5. CHAPTER-II

EVPILUATION OF E. GRAA'LrLOSUS RECOMBINANT

TIGE GENS IN IMMUNODIAGNOSIS OF CYSTIC

ECHINOCOCCOSIS IN HUMANS

Chapter 11

Early diagnosis and treatment of CE is most important for management of the CE

(Amrnann et a]., 1990). The diagnosis of CE based on clinical grounds is frequently

difficult. D~agnosis of CE is primarily based on imag~ng techniques and serological

methods (Pawlowski et al., 2001). The diagnosis of CE by imaging methods is too

expenstve and inaccessible in most endem~c areas. The interpretation of results by imaging

methods also found to be difficult in case of small leslons andlor atypical Images

(Pawlowski et a]., 2001). Therefore, reliable and economtcal nnmunological assays are

most useful for diagnosis of CE (Parija, 1994).

The serodiagnosis of CE based on serum antibody or antigen detection IS of immense value

to c o n f m clinical findings or render diagnostic help in radiologically unclear cases

(Cottstein, 1992). Earlier, several nu nu no logical methods have been developed and

evaluated for the detect~on of hydat~d ant~bodies In serum wlth varying sensitivity and

apec~ficlty for the diabvosis of CE. These serodiagnostic methods ~nclude; compliment

fixation test (CFT) (Mahajan et al., 1976). agar-gel-precip~tation test (Mahajan et al.,

1976), bentonite flocculation test (Mahajan et a].. 1973; Rotros et a].. 1975).

~mmunoelectrodifhs~on (IED) (&chard-Lenohle et al , 1978). ~ n d ~ n c t ~mmunofluorescent

test (IIFT) (Mahajan et al.. 1976) and rad~oimmunoassay (RIA) (Richard-Lenoble et al.,

1978), indirect haernagglutination (IHA) test (Mahajan et a]., 1973; Picardo and Gutsantes,

198 1; Parija and Ananthaknshnan, 1985; Parija et a]., 1986; Parija and Rao. 1987; Todorov

et L;., 2003), rapid H A (Parija and Rao, 1986; Parija et a]., 1987). latex agglutinat~on test

(LAT) (Picardo and Guisantes, 1981) and countercurrent immuno electrophores~s (CIEP)

(Mahajan et a]., 1976; Picardo and Guisantes, 1981; Parija, 1991).

The enzyme immunoassays evaluated for detection of hydatid antibodies in serum mclude,

IgGenzyme-linked immunosorbent assay (ELISA) (Kanwar and Vinayak. 1992; Kaur et

a]., 1999), IgM-ELISA (Wattal et al.. 1986), IgE-ELISA (Wattal et al., 1988) and enzyme

linked ~mmuno-transfer blotting (EITB) (Maddison et al.. 1989, Siracusano et a]., 1991;

Leggatt et a]., 1992; Crag, 1993: Porett~ etal., 1999).

ELISA has been used more frequently than other serological tests for diagnosis of CE

because of its high sensitivity in CE (Force et al., 1992; Kaddah etal., 1992; Ramzy et a].,

1999; Kaur et a1.,.1999). However, ELlSA is a technically cumbersome procedure, needs

Chapter I1

technically sLilled personnel and could be camed out only m well equipped laboratones.

Therefore, a rapid Dot-ELISA was developed for detection of hydatid antibodtes m serum

for diagnosis of CE wlth a sensitivity of 94% and a specificity of 90.3% (Rogan et al.,

1991). The Dot-ELISA was found to be rapid, inexpenswe and simple to perform and

considered to be a useful supplement to the US scanning (Rogan et al.. 1991).

Recently, ElTB has been increasingly used In the d~agnosis of CE (Maddison et a]., 1989;

Stracusano et al., 1991; Leggatt et al., 1992; Craig, 1993; Poretti et a]., 1999). The ElTB

usmg native antigens has been reported to show hrgher sens~tivlty and specific~ty than :lie

ELSA for detection of antibody in serum (Leggatt et al., 1992; Cra~g. 1993; Porettl et al..

1999). The EITB using hydatid cyst fluid antigen, showed a sensitivity of 90% and a

spec~ficity of 100% for diagnosis of hepatic hydatidosis, but it was less sensitive for

detection of uncomplicated cysts located In the lung and brain (Maddison et al., 1989). In

another study, the EITB uslng E. granulosus antigens showed a sensltlvlty of YO%, as

compared to 78% w~th the immunoelectrophoresis (Siracusano et al.. 199 I). However, the

low sensitivity and low spec~fic~ty have been the major problems associated with the

sero&agnostic tests employed for detecting hydatid antibodies for diagnosis of CE

(Shepherd and McManus, 1987).

Detection of hydatid antlgens in serum, unne, hydatid cyst fluid and other body flulds is

another approach for diaglosis of CE. These include, rapid and simple immunoassays for

detection of hydatid antigen In serum by ClEP (Shariff and Parija, 1991), co-agglutrnation

(Co-A) test (Shanff and Partla, 1993). LAT (Sheela Devi and Panja, 2003); and Ag-

ELlSA (Craig et al., 1986, Kanwar et al., 1994) and Ag-Dot-ELISA (Wang et al., 2002).

'These also include antigen detect~on in unne by ClEP (Panla et al., 1997) and Co-A test

(Ravinder et al., 2000), and antigen in the hydatid cyst fluid aspirates by Co-A test (Panja

et al., 1996), LAT (Sheela Devi and Parija, 2003). CIEP (Ravinder and panja, 1997) and

reverse latex agglutination (RLA) test (Mahmoud and Ezzat, 1999).

Despite the development of different techniques with variable sensitivities and specificities

the lmmunodiagnosis of CE in clmical practice still remains a diagnostic challenge. This is

mainly due to the use of native hydatld antigens in various serlogical tests (Babba et al.,

19~4). The false-pos~t~ve (non-specific) reactions with sera from other parasitic diseases,

malignancies (Dar et al., 1984). and liver curhosis (Iacona et al., 1980) is a noted problem

associated with these serological tests. The analysis of E. granulosw. native antigens by

using one-dlmensionavtwo-dimensional electrophoresis and micro-sequencing by Zhang

and McManus, (1996) have also suggested that both antigen B (A@) and antigen 5 (Ag5)

components of HCF are composed of a family of pmtems, but not a single protein. This

Indicates the possibility of cross-reactions in the diagnos~s of CE using native antigens.

Therefore. E granulosur recombinant antigens (Helbig et al.. 1993; Femandez et al.. 1996;

Gonzalez-Sapienza et al., 2000; Ron et al., 2000; Kittelberger et al.. 2002; Virginlo n al.,

2003; Li et al., 2003; Lorenzo et al., 2005a) and synthetic peptides (Facon et al.. 1991;

Chamekb et al., 1992; Gonzalez-Sapienza et al., 2000) have been evaluated in many

~mmunod~agnostic tests to enhance specificity as well as sensitivity of these tests .

More attention has been focused on the development and evaluation of ~mmunodiagnostic

assays using E. granulosus recombinant AgB fractions for diagnosis of CE both in humans

and animals. The E. granulosus recombinant AgB fractions werc found to be h~ghly

lmmunogenlc and specific in diagnosis of CE (Lormzo et al., 20058). The recombinant

A f i fractions, EGSS and EM 10, of the E. grunulosus and E, rnulrilorulan.s metacestodes.

respectively, were Sound to be species spec~fic for serodiabmos~s and serolog~cal

d~fferentiatlon of CE and alveolar echlnococcos~s (AE) (Helbig et al., 1993). However. In

t'ew cases, the E grunulouus recombinant AgB fractions also appeared to be genus

specific, but not species spec~fic (LI et al., 2003). Only few stud~es evaluated the

immunodiagnostic assays using E gronulosus recombinant Ag5 (Facon et al., 1991;

Gonzalez-Sapienza et al., 2000), probably because of its low specificity (Facon et a]..

1991).

Rccently, two E. grunu1osu.s recombinant antigen B fractions including, Rec Eg-AgBBII

and Rec Eg-AgB812 were found to be the most efficient and relavent diagnostic antigens

for diagnosis of CE (Virginio et al., 2003). The ELlSA using Rec Eg-AgB8ll and Rec Eg-

AgB812 demonstrated that Rec Eg-AgB8I2 provides better performance In serodagnos~s of

human CE than does the Rec Eg-AgBBIi (Rott et al., 2000; Virginio et al., 2003).

Subsequently, Li et al. (2004) ~dent~fied and evaluated a recombinant antigen, EpCI, for

dragnosis of CE. The EpCl recombinant antlgen showed a sensitivity of 92.2% and a

specificity of 95.6%.

In the fust phase of the present study, two diagnostic relevant recombinant antigens, Eg-

crV12 and Eg-rCW24, were identified by immunoscreen~ng of the ZAP Express cDNA

ltbrdl~ obtained from E. grunulosus metacestode germ~nal membrane mRNA. Both

recombinant antigens, Eg-rCWI2, Eg-rCW24, showed 100 % sensitivity and specificity in

L""ial screening by ELISA and EITB as described earlier in the chapter 1. In the f i s t phase

of the present study, Rec Eg-AgB812 was also produced by cloning and expression of

corresponding genomic DNA fragment. Both ELISA and EITB using Rec Eg-AgB812 also

demonstrated 100 % sensitivity and specificlty in initial screening.

merefore, in the second phase of the present study two simple, rapid and reliable

serodiagnostic assays such as ELlSA and Dot-ELISA were evaluated by using the E

Lmnulosus recombinant antigens. Eg-rCW24. Eg-CWIZ and Rec Eg-AgBXIZ, for

dltection of hydatid ant~hodies in serum for diagnosis of CE.The polyclonal antibodies

rnlsed against E, granulosus recomb~nant antigens, Eg-rCW24. Eg-rCWI2 and Rec Eg-

AgBX12, were also evaluated for use in ELlSA and Dot-ELISA for detrection of hydatid

antigen in the serum.

The objectives of this phase of the study are:

I. To develop and evaluate enzyme-linked imrnunosorbent assay (ELISA) and Dot-

enzyme linked inmunosorbent assay (Dot-ELISA) usmg E. granulosus recombinant

antigens (Eg-rCW12, Eg-rCW24 and Rec Eg-AgBBR) to detect hydatld ant~bodies in

serum for diagnosis of cystic echinococcosts

2. To develop and evaluate ELISA and Dot-ELISA using affinity punfied anti-Eg-

rCW12, anti-Eg-rCW24 and anti-Rec Eg-Ag8812 antibodies to detect hydatid

antigens in serum for d~agnosls of cystic echmococcosis.

Chapter II

shldy Groups

Group-I: Surgically conjirmed and ultrasound drugnosed cysfic echinococcosis (n=100)

71us group included 45 cases of surgically confirmed cases of CE and 55 cases of

radiological confmed cases of CE. The cysts removed during surgery were confmed to

be of hydatld cyst etiology by histopathological evidence of germinal layer and by

demonstration of scolices and hooklets in the asptrated HCF. The rad~ologically-diagnosed

CE cases included un-operated cases of CE but diagnosed by ultrasonography

(US)lcomputed tomography (CT)/magnet~c resonance imaging (MRI)

Group-II: Presumptive or suspecled cases of cystic echinococcosis (n=60). This group

lncluded clinically diagnosed (presumpt~ve) cases of CE. These patients presented with the

cllnical signs and symptoms of CE but they are proved neither surg~cally nor by radio

diagnosis.

Group-III: Conrruls wirh other puru.rrtrc di~eu.se.s (n=60): T h ~ s grcup Included patients

uwth other parasltlc diseases like amoeblasls (n=8), filanasis (n=IO), neurocyst~cercosis

(n=30), toxoplasmos~s (n=9) and sch~stosom~asis (n=3).

Group-IV: Henlrhy control^ (n=30). Th~s group Included healthy adults (blood donors and

students) who have not suffered from CE or any other dlsease In the recent past.

Specimens

Serum

F~ve milliliters of venous blood was collected from all patients (n=220) and healthy

lnd~viduals (n=30) under aseptlc precautions and was allowed to clot. The serum was

separated and stored in allquots at - 2 0 ' ~ till use.

Preparation of E. granulosus recombinant antigens

The E granulosus recombinant antigens Eg-rCWI2, Eg-rCW24 and Rec Eg-AgB812, were

synthesized and purified es described earlier (Chapter-I).

Chapter I1

preparation of polyclooal antibodies against E. granulosus recombinant antigens In

rabbit

polyclonal antibodies were ra~sed in rabbits against each E, granulosus recombinant

antigen (Eg-CWIZ and Eg-CW24 and Rec Eg-AgB8iZ) as described earlier (Chapter-1).

Cbromatographic purification of total IgC antibodies from rabbit s e n

The anti-Eg-rCWI2, anti-Eg-rCW24 and anti-Rec Eg-Am812 IgG antibodies were

pnfied by using commercially ava~lable protein A-Sepharose CL 48 column (Gene',

brdia) as per the method described by Choromansky et al. (1990).

Bnetly, the pH of the each serum was adjusted to 8.0 by adding 1.0 M Tris drop by drop.

The antibody solution was passed through the pre-packed protein A-Sepharose CL 49

column (Genei. India). The column was washed sequentially with approximately 500 p1 of

100 m M glycine (pH 3.0) per sample in stepwise. The IgG fraction was analyzed by

reading at 280 nrn m a spectrophotometer (Sysfronics, India). Analysls of IgG fraction was

done ln the elute, wash a d flow-through. Finally, the elute was subjected to SDS-PAGE m

order to estlmate the purification process. Sunilarly the wash and the flow-through were

also subjected to SDS-PAGE. The affin~ty punficd IgG antibod~es were concentrated by

ultra membrane filtrat~on using Centricon concentrator tubes of 3 kDa cut-off (Amicon.

LrSA).

Detr.?tion of bydatid antibodies in serum

ELISA for detection of hydarid antibodies in serum

Ibe ELISA was evaluated by using E. granulosus recombinant antigens Eg-rCW12, Eg-

CW24 and Rec Eg-AgB812 separately for detection of QG antibodies m serum from cases

of CE and control subjects. All tests were performed three tlmes to exclude vanations from

day to day.

Srandardization of ELISA: The optimum antigen concentration, for coating mi~r~ t i t e r

wells, and serum dilution was standardized by checkerboard titration. The concentration of

mtigen and the dilution of semm, which gave the maXlmM ratio of ODIW values wlth

known positive serum and that with negative serum, were considered to be the optimum

conditions for the test. ~ l l the test sera were analyzed at 1:ZOO dilutions in PBST.

ELISA using E. grnnulostrr recombinant antlgens for detection of IgG antibod~es in selum

consisted of the following steps:

1. Anligen coating: Polyvinyl high binding microtiter plates (NUNC. New Zealond)

were coated w~tb 100 pl/well of appropriate dilution of the E. granulosus

recombinant ant~gen (0.5 pg/well) In carbonate bicarbonate buffer (pH 9.6) and

incubated overnight at 4% undisturbed.

2. Washing: The un-adsorbed antlgen was removed by washing the plates with PBST

(sterile PBS (pH 7.2) containing 0.1% Tween-20).

3. Blocking: The uncoated reactive sltes in the wells were blocked using PBS (pH 7.2)

containing 2% BSA by incubating far 3 h at 37 '~.

4. Washing: Plates were washed three times wlth PBST as descnbed before.

5. Sample serum dilutron und rncubuiion: Appropriate dilution of the patlent sera

(1:200) was prepared In PBST and 100 p1 of each dlluted scmm was added In

duplicate and incubated for 1.5 h at 3 7 ' ~ .

6. Washing. The plates were washed three tlmes w~th PBST as before to remove

unbound antibodies In serum.

7. Second antibody (conjugate) incubation: Rabb~t anti-human-IgG-HRP conjugated

second antibody (Genei. Indiu) was used as per the manufacturers insmctlon

(1.2000) with PBS (pH 7.2) containing Tween-20 (0.05%) and 100~1 volume was

dispensed to all wells and rncubated for 0.5 b at 37% in dark.

8. Washing: Plates were washed three tlmes w~th PBST as before to remove unbound

conjugate.

9. Colour development: Substrate solution was prepared freshly by add~ng 6 mg of

OPD (S.D. Fine Chrrnicols. bdra) in 10 ml of PBS (pH 7.2) containing 0.05%

Tween-20 and 10 of pl H202 was added just before adding to the wells. Finally. 100

pllwell of the substrate solut~on was dispensed to all the wells and ~ncubated for 15-

20 nun at 37°C in dark.

10. Stop reaction The reaction was stopped by adding 50g1 of 2N HISOI per well. The

absorbance value was recorded at 492 nm using ELISA reader (Bio-Rad USA).

The same ELISA procedure was performed by using Eg-rCW12, Eg-rCW24 and Rec Eg-

AgB812 for detection of hydatid IgG antibodies in the same batch of serum samples from

Cases of CE and control groups as mentioned earlier. Various controls such as antigen

blank, fmt antibody blank, conjugate blank, and substrate blank, healthy normal serum

Chapter I1

blank were used during the standardization of the ELISA. Antibody blank, positive contml

senun and negative control senun were used in every run for the validation of the test.

Dot-ELISA for detection of hydatid antibodies in serum

The Dot-ELISA was evaluated by using E granulosus recombinant antlgens Eg-rCWI2,

Eg-KW24 and Rec Eg-AgB812 separately for detectton of hydatid antibodies in in the

same batch of sera from cases of CE and control subjects. The Dot-ELISA was optimized

to est~mate the concentration of antigen to be dotted and the dilutlon of serum to be used

by checkerboard titration. All tests were performed three times to exclude vanattons from

day to day.

Dot-ELISA using E granulosus recombinant antlgens for detection of hydatld antibody

consisted of the following steps:

I. Antigen sporting: A nitrocellulose membrane WCM) (Hybond ECL, Amershum

bioscience. Germuny) of size 0.5 x 0.5 cm was cut and mounted onto a plastic stnp

(0.5 cm x 5cm). A volume of Zpllstrip (0.2 pustrip) of antigen was dotted on to

Indimdual strip and air dried for 30 min.

2 Whshing: The un-adsorbed antigen was removed by wash~ng the strips with PBST

(ster~le PBS (pH 7.2) contaming 0.1% Tween-20).

3. Blocking: The uncoated mactlve sltes on the strips were blocked wlth PBS (pH 7.2)

containing 2% BSA by incubating for 3 h at 37"c wlth constant shaklng.

4 Washing: Strips were washed three times w~th PBST as described before.

5. Sample serum dilutton and incubation: A 1:1000 dilution of the patlent sera was

prepared in PBST and the strips were incubated for 1.5 h at RT with constant

shalung.

6 Washing: The strips were washed three times with PBST as before to remove

unbound antibodies in the sample serum.

7. Second antibody (conjugate) incubation: A 1:2000 dilution of rabblt anti-human-

IgG-HRP conjugated second antlbody (Genie. Indza) was used as per the

manufacturers' instructions with PBS (pH 7.2) containing Tween-20 (0.1%) and

Incubated for 30 min at RT in the dark with constant shaking.

8 . Washmg. Strips were washed three times with PBST as before to remove unbound

conjugate.

9. Colour development: Substrate solution was prepared freshly by adding 3 mg of 3.

3' diaminobemidine (DAB) (Sigma. USA) in 5 ml of PBS (pH 7.2) containing 0.1%

Chapter I t

Tween-20 and 5pl Hz02 was added just before adding to the plastic tray. The stnps

were dispensed and incubated for 15-20 min at 37'c m dark under constant shaking.

10. Stop reocfion: The reactlon was stopped by washing the strips with double distilled

water.

The posltive reactlon was mdicated by the development of brown coloured spot In 2-5

min. When the reaction was complete, the strips were washed with distilled water and

allowed to dq'. Once dried, the strips were stored m the dark.

Detedion of hydafld antigens In serum

ELISA for detection of hydotid antigen in serum (Ag-ELISA)

Srundordimtion of Ag-ELISA: The optlmum concentration of capturing ant~hody (affinity

prified polyclonal antibodies ra~sed agalnst E granulosus recomblnant cyst wall antigens)

and optimum dilution of the detecting antibody (hyperimmune polyclonal antiserum ra~sed

against E. granulosus recombinant antigens) were initially standardized by checkerboard

laration.

The Ag-ELISA for detection of hydatid antigen in serum consisted of the following steps:

I . Capturing unfibody coating: Poly-finyl high binding microliter plates (NUNC. New

Zealand) were coated with 100 pIfwell(4 concentrations of 18 pg, 28 pg, 48 pg and 8

;g/well) of the affmity purified antibody (antl-Eg-CWI?, anti-Eg-rCW24 and anti-

Rec Eg-AgB812 antibodies) In carbonate bicarbonate buffer (pH 9.6) and incubated

overnight at 4 ' ~ undisturbed.

2. Wushmg. The un-adsorbed antrbodles were removed by washlng the p k e s with PBST

(sterile PBS (pH 7.2) containing 0.1% Tween-20).

3. Blocking: The uncoated reactlve sites in the wells were blocked by PBS (pH 7.2)

containing 2% BSA by incubating for 3 h at 37'C.

4. Washing: Plates were washed three times with PBST as before.

5 . Dzlution and incubation of E granulosus recornbmuni antigens: Known quantities of

E granulosur recomblnant antigens (Eg-rCW12. Eg-rCW24 and Rec Eg-AgB812)

were prepared in PBST. A concentlation of 400 ngilOO pI of each diluted antigen was

added in duplicate and incubated for 1.5 hat 37%.

6 . Washing: The plates were washed three times with PBST to remove unbound antigen

molecules.

Chapter N

7 . Detecting antibody: Polyclonal hypenmmune mum was diluted by serial dilution

(1:25 to 1:3200) in PBS @H 7.2) and 100 @I volume was added to each well.

8. Washing: The plates were washed three times wrth PBST to remove unbound detecting

antibodies.

9 Second antibody (conjugate): Goat anti-rabbit-IgG-HRP conjugated second antibody

(Genei. India) was used as per the manufacturers instruction (1:2000) with PBS (pH

7.2) containmg Tween-20 (0.05%) and 100 p1 volume was d~spensed to all wells and

incubated for 30 min at 37% in dark.

10 Washing: Plates were washed three tlmes with PBST to remove the unbound

conjugate.

I I. Colour development: Substrate solution was prepared freshly by adding 6 mg of OPD

(SD. Fine Chemicals, India) in 10 ml of PBS containing 0.05% Tween-20 and 10 p1

H202 was added just before adding to the wells. 100pl volume of the substrate solution

per well was dispensed to all wells and incubated for 15-20 min at 3 7 ' ~ in dark for the

development of optimum colour.

12. Stop reaction: The reaction was stopped by adding 50pl of 2N H,SO, per well

13. The absorbance values were recorded at 492 nm using ELISA reader (Bio-rad, USA).

The same Ag-ELlSA procedure was performed by usrng antr-Eg-rCWI2, antl-Eg-CW24

and anti-Rec Eg-AgB812 monospec~fic antibodies for detect~on of hydat~d anttgens rn the

same batch of serum samples tiom cases of CE and control groups as mentioned earlier.

The microliter plates were coated wlth 4 pglwell of the capturing antibody (anti-Eg-

tiW12, anti-Eg-rCW24 and anti-Rec Eg-AgB812 antibodies). The test sera and detect~ng

antibody were used at dilutions of 1:100 and 1:200 respectively. Blank, posltrve control

serum and negative control serum were used In every run for the validation of the test All

tests were performed three times to exclude variations from day to day.

Estimation of antigen derecrion limit ofAg-ELISA

The Ag-ELISA using anti-Eg-rCW 12, anti-Eg-rCW24 and anti-Rec Eg-AgB812 antibodies

for verification of the antigen detection limit consisted of the following steps:

In atep-1 of the Ag-ELISA protocol as described above, the polyvinyl h~gh binding

mlcrotiter ELlSA plates were coated w ~ t h 100 j11 (containing 4 pg) of the affinity purified

antibody (anti-Eg-rCWI2, anti-Eg-rCW24 and anti-Rec Eg-AgBR ant~bodies) in carbonate

bicarbonate buffer (pH 9.6.). Step-2 to Step-4 was same as described in Ag-ELISA. ,

Chapter I1

In the Step5 of the above Ag-ELISA protocol, different concentrations (2 pg, I pg, 800

ng, 400 ng, 200 ng, 100 ng, 50 ng, 25 ng and 12.5 nglml) of each antigen preparation were

added. The plates were incubated for 1.5 h at 37%. The plates were washed for removing

unbound antigens (Step-6). In Step-7 of the above Ag-ELISA protocol, the polyclonal sera

(detecting antibody) at appropriate dilution (standardized by checkerboard titration)

estimated in the previous step were added. The plates were developed using anti-rabbit-

I&-HRP conjugate and substrate solution as described before (Step-8 to Step12 of the

A ~ E L I S A protocol).

Dot-ELISA for defection of hydatidanfigen in serum

The Dot-ELISA using antl-Eg-rCW12, anti-Eg-rCWZ4 and anti-Rec Eg-AgBBR

monospec~fic antibodies, was evaluated to detect c~rculatlng E, granulosus ant~gens In

serum of the cases with CE and controls. The Dot-ELISA was optimized to estlmate the

concentration of coating antibody to be dotted and the dilution of test serum to be used by

checkerboard titration All tests were performed three times to exclude variations from day

to day.

Dot-ELISA procedure cons~sted of the following steps:

1. Capturing antrbody spottrng: A NCM (Hybond ECL, Amrrshorn bioscit~nce,

Germanj,) of slze 0.5 x 0.5 cm was cut and mounted onto a plast~c strip (0 5 cm x

5cm). A volume of 2p1 (2 &strip) of affinity punfied IgG fraction of ant,-Eg-

rCWL2 IgG, anti-Eg- rCW24 IgG and anti-Rec Eg-AgB812 antibody was dotted on

to individual strips and air dr~ed for 30 min.

2. Washing. The un-adsorbed antibodies were removed by washlng the strips with

PBST (sterile PBS 7.2 contalnlng 0 1% Tween-20).

3 Blochng: The uncoated reactlve sites on the stnps were blocked uslng PBS (pH 7.2)

containing 2% BSA by incubating for 3 h at RT on rocker shaker.

4. Washing: Strips were washed three times with PBST as described before.

5. Sample semm drlutron and incubation: One ml of 1:10 dilution of the patient sera

was prepared in PBST and the stnps were incubated for 1.5 h at RT wlth constant

shaking.

6. Washmg: The strips were washed three times with PBST as before to remove

unbound antibodies In sample semm.

7. Second antibody (conjugate) mcubation: Rabbit anti-Eg-rCW12 IgG, anti-Eg-

rCW24 IgG and anti-Rec Eg-AgB812 IgG coupled to HRP was used at a dilution of

1:400 in PBS @H 7.2) containing Tween-20 (0.1%) and the strips were incubated

for 1 hat RT in the dark with constant shaking.

8. Wushing: Stnps were washed three times with PBST as before to remove unbound

conjugate.

9, Colour development: Substrate solut~on was prepared freshly by adding 3 mg of 3,

3' diaminobenzidine (DAB) (Sigma. USA) in 5 ml of PBS (pH 7.2) contaimng 0.1%

Tween-20 and 5 pl H202. The strips were dispensed and Incubated for 15-20 min at

RT in dark under constant shaking.

10. Stop reaction: The reaction was stopped by washmg the strips with double distilled

water.

The positive reaction was indicated by the appeamnce of brown coloured spot in 2-5

m~nutes. When the reaction was cumplete, strips were washed with d~st~lled water and

allowed to dry. Once dned, the strips were stored in the dark.

Statistical analysis

The sensit~vlty, specificity, posltive predictive value (PPV), negatlve pred~ct~ve value

(NPV), and effic~ency of the d~agnostlc methods were calculated as per the standard

method (Park and Park, 2001). Statistical analysis for test of s~gnificance (chi-square test)

for comparing the subgroups was done using the software Epi-info 2001 and Graph Pad

(Online version) softwares. "P-value" e0.05 was considered as significant. The mean,

median, standard deviation calculat~ons were perfonned using Windows Mlcrosoft Excel,

2002 software.

Chapter N

Cllnlcd proNe of the patients

The CE was more frequently observed In the age group of 40-60 years (46.87%) followed

by the age group 20-40 years (38.75%). The age group ranged from 41-60 years (m~ddle

age people) appeared to involve highest by CE lnfect~on in males (51.38%) and followed

by 2040 age group (29.16%). However. the age group 21-40 appeared to involve highest

(46.59%), followed by the age group 40-60 years (43 18%) respect~vely, in females. The

sex and age d~stnbutions among confirmed and suspected cases of CE were summarized In

Tabled-1.

Tables-1: Age and sex distribution of Group-I and Group11 cases of CE

In the present study. 83.00% confirmed CE cases and 75.00% suspected CE cases

presented with pain. 48.00% and 48.33% of cases presented with swelling, 24.00% and

?1.(5% of cases with vomit~ng. 24 00% and 33 33% of cases with we~ght loss, 19.00% and

31.66% of cases presented w ~ t h cough, 10.00% and 3.33% cases w~th hemoptysis out of

100 confirmed CE cases and 60 suspected CE cases respectively. A total 25 of 100

confirmed CE cases showed other complications such as cyst rupture m 5 (5.00%) cases, 7

(7 00%) cases w ~ t h calc~fied cyst, 5 (5.00%) cases with infected cyst. one cases cach of

renal calculus, carcinoma cervix, diabetes, Nberculosn, gallbladder calculus, hem~plegia,

pleural effusion, pregnancy termination and one (1.00%) cases with T versrcular. A total 5

of 60 (8.33%) suspected cases of CE showed other complicat~ons such as tuberculosis,

ascites, neck swelling, pleural efision and renal calculus.

The predilection of hydatid cysts towards different body p m s in confirmed CE cases and

In suspected CE cases was summarlzed m Table-5-2. The predilect~on of hydatid cysts

among the South Indian patients involved in this study, were found more frequently in

Chapter tl

76% of c o n f m e d and 41% of suspected CE cases in liver, followed by lungs (9% and

16%) respectively. Multl O r p location of hydat~d cysts was found in 7 % of confimed

and 1.66% of suspected CE cases.

Table-5-2: PredUectlon of hydatld cysts towards different body par ts in patlents wlth

CE

[Group-I: Casc.ier of CE with confirmed dlngnaals; Group-ll Cuer of CE with prrbumptivc diilgnos~r]

Site of the bydatid cyst

The occupational profile of the study cases were summarized In Table-5-3. The CE was

prevalent more frequent among the agricultural workers (about 41.41% and 43.33% of

confmed and suspected cases of CE respectively), followed by house wives (with a

frequency of 26.26% and 28.33% of confirmed and suspected CE cases respectively) and

public service undertakinglcorporate sector employees (22.22 % and 16-66 % of confirmed

and ~uspected C E cases respectively).

Table-5-3: Occupational profile of patients wlth CE

Group-1 No (%) (n-100)

No (% ("-159 - (n;99*) (;jo: I 0 A rieulture 41 (41.41) 26 43.33) 67 (42 13) PSUlcor rate sector 22 (22.22) 10 (16.66) 32 (20.12) House wife 26 (26.26) 17 (28.33 43 (27 04 Meebanidmnid 2 (2.02) 2 (3.33) 4 2.51) Butcher I 1.01) 1 (0 62) SLudent 7 7.07) 5 8.33) 12 (7 54 [Gwup-I: Cases of CE with confirmed dinanosir. Group-11: Cuer of CE wtth presumptive d~egnurir; PSU-

Group-11 No (%) (11-60)

Publrc s m c r undnraking; * One case with age less thnn 5 years1

Total number of cases

No (%) (11-160)

Total number of eases

1 Group-1 Group-I1 Number (%) / Occueation Number (%)

Detection of E. g r ~ n ~ l 0 ~ I d S IgG antibodies in serum by ELlSA

Standardization of ELISA

The optimum concentration of the purified recombinant anttgens. Eg-rCWI2. Ep-rCW24

and Rec Eg-AgB812, for coatlng mlcrotiter wells were estimated by checkerboard titration

to be 0.5 pg per well. The optlmum dtlution of serum for the ELISA ustng each of the

recombtnant antigen preparations for detection of IgG ant~bodies was standard~red by

checkerboard titration to be 1:200.

ELISA for detection of E. granulosus IgG antibodies in serum

'The cut-off OD492 values of the ELISA uslng E. granu[usus recombmant antigens (Eg-

rCWL2, Eg-rCWZ4 and Rec Eg-Agl3812) for detection of IgG antibodies in serum were

summanzed in Table-5-4.

Table-5-4: Estimation of the cut-off ODdg2 values for the ELlSA for detectioo of E

gronulosus IgG antibodies in serum

OD,9, of normal sera

(n = 30)

Absorbance range

The results of ELISA using E. ~ranulosus recombinant antigens Eg-rCW12, Eg-rCW24

and Rec Eg-AgB812 for detection of IgG ant~bodies in serum from the cases of CE and

controls were summarized in Table-5-5.

I I 1

ULISA using Eg-CWlZ demonstrated diagnostic level of antibodies in 92 of 100 (92.00

%) sera of confirmed CE cases (Group-I) (Table-5-5). The assay also demonstrated a

diagnostic level of antibodies in 31 of 60 (51.66 %) serd of Group-I1 cases. The test

detected antibodies in 5.00 % sera from controls with other parasitic diseases (Group-Ill),

but did not show any diagnostic antibodies in the sera of healthy controls (Group-N).

Detection of anti-E. granulosus antibody in serum using

0.110223 Mean average OD

Rec Eg-AgBBIZ Eg-rCW1Z

Standard deviation 1 0.019237 1 0.016552 1 0.037442

0.062-0.129 / 0.056-0.128 1 0.082-0.297

Eg-rCWZ4

0.101533 0.101867

ELISA using Eg-rCW24 demonstrated diagnostic level of antibodies in 95 of 100 (95.00

%) sera of confirmed CE cases (Group-I) (Table-5-5). The assay also demonstrated a

diagnostic level of antibodies in 36 of 60 (60.00 %) sera of Group-I1 cases. The test

detected antibodies in 6.66 % sera from controls with other paras~trc diseases (Group-III),

but &d not show any d~agnostic antibodres in the sera of healthy controls (Group-N).

Table-5-5: ELISA using E. granulosus recombinant antigens for detection of E.

granulosus lgG antibodies in serum from cases of CE and controls

I Study 1 groups

Group1

ELISA uslng Rec Eg-AgB8i2 demonstrated diagnoatlc level of antibodies In 94 of 100

(94 00%) sera of confirmed CE cases (Group-I) (Table-5-5). The assay also demonstrated

a d~agnostic level of antibodres in 39 of 60 (65.00 %) sera of Group-11 cases. The test

detected antibodies in 8.33 % sera from controls with other parasitic diseases (Group-111).

and 3.33 %sera from healthy controls (Group-IV).

/ Group-I1

Group111

Group-IV

Ihe sensitivity, specificity, PPV, NPV, and efficiency of the ELlSA using E grunulosus

recombinant antigens Eg-rCWI2, Eg-rCW24 and RecEg-AgB812 for detect~on of E.

granulosus IgG antibodies in serum were presented in Table-5-5. The scatter plots showing

relative distribution of ODIY2 values in the ELISA using Eg-CW12, Eg-rCW24 and

Re~dg-AgB812 for detection of E, gronulosus IgG antibodies in serum from cases w~th CE

and conh.ols were presented in Fig 5-1 to 5-3. The difference in the mean OD492 V ~ I U ~ S

between groups (Group-I and N ; Group-Il and N ) was analyzed by Mann-Whitney test.

No of subjects

100

Number (%) of sera positive for E. granulosus IgG antibody by ELISA uslup,

Eg-rCW1Z I Eg-rCW24 1 RecEg-AgBBR 92 (92.00) 1 95 (95.00) 1 94 (94.00)

60

60

30 Sensitivity (%) Specificity (%)

Positive Predictive Value (%) Negative Predictive Value (%)

I Efflclency (%)

31 (51.66)

3 (5.00)

0 (0)

[Group-1. Cwes of CE with continned d~npnosir; Group-11. Cases of CE with prwurnpttve diagnosis.

Grouplll: Controls with othrrpurnsttic diswscs, GmuplV' Healthy controls]

92.00 96.66 96.84 91.57 94.21

36 (60.00)

4 (6.66)

0 (0)

39 (65.00)

5 (8.33)

I (3.33) 95.00 95.55 95.95 94.50 95.26

94.00 93.33 94.00 93.33 93.68

I

GROUP l (n-100) GROPU 11 ("-60) GROUP-Ill in-60) GROUP IV ("-30) - Fig 5-1: Scatter plots showtnb: rrlntlvc d~smbutlon of OD1*: values on thc ELlhA usmg E g,onulo.vur rciwlblnant nntlgen Eg-rCWI? for dacction of antlbodlrr I" srmm trom c a s o n ~ t h CE and CL~ITOIS. Tllr mean ODne2 valuer of the ELlSA ~n Group-1 crser (p=0.0001) and Croup-11 (p=O.OODI) cuic, wa, spn~ticunrly d~ftment compared to heollhy controls The p valuca were esttlnoted urlng Cmph Pdd ,ofiwnrc (Onllnc i~rs l l ln )

o GROUP 1 (n.100) GROPU ll (n.60) GROUP 111 In-60) GROUP IV (n=3O)

Fig =2: Scatter plots showng reletivr distnbutlon of OD,,? values ~n the ELISA using E ~runc, loru~ r=ombmm( antigen Eg-rCw24 for dctrction of nntibodtcs III s m m horn cnscs with CE and controls Thr

OD4*: values of thr ELISA tn c r a ~ p - l csses ( p O O U ~ I ) and Group-I1 (pi0 0001) ciu* wn, significantly different compared to h d h y C O ~ ~ O I S . me p values were r ; t~maed using Cmph Pad software IOnltnc ,ernon).

~ i g 5-3: Scaun plots showing relative distribution of OD,,, values ~n the ELlSA using E gronuloru rscombinant antrgen B fraction R e Eg-AgBBl2 far dstoctlon of anribodler i~ s m m from cases wlth CE and cunrrols The mean ODls2 values of the ELlSA in Group-l casea (p-0.0001) m d Croup-Il (p=OU001) EUJCS

war stgnificmtly different c<>mpored to hcuithy controls The p values were csrtmuted using Graph Pad satlam (Onlinc version)

Dot-ELISA for detection of E. granulosus IgG antibodies in serum

Dot-ELISA using Eg-rCWI2 demonstrated diabmostic level of antibodies In 88 of 100

(88.00 %) sera of confirmed CE cases (Group-I) (Table-5-6). The assay also demonstrated

a diagnostic level of antibodies in 34 of 60 (56.66 %) sera of Group-11 cases. The teat

detected antibodies in 6.66 % sera from controls wlth other parasit~c d~seases (Group-Ill),

and 3.33 % sera from healthy controls (Group-N).

Dot-ELISA uslng Eg-rCW24 demonstrated diagnostic level of antibod~es m 91 of 100

(91.00 %) sera of confirmed CE cases (Group-I) (Table-5-6). The assay also demonstrated

a d~agnostic level of antibod~es in 36 of 60 (60.00 %) sera of Group-11 cases. The test

detected antibodies in 6.66 % sera from controls with other parasitic diseases (Group-111).

and 3.33 % sera from healthy controls (Group-N).

DO~-ELISA using Rec Eg-AgB8R demonstrated diagnostic level of antibodies in 90 of 100

(9C 10 %) sera of confirmed CE cases (Group-I) (Table-5-6). The assay also demonstrated

a dlagnost~c level of antibodies in 38 of 60 (63.33 %) sera of Group-I1 cases. The test

detected antibodies in 8.33 % sera from controls with other parasitic diseases (Group-III).

and 3.33 %sera from healthy controls (Group-N).

Chapter 11

The smsltivity, specificity, PPV, NPV, and efficiency of the Do t -ELls~ using Eg-rCw12

and Eg-rCW24 in wmparison to RecEg-AgB8!2 for detection of E grunulo.~~.~ I&

antibodies in serum were presented In Table-5-6.

Tables-6: Dot-ELISA using E. granulosus recombinant antigens for detection of lgG

antibodies in sera from cases of CE and controls

111 Connols wth other pnrasltic d~seuses; Groug-IV. Healthy conools]

Detection of E. granulosus antigen iu serum by Ag-ELISA

I Group-n'

The optunum concentration of each of the affinity purified ant-Eg-rCW12, anti-Eg-rCW24

and anti-Rec Eg-AgB8!2 monospecific IbG antibodies (coating antibody) was found to be

4 pdwell. The optimum dilution of each of the affinity purified ant-Eg-rCWI?, anti-Eg-

CW24 and ant iaec Eg-AgB812 monospec~fic IgG antibodies (detect~ng antibody) was

found to be I : 100. The detection limits of the E granulosus antigens were 50 ndml to 100

ngml for the ApELISA using all three anti-Eg-rCW12 (Rg 5-Sa), anti-Eg-rCW24 (Fig 5-

5h) and anti-Rec Eg-AgB8I2 (Fig 5-5c) antibodies.

[Group-l Cmu of CE with confinned diaposis: Group-ll Cnses of CE with provlnpt~ve dtapr~osts. Group

30 1 (3.33) 88.00 94.44 94.62 87.62 91.05

I Sensitivity (%) I Specificity (%)

Positive Predictive Value (%) Nezative Predictive Value (%)

Efficiency (%)

1 (3.33) 1 1 (3.33) - 91.00 94 44 94 79 90.42 92.63

90.00 93.33 93.75 89.36 91.57

Fig 541: Chromatogram showing elution of IgG fract~on from rabb~t immune sera raised against Eg-rCW 12 antigen

big 5-4b: Chromatogram showtng elutlon oi'lgG fract~on iiom rabbzt xmmune aera ralsed aga~nst Eg-rCW24 antigen

Fie 5 - 4 ~ . Chromatogram showing elution of IgG fraction from rabbit immune aera ra~sed agalnst Rec Eg-Ag8812 anagen

" C . - - I U * r * W U ".". -*4" I.""

~ i g 5-5s: Estimation of antlgen detect~on llmlt for the Ap-ELISA using ant,-Eg-rCW12 anribody HS llpenmmune s e m rarsed against Eg-KWIZ antigen; NS: N o m l rabblt serum as canvol

Anlkgsn dstect#on llmll of the Ag-ELISP using anla-rCW24 ant~bcdy

Fig 5-Sb: Estimation of anttgen detection limit for the Ag-ELISA vslng antr-Eg-CW24 ant~body. HS h>penmmvne s e m raised against Eg-rCW24 antigen; NS. Nonnal rabbit serum ay control

5-5c: Estimation of antigen detection limn for the Ag-ELISA using ant,-Rec EpAgB812, antibody. Srnes I . h y p e n m U e serum raised against Rec Eg-~gB8!2 antigen; Series 2: Normal rabblt seturn as control

Chapter II

&-ELISA for detzction of E. g~anulosus antigens in sera from caves of CE and controls

The cut-off 0D.w values of the Ag-ELISA using anti-Eg-rCW 12, anti-Eg-CW24 and ant,-

RE Eg-AgB812 ant~bodies for detection of ant~gen In serum were estimated to be

0 118201,0.118969 and 0.117355 respectively (Table-5-7).

Table-5-7: Estimation of the cut-off OD,,, values for the Ag-ELISA for detection ofE.

granulosus antigens in serum

Detection of E. granulosus antigen in serum by ApELISA

using

Eg-rCW12

antibody antibody antibody

0.075-0.1 17 0.085-0.1 14 0 075-0.1 16

The results of Ag-ELISA using E grunulosu~ Eg-KW12 antibody, Eg-rCW24 antibody

and Rec Eg-AgB812 ant~body for detection of E granulosus antlgens in serum fiom the

cases of CE and controls were summarized in Table-5-8.

/ Mean average OD191

7 Standard deviation

/ Cut-off

The Ag-ELISA using anti-Eg-CW12 monospecific antibody demonstrated E flunulo.~us

antigen in 80 of 100 (80.00 $6) sera of confirmed CE cases (Group-I) (Table-5-8). The

assay also demonstrated the antigcn m 23 of 60 (38 33 %) sera of Group-I1 cases. The test

detected antigens in 2 (3.33 %) sera from controls with other pms~ t i c d~seases (Group-111).

but did not show any E. gronulosus antlgens in the sera of healthy controls (Group-N).

The Ag-ELISA using anti-Eg-CW24 monospecific antibody demonstrated E. granulosus

antigen in 83 of 100 (83.00 %) sera of confirmed CE cases (Group-I) (Table-5-8). The

assay also demonstrated the antigen in 23 of 60 (38.33 %) sera of Group-11 cases. The test

detected antigens in 2 (3.33 %) sera from controls with other parasitic d~seases (Group-Ill),

but did not show any E. granulosus antigens in the sera of healthy controls (Group-N).

The A ~ E L I s A using anti-Rec Eg-AgB8i2 monospecific ant~body demonstrated E.

grilnulosus antigen in 83 of 100 (83.00 %) sera of confirmed CE cases (Group-I) (Table-5-

0.095833

0.010761

0.1 17355

0.097367

0.010417

0.118201

0.101267

0.008851

0.1 18969

Chapter I t

8). The assay also demonstrated the antigen in 23 of 60 (38.33 %) sera of Group11 cases.

he test detected antigens in 1 (1.66 %) serum specimen from controls wlth other parasitic

drseases (Group-In), hut did not show any E, granulosur antigens in the sera of healthy

controls (Group-N).

The sensitivity, specificity, PPV. NPV. and efficiency of the antlgen-ELISA using anri-Eg-

s W 1 2 antibody, anti-Eg-rCWZ4 antibody and anti-RecEg-Ag8812 antlbody for detect~on

of E. granulosur antlgens In serum were presented In Table-5-8. The scatter plots sllowing

relative dlstnbut~on of OD,,> values in the Ag-ELISA using anti-Eg-KWIZ. anu-Eg-

rCW24 and anti-Re&-AgB812 antibod~es for detection of E punulosus antlgen In serum

from cases with CE and controls were presented in Fig 5-6, Fig 5-7 and Fig 5-8. The

difference in the mean OD192 values between groups (Group-1 and IV; Group-I1 and IV)

was analyzed by Mann-Whitney test.

Table-5-8: ApELISA In serum using anti-Eg-rCW12, antCEg-rCWZ4 and anti-Rec

Eg-AgB812 antibodies for detection of E. graranulosus antigen in sera from cases with

CE and controls

[(jmupl Cases ofCE with confimd dlaynosis; Group-11: Ciws of CE with presumptive dlagno,ls; Croup-Ill. c""rols with other parasllic diseases; Group-IV: Hsalthy connols]

Chapter I f

AD-ELISA uriog anti-Eg-rCWl2 antlbod) O l O W l (IsOPU.II Ono"P,,, W W P ,u

l a , ,

Fig 5.6 Scatter plots show~ng relarive dlatrlbullan o f ODlv: viilust m the 48-ELISA using ilnll-Eg-CWIZ alltlhody for derection o f wrigsns in rcra frotn cuacl with CE and conlrols The diffrmmcc mran OD,,,: i,lucl of thc AgELISA betwern Group1 case, and healthy ~ndirtdunls (p0.00il l) A, well na hetweco Group- I! and hwlthy indlv~dualr (p=O.MlOlI werc found to be stilt~sricnlly stgntficont The p values were cstzmdrcd using m p h Pad soflwurr (Online vaswn)

As-ELISA using nntl-El-rCW24 antibody

.,. OSOVPl OWP" ,I DllOUP,,, DROYCI" ~

k g 5-7. Scalar plats re lat l~r distrihulion of ODle2 values m the APELISA urlng ontl-Eg-CW24

dnllh~dy for detsction of ,,,,tigens in sad from citjcs ~ , t h CE and connolr. The dtftbrence rn mean ODIV~ -1.u of the A ~ E L I S A betw- c ~ o u ~ . ~ wsrs and healthy indidduals (p=O.OOO1) a. wd l as between Group- 11 and healthy indlvtduals (p=0.0002) wnc found to be stetlst~cally s~gnificmt. The p values war sumat*

Graph Pad mftware (Online vmian).

0 . -~ - - - - - ~ - - - - -

o GROUP-I In-1001 GROPU-11 ln=60) GROUP-Ill (n=BO) G R O U P - ~ ~ (n-w) r

Fig 5-8: Scalta plols sharing relative d~slnbution of ODrv2 values an the Ag-ELISA urtng ant,-Ref Eg-~gBi2 - ~

,nc hJ) L.r dclcl~uli . ,I mtlgrn. on Gcru lroal .&re.; ulln CI UJ c ~ a t r u l * Tls J8iicrcn.r tn lncdn OD..: ..,.L~. oithc A x - C L I S . hetucecl C I J L ~ I c*.L.\ dno l.~llh) ~nd.r.~Ju.~l\ (p=lr $11, r l A. uca. iri hnu-1. Grvrp .I AIJ h c ~ l l h ) ~nJ~.'~audl, lp=(. bltul, u a c IdunJ I. hc rrrlc.lt.all) .~/s~A.unt Thr p \ ~ l u c \ *err r . ~ n , . t c ~ u\ng Graph Pad saRware (Online vrraon).

Dot-ELISA for detection of E. granulosus antigens In serum

The Dot-ELISA using anti-Eg-rCW 12 monospecific ant~body demonstrated E grunulosus

antigen in 76 of 100 (76.00 %) serum samples of the cases with confirmed CE (Group-I)

(Table-5-9). The assay also demonstrated the antigen in 22 of 60 (36.66 %) sera of Group-

I1 cases. The test detected antigens m 3 (5.00 9.0) serum specimens from controls with other

parqsitic diseases (Group-111). hut d ~ d not show any E grunulusw antlgens in the sera of

healthy controls (Group-1V).

The Dot-ELISA using anti-Eg-rCW24 monospeclfic antibody demonstrated E grrrnulosrir

antryen in 83 of 100 (83.00 %) serum samples of the cases with confmed CE (Group-I)

(Table-5-9). The assay also demonstrated the antigen in 23 of 60 (38.33 %)sera of Group-

11 cases. The test detected antigens in 1 (1.66 %) serum specimen from controls wlth other

Pamsitrc diseases (Group-III), but did not show any E granulosus antigens in the sera of

healthy controls (~roup-IV)

DO~-ELISA using m t i - ~ e c Eg-As812 monospecific antlbody demonstrated E

Krunulosus antigen in 82 of 100 (82.00 %) serum samples of the cases w~th confirmed CE

(Grou~-I) (Table-5-9). ~h~ assay also demonstmed the antigen in 21 of 60 (35.00 %) sera

Of G r o ~ ~ - n cases. The test detected antigens in 3 (5.00 %) serum specimens from controls

~ l t h other parasitic diseases (Group-El), but did not show any E gronulosur antigens in

the sera of healthy controls (GroupIV).

The sensitivity, specificity, PPV. NPV, and efficiency of the Dot-ELISA using ant,-Eg-

KWi2 antibody. anti-Eg-rCW24 antibody and anti-Rec Eg-AgBI2 antibody for detection

of E. granulosus antigens in serum were presented in Table-5-9.

Table-5-9: Ag-Dot-ELISA using anti-Eg-rCW12, anti-Eg-rCW24 and anti-Rec Eg-

AgB812 antibodies for detection of E. granulosus antigen Lo sera of cases wlth CE and

controls

No (%) of sera positive for E. granulosus antigen by

Auti-Eg-

82 (82) 76 (76) 83 (83)

1 Group11 21 (35)

1 (1 66)

Sensitivity (X) k Specificity (%) 1 Positive Predictive Value (%) I Negative Predictive Value (%)

Efficiency (%) [Group-1. Cases of CE with custirmul dtagnor~r. Group-11. Cases of CE witti prcsulnptivc diupnoar, Group-

82.00 96.66 96.47 91.57 88.94

76.00 96.66 96.20 78.37 85.78

83.00 98.88 98.80 83.96 90.52

Chapter II

CllnieaI rnaolfestfitions in patients with CE

Cystic echinococcosis is an important public health problem tn many regions of the world,

both m areas where the disease is endem~c, and m reglons where the disease is emerglng or

reemerging (Craig et al., 1996). Though CE 1s preventable by efficient and sustained

control campaigns (Torgemon and Heath, 2003). numerous reports indicated that its

~nc~dence has been increasing in various regons of the world (Eckert et al.. 2000)

lnclud~ng lndia (Parija, 1994; Khurana et al., 2007). The accurate assessment of the

prevalence of CE is therefore a major element to expose the magnitude of the problem and

evaluate the success of the control strategy. Th~s ~nvolves clintcal diagnosis of the disease

and epidemiological surveillance of high-nsk populations (Paija, 1994).

CE 1s a chronic zoonosls, usually charactenzed by slow growing flu~d-filled cysts In the

Ilvrr, lungs or other organs (Pawlowsk~. 1993). The cl~nlcal d~agnos~s of CE IS invariably

made when the disease is relatively advanced. The slgns and symptoms of CE are also

dependant on the location, number. slze, topographic relationships of the cysts In the

human body (Thompson and Lymbery, 1988; Eckert and Thompson, 1988; Thompson et

al., 1995; Eckert and Thompson, 1997; Rosenzvtt et al., 1999; Kamenetzky et al., 2000,

Kamenetzky et al.. 2002).

The infection generally occurs between 2-5 years of age (mean: 3 years), when children

begin to go upon all fours and become friendly wtth dogs (Thompson et al.. 1995;

Pawlowski, 1997; Ecken and Thompson, 1997). But the clinical symptoms have been

evident afler many years of infection, generally in middle aged patients (Gottstein et al.,

1987; Ammann et al.. 1990; Pawlowski. 1997). Paul and Stefaniak (1997) observed that

females (82.71%) were more frequently infected with CE compared to males (17.28%).

In the present study, CE was more frequently observed in the age soup of 40-60 years

(46 87%) followed by the age qoup 20-40 years (38.75%)(Table-5-1). The age group

"aged from 41-60 years (middle age people) appeared to ~nvolve highest by CE infection

In males (51.38%) followed by 20-40 age p u p (29.16%) (Table-5-1). The age group 21-

40 affected highest (46.59%), followed by the age group 40-60 years (43.18%) in females.

The CE was more frequently observed in females (55.00%) compared to males (45.00%)

Chapter 11

(Table-5-1). However, the difference was n m w in contrast to the observation made by

paul and Stefaolak (1997).

ln CE of humans, primary hydatid cysts have been found most frequently In liver

(approximately 65% ofthe cases). but also in lungs (25%) and other organs such as kidney,

spleen. brain, heart. and bone (Schanu: and Gottsteiq 1986; Thompson et al., 1995; Eckeri

and Thompson, 1997; Rosenzvit et al.. 1999; Kamenetzky et al., 2000). Ln other study.

pnmary hydatid cysts were found most frequently in the llver (58.6 %), followed by lungs

(29.2 %) and spleen (2.4 %) and the multi organ locallon of hydaud cysts were found III

9 8 % of CE cases (Guarnera el al., 2004).

In the present study, the predilection of hydatid cysts among the South lnd~an patlents were

found more frequently in 76 % of confirmed and 41 % of suspected CE cases m liver.

followed by lungs (9 % and 16 %) respectively (Table-5-2). Multi organ location of

hydatld cysts were found m 7 % of confirmed and 1.66 % of suspected patlents w~th CE.

wh~ch were similar to that of prevlous studies (Table-5-2).

The clinical symptoms in patients with liver cyst Include right h~pochondrial paln (48.27

%), vomica (37.93 %), fever (17.24 %). palpable tumor (17.24 %) and very subject~ve

slmptoms like astenia, anorexia and postprandial fullness sensation (Guamera et al.,

2004). The clinical symptoms in patlents with non complicated lung cysts mclude, thoraclc

paln (50 %), cough (37.5 %), vomlts (37.5 %), astenla (25 %) and anorexla (12.5

%)(Guamera el al., 2004). These symptoms were found to be sim~lar In CE patlents w~th

any stram of E, gronulosus (Schantz and Gottsteln. 1986: Pawlowsk~, 1997; Onona el al.,

~303).

In the present study, 83.00% confirmed CE cases and 75.00% suspected CE cases

presented with pan. 48.00% and 48.33% of cases presented with swelling, 24.00% and

21.66% of cases wlth vomiting, 24.00% and 33.33% of cases with weight loss. 19.00% and

31.66% of cases presented with cough, 10.00% and 3.33% cases wlth hen~optysis out of

100 confirmed CE cases and 60 suspected CE cases respectively.

The present study also demonstrated that, CE was prevalent more frequently among the

agricultural workers (about 41.41% and 43.33% of confirmed and suspected cases of CE

respectively), followed by house wlves (with a Frequency of 26.26% and 28.33% of

confirmed and suspected CE cases respectively) and public service undertak~ng/corporate

Chapter 11

sector employees (22.22 % and 16.66 % of confmed and suspected CE cases

respectively) (Table-5-3).

Detection of E. granulosus antibodies in serum

ELISAfor drfecrion of E. granulosus antibodies in serum

Detection of antibodies in serum is found to be the most convenient and commonly used

method for the diagnosis of CE (Siracusano et a]., 1991; P q a , 1994). Anr~body-based

serological tests are useful not only for confirming thc d~agnosis of CE in an ind~v~dual

patlent, but are also helpful in epidemiologic studies. Several ant~body detection assays.

such as IED. ELISA, Dot-ELISA and EITB, have been used to detect hydatld antibodies In

serum for diagnosis of CE (Varela-Diaz, el al., 1978; Coltort~ and Varela-D~az, 1978;

Coltorti, 1986; Maddison et a1 . 1989; Siracusano et a]., 1991).

130th ELISA and Dot-ELISA were found to be more reliable tests, cheap and rap~d, hence

nlost frequently used in recent times (Kanwar et al. 1992; LI ct a].. 2004). The ELlSA has

brrn evaluated for diabmosis of CE by detectlng E gronu1osu.r 1gG antlbod~es ~n serum

(Facon et al.. 1991; Chamekh et al., 1992; Kanwar et al, 1992; LIU et a]., 1993; Helb~g et

al., 1993; Leggatt and, McManus, 1994; McVle st al., 1997; Kaur el al., 19991, 1gM

ant~bodies (IgM-ELISA) (Wattal et al., 1986) and IgE (IgE-ELISA) (Wattal et al.. 1988).

Therefore, the present study was designed to develop and evaluate ELISA using E

g~onulosus recombinant antigens, Eg-CW12 and Eg-rCW24 and Rec Eg-AgB812, to

dctect hydat~d ant~bodies in serum for d~agnosis of CE.

The recombinant A@ fractions were found to he the most relevant dlagnost~c antlgens for

d~agnos~s of CE (Matoss~an el a]., 1972; Maddlson d al.. 1989; Kanwar et al.. 1992; Rott

rt al., 2000; Virginio et a!., 2003; Li et al.. 2003; Li et al., 2004). The recomb~nant anugens

ldent~fied in the first phase of the present study also appears to be A@ fractions.

Therefore, m the present study, the ELISA was developed and evaluated by ustng E

~ . m u l o s u s recomb~nant antlgms, Eg-rCWI2, Eg-rCW24 and Rec Eg-AgB812 for the

diagnosis of CE. For interpretation of seroposltlvlty, a threshold (cutoff) value was

calculated for each antigen using 30 semm samples collected from healthy ~ndlvlduals

(Table-5-4). The mean ODIPl plus two standard deviations was taken as the cutoff.

The ELSA using E. granulosur recombinant antigens, Eg-rCWI2. Eg-rCW24, for

detectton of antibodies in serum, showed a smsitivrty of 92.00% and 95.00% respectively

(Table-5-5). The specificity of the ELSA uslng Eg-rCWI2. Eg-rCW24 antigens for

detection of antiboches in serum was 96.66% and 95.55% respcctlvely (Table-5-5). The

sensitlvily and specificity of the ELlSA ustng E granulosur recombinant antigm8/2 (Rec

Eg-AgBIZ) for detection of antibodes m serum was 94.00% and 93.33% respectively

(TableJJ) . In comparison, the efficrency of the ELlSA using Eg-KW24 (95.26%) Ibr

detection of IgG antibodies In serum was found to be higher than the ELlSA using both

Eg-rCW12 (94.21%) and Rec Eg-AgBi2 (93.68%) (Table-5-5).

In the present study, the sensitivity (92.00%. 95.00'') (Table-5-5) of the ELlSA using Eg-

rCW12 (12 kDa fraction) and Eg-KW24 (24 kDa fraction) appeared to be s~milar to that

of rEpC1-GST (92.20%) reported by 11 et al. (2003), 8 Ld)a subunlt (94%) reported by

Shambesb et al. (19951, an antigen (92%) reported by Ito et al. (1999) and 12-kD subuntt

(90%) described by Leggatt et al. (1992). but higher than the 12-kD subunrt (80%)

reported by loppolo et al. (1996). However, the specrficity of the ELISA using Eg-rCW12

and Eg-tCW24 was in accordance with that of these prevrous studies.

The ELISA using Rec Eg-AS8i2, In the present study, showed a sensittvity of 94.00%

(Table-5-5) which was in accordance w~th previous studies (Rott et al., 2000; V~rg~nlo et

al., 2003). But. the specificity of the ELlSA using Rec Eg-AgB8/2 (93.33%) was found to

be lesser than the prev~ous studies (99.50%)(Rott et al., 2000: Vrrgtnio et al., 2003).

False positive reactions have been the frequently noted problem In serologtcal tests III

patrents infested by tapeworm and other helminths (Guisantes et a]., 1981; Sjolandrr rt al . 1989; Maddison et al., 1989; Force et a]., 1992; Wen and Craig, 1994; Taherkhani et a].,

2007). The serodiagnostic tests uslng E. grunulosus antigens also showed cross reactions

with sera from patients with AE, schistosomiasis and trichinosis (Slolander et a].. 1989;

Force el al., 1992). sera from onchocerc~as~s and ascariasls (Sjolander el a]., 1989) and sera

from cysticercosis (Maddison et al., 1989; Wen and Craig, 1994). The ELISA using

purified E. granulosus anugen also showed cross react~ons with the sera collected from

T~enia suginaru, Enterobius vermiculoris and F a x i o h hepat~co (Gursantes et a1 . 1981).

the present study, the ELlSA using Eg-KW12, Eg-KW24 and Rec Eg-AgBBl2

demonstrated a cross reactivity of 5%. 6.66% and 8.33% respectively (Table-5-5). with

sera from neurocysticercosis cases, which was similar to those of other studies reported

elsewhere (Maddrson et al., 1989; Leggan et al., 1992; Wen and Craig, 1994; Shambesh et

Chapter 11

al., 1995). However, no cross-reactions were observed with sera fmm neumystlcercosls

cases as reported by Ito et al. (1999). The ELlSA using Eg-rCW12. Eg-rCW24 and Rec

Eg-~gB8/2, m the present study, also did not show cross reactlons with sera from patients

w~th other parasitic diseases such as filariasis, amoebiasis, chist to so mi as is and malans

(Table-5-5).

The GST-fusion proteins, even after affinity purification, are h o w to show false-positive

reactions with the E coli-specific antlbodies tn patlent sera (Gasser et al., 1990; Facon et

al., 1991; Helbig et al.. 1993). The use of GST-fusion proteins in srrodiagnosis of CE

have shown false-positlve reactlons with sera from cases of schistosomias~s (Smith and

Johnsoh 1988; Gasser et a].. 1990; Facon et a l , 1991). and 47% of those patients

exhibited IgG antibodies against GST (Aunault et al.. 1990). In the present study, the

recombinant antigens that were cleaved from the11 GST molety usrng Factor X d ~ d not

show cross reactlons wlth sera from schistosomlasls cases, as previously observed by

llclblg et al. (1993) and Li et al. (2004).

The antigen B (8 kDa) subunit of E, granulosus 1s h o w n to produce cross-reactions with

sera from AE cases (Leggatt et al., 1992; Shambesb et al., 1995; Ito et al., 1999). This

would limit its usefulness for serolo@c differentiation of CE from AE. In the present study,

the recomblnant antigens were not evaluated using sera frnm AE and polycystic

cchlnococcosis (PE) cases. Slnce, E mulriloculrrris. E uligarthms and E vogeli infect~ons

have not been prevalent m Indla, these recombinant antigens could be effic~eutly used as

the diagnostic antigens for diagnosis of CE In thls part of the world and other places, where

these parasitic diseases are not commonly prevalent.

In the present study, ELlSA was not evaluated using sera from patlents with liver c~nhosls

(lacona et al., 1980). anti-PI antlbodies (Ben lsmail et al., 1980). and mal~gnanctes

(Varela-Diaz et al., 1978). wblch could also produce false positlve reactions. Although

iunher tests with a significantly greater number of heterologous sera are required. thc

results of the present study (Table-S-S), suggested that any of the three recomblnant

antlgens (Eg-rCW24, Eg-rCW12 and EG-AgB812) evaluated m the present study could be

used for the diagnosis of CE.

In the present study, all sera were tested thnce, including the sera used for determination of

the cutoff values, to exclude variations from day to day. However, all independently

Performed ELISAs showed the same results. This high level of reproduc~bil~ty of these

ELISAS is most imponant and especially useful when there are low serum antibody titers.

In the present study, the efficiency of ELlSA using Eg-rCW12 and Eg-rCW24 (Fig 5-1 and

Fzg 5-2) was sunliar to the prevlous studies where variations in the antibody response to

CE were associated with the location. slze, number and/or condiuon of the hydat~d cysts

(Verastegui el al., 1992; Cralg, 1997). These results were also slmilar to the previous

stud~es where seroconvenion was found to be comlated w~th increased immune

st~mulation with respect to the cyst growth and degeneration (Shambesh et al.. 1999).

Given the diamostic properties of the E, gronuloslrs recombinant antlgens (Eg-rCWI2 and

Eg-CW24) (Table-5-5) evaluated in the present study and the fact that large quantities of

the protein In pure form could be produced, these antigens were found to be of diagnostic

value to develop vanous s~mple, cheap and rapid immunod~agnost~c assays for dlagnosls 01'

CE In both humans as wells as Irvestock.

The Dot-ELISA is a rap~d, inexpenswe and s~mple test to perform (Rogan et al., 1991).

Pappas et al. (1986) also observed that the Dol-ELISA was a rapid and econom~cal enzyme

tmmunoassay whlch was ant~gen-conservative and serum-conservat~ve. Such sltnpler

assays have been developed for the detect~on of antibod~es against a range 01' ~nl'ecr~ous

agents including Schlsrosonra (Zhu et al., 2002) and Leishmania (Schall~ng et al.. 2004).

Therefore, the present study was further extended lo evaluate Dol-ELISA uslng these E

granulosuv recombil~ant antigens (Eg-rCWI2 and Eg-rCW24), which were found to bc

dlabmostlc.

Dot-ELISA for detection of E. granulosus antibodies in serum

A rap~d, simple, cheap and relevant ant~body-based test uslng recomb~nant E. p n u l o s r l s

antigen is a necessity for diagnos~s of CE In humans and an~mals (Muller et al., 1989:

Froschet al., 1993; Ferreira and Zaha, 1994; Li et al., 2004).

Dot-ELISA 1s a highly versatile sol~d-phase xmmunoassay for detection of ant~body In the

diagnosis of a number of parasitic dlseases includmg, lershman~as~s (Pappas el al., 1983).

toxoplasmosis (Pappas, 1988), malarla (Pappas, 1988), trypanosomlasls (Araujo. 1985).

cysticercosis (Tellez-Giron et al., 1987) and CE (Schantr and Kagw, 1980). The test 1s

found to be su~table for routlne application in the clin~c, laboratory and In the field for

community surveillance surveys (Rogan el al., 1991). The assay uses minute amounts of

reagents d o q d onto solid surfaces such as n~trocellulose or cellulose acetate membranes

which avidly bind proteins (Pappas, 1988). Hence the present study was undertaken to

develop and evaluate Dot-ELISA using Eg-rCW12 and Eg-rCW24 for dialylosrs of CE.

Lo the present study, the Dot-ELISA using E gronulosus recombtnant antigens. Eg-

rCW12, Eg-rCW24, for detection of antibodies In s e m . showed a sensitivity of 88.00%

and 91.00% respectively (Table-5-6). The specificity of the Dot-ELISA usiog either Eg-

KW12 or Eg-rCW24 antigens was observed to be 94.44%. The scnsit~vity and specificrty

of the Dot-ELISA uslng Rec Eg-AgBR for detection of antibodies ~n serum was observed

to be 90.00% and 93.33% respect~vely (Table-5-6). In companson, the efficiency of the

Dot-ELISA ustng Eg-rCW24 (92.63%) for detection of lgG ant~hod~rs in serum was

observed to be higher than the Dot-ELISA using both Eg-rCW12 (91.0S0/0) and Rec Eg-

AgB/2 (91.57%) (Table-5-61,

In the present study, the sensitrvlty and specificity of Dot-ELISA ustt~g Eg-KW12

(88.00%, 94.44%), Eg-KW24 (91.00%, 94.44%) and Rec Eg-AgB812 (90.00%. 93.33%)

(Table-5-6) and is In agreement wlth the studles of Romia et al. (1992) who demonstarted

a sensitlwty of 88.9% and a specific~ty of 96.9% by Dot-ELISA for diagnosis of CE. The

sensitivity of Dot-ELISA obtained In the present study, however, was sl~ghtly lesser than

thc Dot-ELISA using sheep HCF antlgen, which showed a sensitiv~ty of 96% and

spec~ficity of 98% (Table-5-6) (Pappas et al., 1983; Mistrello el a1 , 1995).

Kogan et al. (1991) reported lower specific~ty (52%) and higher sensitivity (97%) by uslng

crude sheep HCF as diagnostic antlgen. It was suggested that the use of more pur~fied

antigen preparation, greatly unprove the assay reltabiltty by reduclng false-posltive

reactions (Schantz and Kagan. 1980. Rogan et al.. 1991; Wen and Crag. 1994). In the

present study, the Dot-ELISA ustng reco~nb~nant antlgens (Eg-rCW12 and Eg-tCW24)

demonstrated high specific~ty (94 44%) (Table-5-6).

In the present study, the Dot-ELISA showed some problems associated wlth dlfferentlat~ng

weak posltive reactions from negatlve ones. In some cases the repetltlon of the tests uslng

freshly collected serum samples produced clear results. Similar results were also observed

in prenous studies, where they suggested that the use of adequate and freshly collected

negative and positive control sera could minlmize these problems (Pappas et al.. 1984;

Rogan et al., 1991).

The antibody detection assays (ELISA and Dot-ELISA), as demonstrated m the present

study, were found to be useful for serod~agnosts of CE. However, the tnability to

discriminate between recent and old infections and false-negativity were the major

d~sadvantages of these immun0aSSays. Some of the reasons for the occurrence of such false

negative reactions were due to (1) secretion1 excretion of high levels of c~rculat~ng antigen

by the parasite which generate circulating Immune complexes after reacting wlth

ant~hodies (Craig and Nelson, 1984; Craig et al.. 1986). (ii) the parasite with dormant or

calcified cyst that may cease to produce antibodres (Biffm et al., 1993). (lii) antibody

mopping by circulating antigen (Cmg and Nelson, 1984). and iv) some of the cysts of

lung associated with low serum antihod~es and hydatld cysts of the eye and brain produce

no or very low serum antibodies (Maddison et al.. 1989; Siracusano et al.. 1991).

Detection of E. granulosus antigens In serum

Ag-ELISA for detection of E. granulosus antigens in serum

The antibody detect~on assays In CE were nelther sensitive nor very specific and there was

no correlat~on between the levels of anti-liydat~d antibodies and the burden of the dtsense

(Rlckard et al., 1984). Moreover, the persistence of antl-hydatld ant~bodles for years after

surgical removal of the cyst (K~ckard el al., 1984) and the cross-reactlon with other

parasitic infections (Larralde et a1 , 1989) further questton their sultablldy for diagnosis of

CE.

The hydatid antigens are generdlly excreted lnto serum during the actlve infectlo" and

hence detection of serum hydatld antigen always indicates actlve or recent lnfectlon

(Shmff and Parija, 1991). Some studies have also shown the usefulness of the serum

antigen detection for monitoring the post-treatment evaluation of the CE cases (Candolfi el

al.. 1985; Craig et al., 1986; Ravinder et al., 1997).

Several serodiagnostic tests have been evaluated for detection of hydatld antlgen In the

serum and other body flulds for dlagnosls of CE. These tests Include ClEP (Shanff and

Parha, 1991), Co-A (Shanff and Panla. 1993). LAT (Sheela Dew and Parlja. 2003).

ELlSA (Gottstein, 1984; Candolfi et al , 1985; Cra~g et a]., 1986; Kanwar and Vlnayak.

1992) and Dot-ELISA (Romla et al., 1992). These tests demonstrated varied sensitivlt~es

and specificities, whlch were unsatisfactory. Therefore, the ELISA was developed and

evaluated by using affinity purltied ant,-Eg-rCW12, anti-Eg-rCW24 and anti-Rec Eg-

AgB812 IgG antibody for detection of hydatid antigen in serum for the diagnosis of CE.

Antigen-ELISA is a specific test whch enables qualitative or quantitative determination of

specific antigen through chromogenic reaction (Gottstein, 1984). Ag-ELISA for

quantification of antigen is especially valuable when the concmmtton of antigen is low

andlor they are contained lo high concentratlon of contamlnatmg protein (Constein, 1984;

Candolfi et al., 1985; C m g et al., 1986; Kanwar and Vinayak, 1992).

In the present study, the Ag-ELISA uslng anti-Eg-CW12 and anti-Eg-CW24 anthodies

for detection of hydat~d antlgens in serum, demonstrated a sensltlvlty of 80.00% and

83.00% respectively, w ~ t h a spec~fic~ty of 97.77% (Tahle-5-8) The Ag-ELISA usrng

affinity purified anti-Rec Eg-AgBI2 ant~bodies for detection of hydatld antlgens In serum.

showed a sensitivity of 83.00% and a spcclfic~ty of 98.88% (Table-5-8). The cut-off pomt

was calculated from the mean ODIY2 of the Ag-ELISA uslng 30 healthy ~ndiv~duals (Table-

5-7). The mean OD192 plus two standard dev~at~ons was taken as the cutoff

The hy&tid antlgen In the serum was also demonstrated, m our laboratory, by other simple

assays using polyclonal ant~bodies. Thr ClEP test for detectlon of serum antlgen, showed a

moderate sensltiv~ty of 55.55% In surg~cally proved and high sensltlvity of 100% in

ultrasound proved CE cases for the dlabmosis of CE (Shariff and Parija. 1991). The Co-A

test showed a sens~tivity of 95% and spec~fic~ty of 89% for the dlaposls of CE by

detection of serum antlgen. False posltlve rate of 18.5% was also observed w~th control

sera from patients wlth various other paras~tic d~seases by the Co-A (Shanff and Parqa,

1993). The LAT showed a sens~ t~v~ ty of 72% and a apecific~ty of 98% for the dla&mos~r of

CE (Sheela Devl and Panja, 2003). h~ companson, ~n the present study, the ~Sficbcncy of

the Ag-ELISA uslng ant,-Eg-rCW24 (90 00%) and antl-Rec Eg-AgB12 (90.52%)

rntibodes for detectlon of hydatid antlgcn In serum war observed to be higher than the

ELISA uslng anti-Eg-CW12 antibody (88.42%) (Table-5-8).

In the present study, the sensit~vity and speclfic~ty of Ag-ELISA using anti-Eg-rCW24 and

antt-Eg-CWIZ antibodles (Table-5-8) were sim~lar to the studles reported by Kanwar and

Vinayak, (1992) In the~r study the ELISA uslng monospec~fic ant~hod~es agarnst speclfic

hydatld antigens (8 kDa antlgen and 116 kDa ant~gen) showed a sensitivity of 85% and

90% respectively for detection of hy&tid antigen in serum for diagnosis of CE (Kanwar

and Vinayak, 1992).

Gonstem (1984) demonstlated circulating antlgen (concentratlon of 355-580 ng/ml) In

only 40% of CE cases before surgery by employing double-ant~body sandwrh ELISA

uslng polyclopal antibodles. Candolfi et al. (1985) demonstrated c~rculat~ng antlgens In

only 21% of sera form patients with CE. The low sensitivity might be due to ather low

level of protem excretion or due to the formation of immune complexes (d'Amel~o et al.,

1983; Gonstein, 1984; Candolfi et al., 1985). Craig (1986) observed that the sensltmty of

ELSA was 90% for Turkana patlents hut as low as 50% for UK patients. The Ag-ELISA

usmg anti-Eg-rCW24 antibody demonstrated a sensitivity of 83.00% in the present study.

In the present study, the antigen detection limt of Ag-ELISA using all three monospecific

antibodies was I00 nglml to 50 ng/ml (Fig 5-5a. 5-5b. 5-5c). This was much lower than the

antlgen detection limit of 270 nglml reponed by Gottstein (Gottstem, 1984). The possible

reason for difference in the detection l~mit of antigen might be due to qualitative and

quantitative difference in the concentration of coating antibody and the quality of the

serum sample (Cra~g, 1986).

The host antlgens are also found to interference with the antlgen detection assays

producing false positive reactions (Gottste~n, 1984; Craig, 1986). However, In the present

study, the use of monospec~fic ant~bodles against the E yronulusus recornb~nant antigens

eliminated the interference of host components. The Ag-ELISA using antl-Eg-rCW24

showed false posilive reactions in 2 (6.66%) of 30 patients with cysticercosis. The Ag-

ELISA using anti-Eg-rCWI2 showed false positlve reactions in only one (3.33%) of 30

patlents with cystrcercosis (Table-5-8). The Ag-ELISA did not show cross reactions wlth

sera from patients with other pamsit~c diseases and healthy controls (Table-5-8).

Dot-ELISA for detection of E. granulosus antigens In serum

Although, the Ag-ELISA is sensltlve and specific, ~t is expensive, requ~red technical

expertise, perishable reagents and IS d~fficult to adopt In small poorly equ~pped

laboratories. Therefore, the present study was undertaken to develop and evaluatc Dot-

ELISA using affin~ty pur~fied monospecific ant~bodies for the detect~on of hydatid antlgan

In the serum.

In bancroftian filanasls, Dot-ELISA was compared w~th sandwich ELISA for the detect~on

of antigen m Serum (Zheng et al., 1990). The result showed that In Dot-ELISA, 67 of 70

snwn samples from microfilaremic patlents were positive at a dilutlon of 1:50. In

sandwich ELISA, 64 of the 70 serum samples were positrve at a dllution of 1:10. The

specificity of both assays was over 91% but their sensitivity was markedly d~fferent. Dot-

ELlSA could detect as little as 0.055 nglml m~crofilarial autigens added to normal human

serum, whereas the lower limit of detection by sandwich-ELISA was 10 nglml paras~te

antigens (Zheng et al., 1990).

In the present study, the Dot-ELISA using affmlty purified anti-Eg-tiW12 and ant,-Ey-

rCW24 monospecific antibodies for detect~on of hydatid ant~gens m serum, showed a

sensitivity of 76.00% and 83.00% respectively, and a specificity of 96.66% and 98.88%

respectively (Table-5-91, The Dol-ELISA using affinity punfied anti-Rec Ey-AgBI2

monospecific antibodies for detectlon of hydatid antlgens In serum, showed a sensit~vity of

82.00% and a spec~fic~ty of 96.66% (Table-5-9). In companson, the effic~ency of the Dot-

ELISA using anti-Eg-tiW24 antibodes (90.52%) for detection of hydatid antigens in

senun was observed to be hlgher than the Dot-ELISA using both anti-Eg-tiW12 (85.78%)

and anti-Rec Eg-AgB/2 (88.94%) ant~bod~es (Table-5-9).

fhe results of the present study were m contrast to the study reported by Romia et al.

(1992). In their study the Dot-ELISA using anti-HCF hperimmune rabbit serum

demonstrated less sensitivity (55.6%). Low sens~tivity 1s attributed to the small amounts of

circulating antlgens andlor Immune complexes formation (Romia et al.. 1992).

In conclusion, in thrs study, both Ag-E1.ISA and Dot-ELISA uslng affin~ty purified ant]-

Eg-CW24 and anti-Eg-rCWI2 monospeclfic antlbohes for detectlon of hydatid antlgens

in serum showed high sensit~v~ty and h~gh spec~ficity (Table-5-8 and Table-5-9). Hence

both the techniques could bc used for dlabmosls of CE. The Dot-ELISA, has the addded

advantage of being more convenient, s~mple, rapid. cheap and relrable, does not require

costly equipment and requlres only lesser amounts of specimens and reagents.

EvJPlSion of ELISA and Dot-ELISA lulnp E prrurulosys recombhunt antlgcns to

detcd byd8tid antibodl~ in serum for diagnwb of cy8Uc eehinoeoeeorir

The ELSA using E. granul- r coombi i t antigcns, Eg-rCW12. Eg-rCW24, for

detection of antibodies in sorum, showed a sensitivity of 92.00?4 and 95.00% respcctively.

The specificity of the ELISA using Eg-rCWIZ, Eg-rCWZ4 antigens for detection of

antibodies in senun was observed to be 96.66% and 95.55% respectively. The sensitivity

and specificity of the ELlSA using E. p n u l o s u s recombinant antigen-Bi2 (Rec Eg-

AgB812) for detection of antibodies in serum was observed to be 94.00% and 93.33%

respectively. In comparison, the efficiency of the ELISA using Eg-rCW24 (95.26%) for

detection of IgG antibodies in serum was observed to be higher than the ELISA using

both Eg-rCW12 (94.21%) and Rec Eg-AgB812 (93.68%).

The Dot-ELISA using E. grunulosus recombinant antigens, Eg-rCW12. Eg-rCW24, for

detection of antibodies in serum, showed a sensitivity of 88.00% and 91.00% respctively.

The specificity of the Dot-ELISA using either Eg-rCW12 or Eg-rCW24 antigens was

observed to he 94.44%. The sensitivity and specificity of the Dot-ELISA using E.

grundosus recombinant antigen-Biz (Rec Eg-AgB812) for detection of antibodies in

s e m was observed to be 90.00% and 93.33% respectively. In comparison, the efficiency

of the Dot-ELISA using Eg-CW24 (92.63%) for detection of IgG antibodies in serum

was observed to be higher than the Dot-ELISA using both Eg-CW12 (91.05%) and Rec

Eg-AgB812 (91.57%).

The Dot-ELISA using Eg-rCW24 showed the highest efficiency in comparison to other

two antigens (Eg-CW 12 and Rec Eg-AgB812) for demonstration of hydatid antibodies in

serum. Tho ELISA using Eg-rCW24 however, showed a sensitivity similar to that of the

ELISA using other antigens (Eg-rCWl2 and Rec Eg-AgB8LZ). The Dot-ELISA. therefore.

using Eg-CW24 showed to be a simple and rapid diagnostic test for diagnosis of CE.

Chapter I1

E v d ~ U o n of ELISA m d Dot-ELISA to detect hyd~t id antigens Ln serum by ushg

amaity purified antibodies raised against E. grmulosus momblnant antigeos for

&go& of cystlc echlnococcosls

The &-ELISA using aflinity purified anti-Eg-rCW12 and anti-Eg-rCW24 monospec~fic

antibodies for detection of hydatid antigens in sawn, showed a sensitivity of 80.00% and

83.00% mpectively with a specificity of 97.77%. The Ag-ELISA usmg a f f i t y purified

a n t i - h Eg-AgB812 monospecific antibodies for detection of hydatid antigens in serum,

showed a sensitivity of 83.00% and a specificity of 98.88%. lo comparison. the efficiency

of the Ag-ELISA using anti-Eg-rCW24 (90.00%) and anti-Re. Eg-AgBBR (90.52%)

antibodies for detection of hydatid antigen in serum was observed to be higher than the

ELISA using anti-Eg-rCW12 antibody (88.42%).

The Dot-ELISA using affmity purified anti-Eg-rCWI2 and anti-Eg-rCW24 monospec~fic

antibodies for detection of bydatld antigens in serum, showed a sensitivity of 76.00% and

83.00% respectively and a specificity of 96.66% and 98.88% respectively. The Dot-

ELISA using affinity punfied anti-Rec Eg-AgBBIZ monospecific antibodies for detect~on

of hydatid antigens in serum, showed a sensitivity of 82.00% and a specificity of 96.66%.

In comparison, the efficiency of the Dot-ELISA using anti-Eg-rCW24 antibodies

(90.52%) for detection of hydatid antlgens m serum was observed to be hrgher than the

Dot-ELISA using both anti-Eg-rCWl2 (85.78%) and anti-Rcc Eg-AgB8/2 (88.94%)

antibodies.

The Dot-ELISA using affinity purified anti-Eg-rCW24 antibody showed the highest

efficiency in comparison to other two antibodies (anti-Eg-rCW12 and an t i l ec Eg-

AgB8/2) for detection of hydatid antigens in semm. Tho Ag-ELISA using anti-Eg-rCW24

antibody showed the efficiency similar to that of the Ag-ELISA using other antibodies

(anti-Eg-rCW12 and anti-Rec Eg-AgB812). The Dot-ELISA, therefore, using anti-Eg-

rCW24 antibody showed to be a simple and rapid diagnostic test for diagnosis of CE.

In conclusion, in the present study, both Ag-ELISA and Dot-ELISA using affinity

purified anti-Eg-rCW24 and anti-Eg-rCW12 monospecific antibodies for detection of

hydatid antigens in serum showed high sensitivity and high specificity. Hence both the

techniques could be used for diagnosis of CE. The Dot-ELISA, has the addded advantage

of bwog more convenient, simple, rapid, cheap and reliable, does not mui re costly

equipment and requires only lesser amounts of spccimons and nagants.