The conventional method of del iver ingantituberculosis drugs to the patients took place inthe primary health care centre for a 6-month period,during which the drugs were delivered on a dailybasis to the 86 patients belonging to the controlgroup. Patients from the two groups were followed-up in a timely fashion to measure the smearconversion rates at the end of the first, second, thirdand fifth months, the defaulter rates, non-compliance,cure rates, treatment failures and mortality rates.

Main study findingsThere were no significant differences between the

intervention and control groups regarding age, gender,type of family and occupation. On the other hand,house ownership was significantly higher in theintervention group, and the crowding index wassignificantly higher in the control group. In more thanone-third of patients, a household index case wasidentified, mainly a second-degree relative, comparedto almost one-quarter of patients with a non-householdindex case. The ratio of household to non-householdindex cases was 1.64.

The cure rate was significantly higher in theintervention group (83.7%), compared to the controlgroup (68.6%).

The risk of being smear-positive was significantlyhigher through the period of follow-up in the controlgroup compared to the intervention group.Alternatively, the seroconversion rates weresignificantly higher in the intervention group comparedto the control group, and showed a significant trendwith the duration of follow-up.

Moreover, the noncompliance problem was absentfrom the intervention group supervised by themembers of the IWF. In the control group,noncompliance to treatment varied in durationaccording to the duration of follow-up, but the overallnoncompliance rate was 86%. Noncompliance in thisgroup was not associated with gender, family type,ownership of a house or marital status. On the otherhand, the duration of follow-up was the only significantdeterminant of noncompliance, and noncompliancewas significantly associated with treatment failure.

In fact, the cure of a patient was found to be negativelyassociated with the number of doses missed (P =0.03), and the cut-off point was found to be 8 doses(P = 0.04).

The defaulter and the mortality rates were notsignificantly different among the 2 groups in thisstudy. Defaulter rates in the two groups were 11.18%and 10.6% in the intervention and control groups,respectively. Regarding mortality, 2 cases died, 1 ineach group (1.2%). However, a larger sample sizeis needed before drawing conclusions about defaultingand mortality rates. This is one of the study limitations.

Conclusions and recommendationsThis study provides evidence that involvement ofnongovernmental organizations would significantlyimprove the cure rate, smear conversion rate andthe compliance rate of pulmonary tuberculosispatients, thus reducing infectivity days, preventingthe spread of the disease at the community level,and reducing relapses.

Research on the cost-effectiveness of thisintervention is recommended in order to provideadditional evidence about the importance of itsintroduction within the DOTS strategy in low-incomecountries with a heavy tuberculosis burden.

Tube

rculos

is


of home visits on the compliance andtreatment outcomes of tuberculosis patientswho collected their drugs late. Four hundredand eighty new smear-positive patients whocollected their drugs late from health centresover a period of 6 months were selected andrandomized for either home visiting or non-home visiting.

Results Home visiting was highly effectivein improving the return of late-comers; out of240 patients, 231 returned and 9 did notreturn. The treatment success rate was 94.1%compared to 76.7% in the control group. Thedefaulter rate in the intervention group was0.9% while in the control group it was 10%.Smear conversion at the end of the treatmentwas better in the intervention group (92.9%)compared to the control group (75%).

Conclusion Home visits by trained personnelwould significantly improve the complianceof patients, treatment success rate and smearconversion rate and would reduce the defaulterrate.

Background Successful completion of treatment for

active tuberculosis patients is the singlemost important way to control and preventnew cases. However, treatmentcomplet ion is often delayed or

unsuccessful because it requires patientsto adhere to taking medication for at least6 months. A successful, cost-effective,community-based programme of directlyobserved therapy, utilizing volunteers,clinic and community health workers, hashelped ensure adherence to therapy.

In Iraq tuberculosis is a major publichealth problem. In spite of the 50%increase in notification rate achievedduring the last 4 years, the defaulter andlatecomer rates are relatively high (10%and 20%, respectively).

This study was designed to evaluatethe effect of home visits on the return oflatecomers and the treatment successrate in comparison to the conventionalmethod of directly observed therapy, shortcourse (DOTS).

Materials and methodsThis was an intervention study

conducted in 15 randomly selected healthcentres in Baghdad. A total of 480 patientswho collected their drugs late wererandomized into either home visiting(intervention group = 240) or non-homevisiting (control group = 240). Smear-positive patients were selected due totheir role in the spread of infection andthe fact that treatment outcomes are moreaccurately evaluated by following smearconversion.

Fifteen home visitors were trained onmotivating late comers to adhere to theprescribed regimen. A group of TBcoordinators, each for one district, werealso trained on the identification of latecomers, on how to send home visitorsand on the follow-up of the project'simplementation according to the studydesign. They were also trained on datarecording in order to minimize errors inregistration in treatment cards and books.The records of home visiting were filledin separate sheets away from the patients'records.

Another person was identified and

Does routine homevisiting improve thereturn of late-coming

patients?

IraqBaghdad

Study period:May 2001–2002

Small GrantsScheme

(SGS) 2000 No. 35

Principal InvestigatorDr Ayed Mohan Aldelemi

National TuberculosisControl ProgrammeMinistry of Health

Baghdad, Iraqe-mail:

[email protected]

TuberculosisI r a q


Control

Home visiting of patients by trained personnel has a significantpositive impact on the return of latecomers to continue theirmedication, and on their compliance and treatment outcomes.

Risk factors for noncompliance were: positive family history oftuberculosis, residing at a far distance from tuberculosis centres,social stigma, economic burden, feeling no improvement withtreatment, and negative attitude towards drug intake.

Adherence to treatment was not found to be a significant predictorof treatment failure and mortality from tuberculosis. Otherdeterminants should be carefully investigated.

Home visiting of patients by trained personnel should be anintegral part of DOTS strategy.

trained to fill the data collection form. This personwas blinded about home visiting in order to reducethe possibility of bias. The study was carried out afterreceiving ethical clearance and all individualinformation was strictly confidential.The two groups were matched for the distance fromthe TB center in order to avoid its confounding effect.

Several constraints were met during conductingthe study such as refusal of home visiting by thepatients, their absence during the time of the visit,or incorrect addresses. These constraints were facedby educating the patients about the importance ofthese visits, in case of refusal, or asking the homevisitor to pay another visit to the patient, in case ofhis absence during the initial visit. On the other hand,it was not possible to retrieve the patients with incorrectaddresses and this group was excluded from thestudy.

Definitions:

Latecomer: Any patient who does not come totake medication for at least 3 days after a scheduledappointment. The patient is defined as a latecomeron the first episode of coming late.

Home visiting Visit by a person trained andassigned to visit the home of a latecomer in order tomotivate him/her to attend the health centre daily.

Data collection The home visitors were trainedto visit latecomers under the guidance of thetuberculosis coordinator. They received standardizedinstructions. The treatment completion was recordedfrom the tuberculosis register and from patients'treatment cards at the tuberculosis centre.

The information was checked before entry to thecomputer, edited and double entry was performed inorder to reduce the probabil i ty of error.Data analysis Chi-square test was used to comparebetween the intervention and control groups regardingcompliance to treatment, return of late comers andtreatment outcome.

Main study findingsCases were found to be comparable to the controls

and there was no significant difference between thetwo groups in terms of age, gender and marital status.It is clear from the findings of this study that homevisiting of patients had a significant impact on thereturn of latecomers to continue their medication.Out of 240 patients, 231 (96.2%) returned and 9(3.8%) did not. Regarding the treatment successrate, it was significantly higher in the interventiongroup compared to the control group (94.1% and76.7%, respectively).

Home visiting also had a significant impact inreducing defaulter rates (0.9% and 10%, in theintervention and control groups, respectively).

Although the failure rates and death rates werereduced by this intervention, there was no significantdifference between the two studied groups. Thefailure rate was 5.8% in the control group comparedto 2% in the intervention group. The death rate wasalso higher in the control group (3.4% versus 1.3%,p>0.05).

Noncompliance was found to be significantlyassociated with positive family history, living far fromtuberculosis centres, social stigma, economic burden,feeling of non-improvement with treatment, and refusalto take drugs.

Therefore, home visiting proved to play an importantrole in improving compliance and the treatmentoutcome of new smear-positive pulmonarytuberculosis patients.

Conclusions and recommendationsHome visiting by trained personnel would

significantly improve the compliance of patients, thesuccess rate, smear convention rate and defaulterrate. Accordingly, home visiting could play a majorrole in reducing the infection rate in the community.

The integration of this successful intervention inDOTS strategy is highly recommended.

118 119

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of home visits on the compliance andtreatment outcomes tuberculosis patients whocollected their drugs late. Four hundred andeighty new smear positive cases who collectedtheir drugs late from health centres over aperiod of 6 months were selected andrandomized for either home visiting or non-home visiting.

Results Home visiting was highly effectivein improving the return of late-coming patients,recording a compliance rate of 89% comparedto 80% among the group that did not receivehome visiting. Other determinants of treatmentcompliance were studied but none had asignificant impact on compliance. Non-adherence to treatment was attributed to theseverity of illness, stigma, worries aboutteratogenic effects on fetuses, losing treatmentcards, family circumstances and fear ofdeportation in the case of refugees.

Conclusion Home visiting by trainedpersonnel would significantly improve thecompliance of patients, and consequentlytreatment success rates, smear conversionrates and would reduce defaulter rates.

BackgroundWith an annual risk of infection of 3%,

tuberculosis represents a major publichealth problem in Djibouti. This problemis aggravated by the influx of immigrantsfrom neighbouring countries, whorepresent 50% of smear-positive

pulmonary tuberculosis cases and 40%of all cases. Since 1990, severalconferences and workshops were heldin order to coordinate tuberculosis controlactivities within the Horn of Africa.

Adherence to treatment is thecornerstone for achieving higher curerates, hence tuberculosis control.However, the defaulter rate had reached20%, representing a serious impedimentto tuberculosis control in the country,besides favouring the spread of multidrugresistant strains.

This study was designed to evaluatethe effect of home visiting on the returnof latecomers, and thus, on the treatmentsuccess rate of tuberculosis patients inDjibouti.

Materials and methodsAn intervention study was conducted

in 6 tuberculosis centres in Djibouti. Atotal of 480 patients who collected theirdrugs late were randomized into eitherhome visiting (intervention group = 240)or non-home visiting (control group =240). Smear-positive cases were selecteddue to their role in the spread of infectionand the fact that treatment outcome ismore accurately evaluated by followingsmear conversion.

Home visitors were trained onmotivating late comers to adhere to theprescribed regimen. A group ofTuberculosis coordinators were alsotrained on the identification of late comers,on how to send home visitors and on thefollow-up of the project's implementationaccording to the study design. They werealso trained on data recording in orderto minimize errors in registration intreatment cards and books. The recordsof home visiting were filled in separatesheets away from the patients' records.

Another person was identified andtrained to fill the data collection form. Thisperson was blinded about home visiting

Do routine homevisits improve the

return of late-coming patients?

DjiboutiDjibouti

Study period:January 2000–April 2003

Small GrantsScheme

(SGS) 2000 No. 35

Principal InvestigatorDr Houmed Ali

National Tuberculosis ControlProgramme

Ministry of HealthDjibouti, Djibouti

Home visiting of patients by trained personnel has a significant positive impact on the return of latecomers to continue theirmedication, and on their compliance and treatment outcome.

Noncompliance was justified by: severity of illness, social stigma;economic burden; worries about teratogenic effects on fetuses,losing treatment cards, family circumstances (death, illness, etc.),fear of deportation in case of refugees.

Home visiting of patients by trained personnel should be anintegral part of DOTS strategy.

TuberculosisD j i b o u t i


Control

120 121

in order to reduce the possibility of bias. The studywas carried out after receiving ethical clearance andall individual information was strictly confidential.

The two groups were matched for the distancefrom the TB center in order to avoid its confoundingeffect.

Definitions:

Latecomer: Any patient who does not come totake medication for at least 3 days after scheduledappointment. The patient is defined as a latecomeron the first episode of coming late.

Home visiting: Visit by a person trained andassigned to visit the home of a latecomer in order tomotivate him/her to attend the health centre daily.

Data collection: The home visitors were trainedto visit latecomers under the guidance of thetuberculosis coordinator. They received standardizedinstructions. Treatment completion was recordedfrom the tuberculosis register and the patients'treatment cards at the tuberculosis centre.

Data analysis Chi-square test was used to comparebetween the intervention and control groups regardingcompliance to treatment and return of late comers.

Main study findingsSeventy- five percent of the study population was

younger than 35 years of age. There was no significantdifference between the intervention and the controlgroup regarding: age, sex, residence, nationality,income, existence of household contacts, and thepresence of co-morbid conditions. The interventiongroup had significantly lower educational andoccupational levels and were living at closer distancesfrom the tuberculosis centres.

The results of this study showed that home visitingof patients had a significant impact on the return oflatecomers to continue their medication. Adherenceto treatment was significantly higher in the interventiongroup compared to the control group (89% and 80%,respectively).

Other determinants of treatment compliance werestudied but none had a significant impact oncompliance. These factors were: sociodemographics,educational level, occupation, income, existence ofcontacts, and distance to the tuberculosis centre.However, these negative findings could be explainedby the fact that 90% of the study population wasliving within less than 2 km from the tuberculosiscentres. In addition, they belonged to a relativelyhomogenous social class and were exposed to similarenvironmental and social conditions. While apparentlyreassuring, these negative findings indicate that onlya small proportion of the population is served by thetuberculosis centres, leaving the majority of the

population, nomads or those living in remote areas,unserved.

Patients justified their non-adherence to treatmentby: severity of illness, stigma, worries aboutteratogenic effects on fetuses, losing treatment cards,family circumstances (death, illness, etc.), and fearof deportation in the case of refugees.

Conclusions and recommendationsHome visiting by trained personnel would

significantly improve the compliance of patients,success rate, smear convention rate and defaulterrate. Accordingly, home visiting could play a majorrole in reducing the infection rate in the community.The integration of this successful intervention inDOTS strategy is h ighly recommended.

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AbstractA case-control study conducted in all

tuberculosis centres in Egyptian governoratesaimed at investigating the risk factors oftreatment failure in Egypt. One hundred andnineteen new smear-positive patientsrecording treatment failure at the end of theirtreatment period were enrolled in the study,these were the study cases. An equal-sizedgroup of cured patients, matched with thesenew patients in age, gender, place and timeof treatment constituted the control group.

Both the cases and controls were comparedregarding socio-demographic variables,compliance to treatment, knowledge regardingtuberculosis, accessibility to health care,services provided in the health facility, familyknowledge and support given to the patient.

Results Significant risk factors for treatmentfailure were: missed days (1.4-fold increasedrisk for each missed day of treatment),diabetes mellitus, poor patient knowledgeregarding the disease and deficient healtheducation. Missing at least 8 days of treatmentwould significantly affect the treatmentoutcome and was associated with a 3-foldincrease in the risk of treatment failure.

The most alarming finding of this study isthat 40% of cases with treatment failure werefully adherent to the treatment regimen. Riskfactors for treatment failure in this compliantgroup were diabetes mellitus (10-foldincreased risk) and poor patient knowledge

regarding the disease (9-fold increased risk).On the other hand, there was a significantlylower risk for treatment failure among femalesand for those with a high number of childrenin the family. These factors explained only46% of the variability in treatment failure.Other causes for treatment failure should betherefore investigated in this community suchas drug resistance and drug quality.

Conclusion These results emphasize theneed to strengthen the supervision oftreatment compliance, proper control ofdiabetes, proper health education, and timelyand high quality care to all patients.

BackgroundThe National Tuberculosis Control

Programme (NTP) in Egypt was launchedin 1989 in Cairo and Giza, andsubsequently extended to the entirecountry by the year 1999. The DirectlyObserved Treatment Short course(DOTS) strategy started on a pilot basisin 1996 and reached 100% nationalcoverage in December 2000. Casemanagement according to DOTS entailedregular daily observation of treatment inthe first 2 months of the initial phase,followed by weekly observation duringthe patient's visit to the treating healthcentre, where the patient swallows his/hermedicine and is given medicationssufficient for the next 6 days. Home visitsare carried out for non-compliant patients.Therefore, upon completion, patientsshould record a total of 76 visits to thecentre. The treatment regimen consistsof 2S(E)HRZ/4HR, as recommended bythe national tuberculosis controlguidelines.

Treatment failure is a serious problemfor tuberculosis control programmes inmany countries throughout the world.These patients tend to have highermortality. Moreover, they remaininfectious and hence transmit the diseaseto other members of the community. InEgypt, treatment failure represents 3 to

Irregular treatmentintake as a risk

factor to treatmentfailure among

tuberculosis patientsunder the DOTS

strategy

EgyptAll Governorates

Study period:April 2001–

December 2002

Small GrantsScheme

(SGS) 2000 No. 79

Principal InvestigatorDr Alaa Mokhtar

National TuberculosisControl programme

Ministry of Health andPopulation

Cairo, Egypte-mail: [email protected]

Missing at least 8 days of treatment significantly affects thetreatment outcome, indicating the need to strenghten supervisonthroughout the treatment period of tuberculosis patients.

Poor patient knowledge regarding tuberculosis proved to be asignificant predictor of treatment failure, indicating deficient healtheducation delivered to patients. Strengthening health educationin DOTS strategy is therefore recommended.

Diabetic patients proved to be at a significantly higher risk oftreatment failure and should be subjected to tight blood sugarcontrol and supervision. These results suggest shifting from oralantidiabetic drugs to insulin for tuberculosis patients until treatmentcompletion.

TuberculosisE g y p t


Control

122 123

5% of new smear-positive cases and 13 to 17% ofre-treated cases. This study was carried out toinvestigate the risk factors of treatment failure inEgypt.

Materials and methodsA case-control study was conducted in all

tuberculosis centres in Egyptian governorates thatrecorded treatment failure. All new smear-positivepatients recording treatment failure as a treatmentoutcome in the tuberculosis centres of all governorateswere enrolled in the study and labelled as studycases. A control group equal in size to the new casesconsisted of patients who were declared cured at theend of their treatment. Controls were matched tocases with regard to age, gender, place of treatmentand time of treatment

Both cases and controls were interviewed regardingsocio-demographic variables, compliance totreatment, and knowledge regarding tuberculosis andits medication. The interviews also coveredaccessibility to health care and service provided inthe health facility. A family member was interviewedabout family knowledge and support given to thepatient. The collected information was cross-checkedwith the tuberculosis register, the treatment card andthe supervising nurse.

Main study findingsOne hundred and nineteen patients with treatment

failure were enrolled during the study period, whichrepresents a prevalence of 2.9% (119 treatmentfailures out of 4181 total tuberculosis cases). Therewas no significant difference between governoratesregarding the prevalence of treatment failure, whichranged from 1% to 5%.

As controls were matched to cases, there was nosignificant difference between them regarding socio-demographic variables and treatment time. Two-thirds of cases and controls were males, and morethan half of each group were 25 to 45 years old. Sixtypercent of each group were illiterate and around halfof each group were unemployed.

Regarding the accessibility to health services, 27%of patients with treatment failure were living at adistance of 10 km or more from the tuberculosiscentre compared to 11% of cured patients, and thiswas statistically significant. Diabetes mellitus wasthe only co-morbid condition recorded at a significantlyhigher percentage among cases compared to thecontrols (26% and 7.6%, respectively).

Evaluating patients' knowledge regardingtuberculosis revealed a significantly lower level ofknowledge among failed cases; none were rated ashaving very good knowledge compared to 21% ofcured cases. Expectedly, there was a significantdifference between both groups regarding the

frequency of health education sessions. The sourceof information was mainly the chest hospital and TV.

Adverse treatment effects were recorded in 30%and 23% of failed cases and controls, respectively(p>0.05). Failed cases were less satisfied with thequality of care delivered in the health facilities andby the providers. Almost one quarter of this groupreported waiting for more than half an hour to receivecare.

In addition, families of failed cases were smaller insize and recorded significantly lower knowledgescores regarding the disease and its treatmentcompared to those of cured patients.

Regarding irregular treatment patterns, there wasa significant difference in the number of missed dosesbetween cases and the controls during the second,third, fourth and sixth months of treatment.

Multivariate logistic regression analysis showedthat the significant risk factors for treatment failurewere missed days (1.4-fold increased risk for eachmissed day of treatment), the presence of diabetesmellitus, poor patient knowledge regarding thedisease, and deficient health education. Missing atleast 8 days of treatment significantly affected thetreatment outcome and was associated with a 3-foldincrease in the risk of treatment fai lure.

The most alarming finding of this study is that 40%of patients with treatment failure were adherent tothe treatment regimen. Risk factors for treatmentfailure in this compliant group were diabetes mellitus(10-fold increased risk) and poor patient knowledgeregarding the disease (9-fold increased risk). On theother hand, there was a significantly lower risk fortreatment failure among females and among thosewith a high number of children in the family. However,these factors explained only 46% of the variability intreatment failure. Other causes for treatment failurethat were not investigated in the present study shouldtherefore be investigated in this community, such asdrug resistance and drug quality.

Among the non-compliant group, apart from theinvariable implication of poor knowledge of the causesof their condition, other risk factors emerged such asthe distance to the tuberculosis centre (1.4-foldincreased risk for treatment failure for each kilometrefrom the tuberculosis centre) and poor satisfactionwith care. In addition, younger age and small familysize proved to be significant predictors of treatmentfailure.

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AbstractA cross-sectional study was carried out in

2001 among registered medical practitionersin the public and private health sectors in thenorth-west zone of Somalia. The aim of thestudy was to assess the knowledge andpractices of medical practitioners in thediagnosis and management of tuberculosisusing a structured questionnaire.

Results Only 53 out of 100 registered doctorscould be interviewed. Seventeen reportedworking solely in the private sector, 7 in thepublic sector and 29 in both.Symptoms werecorrectly identified by 66% of the doctors, and60% indicated sputum smear microscopy asthe most important diagnostic test. Only onepractitioner had notified a patient to the NationalTuberculosis Programme, and very fewpractitioners prescribed the correct regimenor advocated direct observation of drug taking.Private practitioners were twice as likely tohave suboptimal knowledge regardingpulmonary tuberculosis diagnosis as thoseworking in the public and private sectors.

Conclusion Tuberculosis patients are mainlymanaged in the private sector, and few doctorsfollow the guidelines of the NationalTuberculosis Programme (NTP) in Somalia.The distribution of the NTP guidelines to allmedical practitioners in the North-west Zone,together with the regular training of doctors inthe diagnosis and case management oftubercu los is were the main s tudyrecommendations.

Impact of research resultson health policy

A workshop on tuberculosis control forprivate practitioners was held in Hargeisa on3-4 November 2002. The meeting was verysuccessful with active participation of allattending doctors. At the end of the meetingthe Minister of Health and Labour issued aMinisterial Decree banning the sale of anti-tuberculosis drugs in private pharmacies.

The KAP study, the workshop and theinterest of the Minister of Health should havea beneficial impact and improve the situationof tuberculosis control in the country.

Countries with similar situations are invitedto use the study recommendations andreplicate this successful experience for bettertuberculosis control.

BackgroundIn the north-west zone of Somalia, the

private sector contributes significantly inthe provision of health care. While manytuberculosis patients are treated withinthe National Tuberculosis Programme(NTP), observations at tuberculosis clinicshave indicated that tuberculosis patientscoming from the private sector were notgiven treatment in line with the nationaltuberculosis guidelines. However, little isknown about the knowledge and practicesof the medical doctors in the north-westzone. Therefore, this study wasundertaken to evaluate the knowledgeand practices of medical practitionersregarding the symptoms, diagnosis andcase management of pulmonarytuberculosis in the north-west zone.

Materials and methodsThe study was a cross-sectional survey

interviewing medical practitioners by astructured questionnaire, and took placebetween July and November 2001. Theeligible population included all the qualifiedmedical practitioners in Somaliland. A listof all medical doctors registered by theGovernment in the north-west zone ofSomalia was obtained from the Ministry

Are medicalpractitioners in

Somaliland followingthe national

guidelines fortuberculosis controlin Somaliland in thediagnosis and case

management ofpulmonary

tuberculosis?

SomaliaNorth-west zone

Study period:July–November 2001

Small GrantsScheme

(SGS) 2000 No. 10

Principal InvestigatorDr Bashir A SuleimanNational Tuberculosis

ProgrammeMinistry of HealthHargeisa, Somalia


Few doctors follow the guidelines of the National TuberculosisProgramme (NTP) in Somalia leading to the inadequatemanagement of tuberculosis patients, mainly in the private sector.

Distributing the NTP guidelines and the regular training of medicalpractitioners in the diagnosis and case management of tuberculosispatients were recommended in order to improve tuberculosiscontrol in Somalia and other countries with similar situations.

TuberculosisS o m a l i a


Quality of Care

124 125

of Health and Labor (2001), including public andprivate practitioners. These consisted of 100 medicalpractitioners after excluding medical practitionersworking for the NTP (7) or had no clinical work.Among this group, only 53 participated in the study.Causes for non-participation was the busy practice(4), travelling abroad (6), mental handicap (3), orinaccessibility due to presence of the health facilityin the war-zone (10).

The questionnaire was prepared in English, andinquiring about general information of the respondingdoctors and their qualifications, number of patientsseen and treated in the last 12 months. Questionsabout the knowledge of the symptoms of pulmonarytuberculosis (PTB) were regarded correct if theyidentified 3 of 6 major symptoms of PTB mentionedin the NTP Guidelines. The knowledge of diagnosticprocedures was regarded correct if they rankedsputum microscopy higher than X-ray and otherlaboratory examinations. The regimen for smearpositive PTB cases was regarded correct if followingthe NTP guidelines (2HREZ/4HR). They were alsoinquired about supervision of the drug taking, tracingof patients who missed appointments, tracing offamily contacts, and notification of their TB patients.The questionnaire was pre-tested and modifiedaccording to the results of the pilot phase. Datacollection was done by two doctors. All towns werevisited except Lasanod, which could not be reachedfor security reasons.

Data management and analysis The twointerviewers scrutinized each other's fil ledquestionnaire before data entry. Data entry was donein Epi-Info version 6.04 by a team of 2 doctors anda data-clerk. Data entry validation was done. Cleaningof data was done before analysis using descriptivestatistics. The potential risk factors for sub-optimalknowledge of symptoms and diagnosis were identifiedby computing their relative risks and corresponding 95% confidence intervals.

Ethical considerations Ethical clearance wasobtained form the Ministry of Health. Written informedconsent was obtained from each doctor before theinterview, and the collected information wasconfidential.

Main study findings Of 100 registered medical practitioners, only 53

doctors could be interviewed. Seventeen medicalpractitioners reported working solely in the privatesector, 7 in the public sector and 29 in both.

Sixty percent of the respondents had treated atuberculosis patient within the last 12 months, 66%indicated a correct combination of symptoms, and60% indicated the correct diagnostic tests, but only1 doctor had submitted notification to the NTP. Only7% of the doctors were able to define the correct

regimen for tuberculosis patients; 45% prescribedanti-tuberculosis drugs on a daily basis, 13% weeklyand 32% monthly. DOTS was performed by only13% of the doctors.

The majority of doctors (71.7%) indicated that PTBwas suspected if the duration of a cough was 4 weeksor more, 11 doctors (20.8%) indicated that patientswho missed their appointments should be traced,and 6 would try to look for tuberculosis among thecontacts of the patients. Only 2 reported that theyhad submitted a report to the NTP after treatingtuberculosis patients.

Practising in the private sector was a significantpredictor for suboptimal knowledge of diagnosis ofPTB compared to practicing in both private and publicsectors (RR 2.1, 95% CI 1.1-4.3).

Conclusions and recommendationsA considerable proportion of tuberculosis patients

are managed in the private sector and few doctorsfollow the guidelines of the National TuberculosisProgramme of Somalia. Training of medicalpractitioners in the diagnosis and case managementof tuberculosis patients is needed to improvetuberculosis control in the north-west zone of Somalia.Distribution of the NTP guidelines to all medicalpractitioners in this zone was also recommended.

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AbstractA cross-sectional study was carried out in

2001 among a sample of 245 registeredprivate medical practitioners in Rawalpindiand Lahore, Pakistan. The aim of the studywas to assess the knowledge and practicesof private medical practitioners in the diagnosisand management of pulmonary tuberculosisusing a structured questionnaire.

Results Fewer than 1% of private medicalpractitioners are aware that coughing for morethan 3 weeks is the main diagnostic symptomsuggesting pulmonary tuberculosis (PTB),and that sputum microscopy is the diagnostictest for a PTB patient. And in spite of the factthat very few doctors have sputumexamination facilities at their clinics and areunable to deliver drugs in their private practice,only a small proportion refer patients to publictuberculosis centres.

None of the private medical practitionersfollow national tuberculosis control guidelinesin prescribing drugs, and a majority admit aninability to categorize a PTB patient fortreatment.

Furthermore, none ensure the intake ofanti-tuberculosis medicines under thesupervision of a doctor or a health worker.Most of them ensure this by personalcounselling, clinical assessment and throughrelatives, and the outcome of treatment ismainly determined by clinical assessmentrather than by sputum microscopy.

Very few private medical practitioners aremaintaining records of pulmonary tuberculosispatients or are trying to contact non-compliersor defaulters. Contacts tracing is performedby enquiring about symptoms instead oftuberculin testing.

Conclusion Private medical practitionersare not following national tuberculosis controlguidelines in Pakistan in diagnosing, treatingand conducting follow-up of pulmonarytuberculosis patients. The study revealed theneed to subject the private sector tocontinuous training on the national tuberculosiscontrol guidelines, as well as the need formonitoring during the whole process ofdiagnosis, treatment and follow-up by theNational Tuberculosis Control Programme.

BackgroundIn spite of the fact that the private health

sector has grown considerably in the lastfew decades, information on the extentand role of the private sector intuberculosis care tends to be very sparse.Limited literature is available, especiallyfrom Pakistan, on the subject ofknowledge and practices of privatemedical practitioners in the diagnosis,treatment and follow-up of pulmonarytuberculosis (PTB) patients. The aim ofthis study was to collect relevant andaccurate information regarding theknowledge and practices of the generalmedical practitioners of the diagnosis,treatment and follow-up of PTB patients,and to employ this baseline informationto plan future interventions in order toimprove tuberculosis care provided byprivate medical practitioners.

Is the private sectorfollowing the

national tuberculosisguidelines in the

diagnosis andmanagement of

pulmonarytuberculosis?

PakistanRawalpindi and Lahore

Study period:July–November 2001

Small GrantsScheme

(SGS) 2000 No. 10

Principal InvestigatorDr Syed Karam ShahNational TuberculosisControl Programme

Federal Ministry of HealthRawalpindi, Pakistan

Private medical practitioners are not following national tuberculosiscontrol guidelines in Pakistan in diagnosing, treating and conductingfollow-up of pulmonary tuberculosis patients. This leads toinadequate management of tuberculosis patients who remain adangerous source of infection in the community.

Only 1 out of 245 medical practitioners mentioned coughing formore than 3 weeks as the main symptom suggestive of pulmonarytuberculosis. Similarly, only 1 practitioner recommended sputummicroscopy as the basis for diagnosing tuberculosis. These arealarming findings indicating inappropriate case detection in theprivate sector. These two particular issues could be the maintheme of an awareness campaign for early case detection.

The private medical practitioners should be trained on the nationaltuberculosis control guidelines. Moreover, functional collaborationneeds to be established between private medical practitionersand the National Tuberculosis Control Programme to providequality tuberculosis care services. Finally, the overall process ofdiagnosis, treatment and follow-up of pulmonary tuberculosispatients in the private sector should be closely monitored.

TuberculosisP a k i s t a n


Quality of Care

126 127

Materials and methodsA descriptive cross-sectional survey was conducted

in 2 major cities in Pakistan, Rawalpindi and Lahore,to determine the knowledge and practices of privatemedical practitioners. The study subjects were thequalified medical graduates who were practisingmedicine on a full- or part-time basis outside thegovernment (public) sector. A comprehensive list ofall private medical practitioners was obtained fromthe medical representative of Wythe-Leaderle. Fivehundred and eighty two were in Lahore and 302 inRawalpindi. The basic criterion for inclusion in thestudy was that the private medical practitioners musthave provided cure to at least 1 PTB patient duringthe last year.

Out of the 884 private medical practitioners in thetwo cities, a sample of 245 was interviewed using asemi-structured study tool. The sample included 37postgraduates and 48 women. Most of them (86%)had provided treatment to 1-10 PTB patients duringlast 3 months.

Main study findings Less than 1% (1 out of 245) of private medical

practitioners are aware that coughing for more than3 weeks alone is the main symptom suggestingpulmonary tuberculosis. A combination of symptomswas mentioned by the rest of practitioners as themain symptoms.

The majority (81%) self-diagnose the patientsand only a small proportion refer them to theTuberculosis Centre.

Less than 1% of private medical practitionersperform sputum microscopy alone in order to diagnosea suspected case of pulmonary tuberculosis.

None of the private medical practitioners take thehistory of any previous anti-tuberculosis treatment.Most of them give priority to family history and socio-economic status.

The majority (96%) of private medical practitionerscannot categorize a PTB patient for treatment.

None of the private medical practitioners followthe national tuberculosis control guidelines inprescribing drugs. The majority (69%) give a fixed-dose combination (FDC) of 4 drugs, and 27% give3 to 4 separate drugs initially for 2 to 6 months. Duringthe continuation phase, 42% give a FDC of 4 drugs,and 29% give a FDC of 3 drugs. The majority (89%)give these combinations until the patient is cured.Ninety seven percent of private medical practitionersonly write prescriptions to PTB patients becausedrugs are unavailable in their facilities, 62% prescribeon a fortnightly basis, and 31% on a monthly basis.

None of the private medical practitioners ensurethe intake of anti-tuberculosis medicines under thesupervision of a doctor or a health worker. Most ofthem ensure this by personal counseling, clinical

assessment and through relatives. Only 3% maintainrecords of PTB patients.

None of the private medical practitioners assessthe effectiveness of anti-tuberculosis treatmentthrough sputum microscopy alone. The majority (76%)evaluate the outcome of treatment only clinically.

Only 2% of private medical practitioners try tocontact a patient on anti-tuberculosis treatment ifs/he does not return.

Less than 3% of private medical practitionershave sputum examination facilities at their clinics.

Almost all private medical practitioners find itsufficient to enquire only about the symptoms of theclose contacts of PTB patients.

The majority of private medical practitioners admitthat they do not know about the national tuberculosiscontrol guidelines, and are willing to attend trainingon the national tuberculosis control guidelines, ifprovided.

Conclusions and recommendationsPrivate medical practitioners are not following the

national tuberculosis control guidelines in diagnosing,treating and conducting follow-up of pulmonarytuberculosis patients in Pakistan. Training of privatemedical practitioners in the in the diagnosis and casemanagement of Tuberculosis pat ients isrecommended.

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AbstractA cross-sectional survey was conducted

on private sector physicians in Teheran,Mashhad and Isfahan. A total of 1079randomly selected physicians completed astandardized questionnaire. The study aimedat determining the knowledge and practicesof private sector physicians, both generalpractitioners and specialists, regarding thediagnosis and management of pulmonarytuberculosis and the adherence of privatesector physicians to the National TuberculosisProgramme (NTP) guidelines. This studywould enable decision-makers to chooseappropriate interventions for more efficientinvolvement of private sector physicians intuberculosis control.

Results Most of the physicians (87.3%)stated sputum smear microscopy as thediagnostic method. Among all privatephysicians, the correct regimen for newpulmonary tuberculosis (combination, dosage,duration) was stated significantly lessfrequently than other answers. Fewer thanhalf of the study subjects who treat the patientsthemselves stated the correct regimen.General practitioners (GPs) had attendedmore tuberculosis training courses than had

specialists. Private physicians who participatedin the courses had more correct knowledgeof the NTP guidelines and DOTS, and weremore aware of the availability of freetuberculosis diagnosis and treatment facilitiesin the public health system, than those whodid not.

Conclusion The recent introduction oftraining courses for private physicians in Iranhad a beneficial impact on their practices.

BackgroundOne of the main factors hampering

efforts in the fight against tuberculosis isthe lack of cooperation and coordinationbetween the National TuberculosisProgramme (NTP) and the private healthsector. It is believed that most tuberculosispatients init ial ly consult privatepractitioners. This issue is especiallyimportant in large cities. Althoughtremendous efforts have been made toexpand the primary health care networkin Iran, health services in large cities aremainly delivered by the private sector.Tuberculosis patients as well as othersare mainly managed by private generalpractitioners (GPs) and specialists inthese urban areas of the country. In spiteof this fact, the knowledge and practicesof private physicians have not beendocumented through proper studies inIran. Therefore, this study was conductedto evaluate the knowledge and practicesof private sector GPs and specialists inthree large cities of Iran in order to providebaseline information for planning futureintervention programmes.

Materials and methodsA cross-sectional survey was conducted

among private sector physicians whowere randomly selected from a list ofprivate sector physicians (GPs andspecialists) prepared by the authoritiesof the Medical Councils. These physicians

Is the private sectorfollowing the

national tuberculosisguidelines in the

diagnosis andmanagement of

pulmonarytuberculosis?


Teheran, Mashhad andIsfahan


Small GrantsScheme

(SGS) 2000 No.11

Principal InvestigatorDr Mohammed Reza

ShirzadiNational Tuberculosis

ProgrammeMinistry of Health and Medical

EducationTehran, Islamic Republic of Iran

e-mail:[email protected]

Tuberculosis


Quality of Care

Compared to other countries, a significantly higher proportionof private practitioners in Iran follow NTP guidelines in the diagnosisand management of PTB patients. This was mainly attributed tothe recent introduction of tuberculosis training courses in theircontinuous education. More specifically, good practice was alwaysassociated with attendance at tuberculosis control workshops.

Forty-Two percent of the private sector physicians have attendedtraining courses organized by the Communicable Disease ControlUnits of the health system at the national or provincial level or inthe universities. Course attendance was significantly higher amonggeneral practitioners than among specialists. These resultsemphasize the need for regular training of private physicians inthe diagnosis and case management of tuberculosis in order toimprove tuberculosis control in Iran.

Establishing functional collaboration between private medicalpractitioners and the National Tuberculosis Control Programmewas the main study recommendation.

I s l a m i c R e p u b l i c o f I r a n

were working in the three largest cities of Iran, eachhaving a population exceeding 1.5 million: Teheran,Mashhad, and Isfahan. Three hundred and eighty,356, and 343 physicians, recruited from Teheran,Mashhad, and Isfahan, respectively, completed thequestionnaires. They were randomly selected fromtwo strata of GPs and specialists through their namesand the Medical Council Numbers List, and thismethod allowed the subjects to be proportionallydistributed over the different graduate years of thepractising physicians. Considering the fact thatdrawing conclusions from certain specialities is notrelevant to the impact of pulmonary tuberculosis(PTB) training courses, the results focused on GPsand relevant specialities such as paediatrics, infectiousdiseases, internal medicine and pulmonary medicine.A total of 732 private physicians were included in thestudy (599 GPs and 133 specialists).

Data collection was performed through a pre-testedstandardized questionnaire that included informationabout the knowledge and practices of practitioners.This was defined as the agreement of the studysubject's knowledge and practices with the NTPguidelines.

Since the majority of the physicians had attendedthe tuberculosis training course after the year 1997,the impact of these courses on their practices wasalso evaluated.

Main study findingsForty-Two percent of the private sector physicians

had attended training courses organized by theCommunicable Disease Control Units of the healthsystem at the national or provincial level or in theuniversities. Course attendance was significantlyhigher among GPs than among specialists.

Out of 712 physicians, 47 (6.6%) did not mentioncoughing as one of the main manifestationssuggestive of pulmonary tuberculosis. Most of theGPs who attended the tuberculosis training coursesmentioned a cough duration of 3 weeks to besuggestive of PTB, compared to more than 4 weeksamong the specialists. Moreover, a higher proportionof GPs recognized sputum microscopy as the prioritydiagnostic test compared to specialists. Thisknowledge was significantly associated with thehigher course attendance by GPs over specialists.

Once tuberculosis was confirmed, 82.1% of privatephysicians referred their patients to public tuberculosiscentres.

Half of the physicians stated the correct drugcombination and drug dosage. Drug combinationsand treatment duration knowledge was significantlyhigher among GPs than among specialists. Theproportion of the physicians who stated the correctdrug regimen and attended Tuberculosis ControlWorkshops was greater than that of those who had

attended the course in other institutions. However,among all physicians, the correct regimen was statedsignificantly less frequently than other answers.

Half of the specialists who treat the patientsthemselves stated the correct regimen compared to35% of the GPs.

Regarding the frequency of anti-tuberculosis drugdispension/prescription for smear-positive patients,most dispense on a monthly basis (41.1%). Dailydispensing was performed in 22% of patients.Physicians who attended the Tuberculosis ControlWorkshops tended to dispense/prescribe on a dailybasis. On the other hand, those who attended thecourses in the universities or through the continuingeducation programmes tended to dispense/prescribeon a monthly basis.

Almost all of the practising physicians ensure theconsumption of all dispensed/prescribed drugs. Theobserver assignment method was more frequentlypractised among GPs while the paraclinical methodswas most frequently used among specialists. Theuse of the observer assignment method wassignificantly associated with course attendance,especially of the Tuberculosis Control Workshops.

Documentation of patient information was performedby 78% of private physicians. The majority of privatephysicians use sputum microscopy to assess theeffect of treatment, most have attended theTuberculosis Control Workshops, and mostrecognized the correct frequency of sputumexamination. Moreover, physicians who attended thecourses refer more of their patients to public healthsystem laboratories. Almost all private physicianswere aware of the availability of free services.

Conclusions and recommendationsTraining courses, particularly Tuberculosis control

workshops, had a benificial impact on the quality ofcare delivered by private physicians to tuberculosispatients.

These results emphasize the need for regulartraining of private physicians on the diagnosis andcase management of tuberculosis. More importantly,functional collaboration needs to be establishedbetween private medical practitioners and the NTPto provide quality tuberculosis care services, therebydecreasing the burden of tuberculosis in thecommunity.

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