SEASONAL MOLECULAR CHARACTERIZATIONS OF AN ASYMPTOMATIC COHORT IN A MALARIA ENDEMIC DISTRICT OF GHANA: UTILIZATION OF THE KINTAMPO DSS

Agyeman-Budu, A.,1 Brown, C.,2 Adjei, G.,1 Randall, A.,3 Dosoo, D.K.,1 Poku Asante, K.,1 Sutherland, C.,3 Wilson, M.,2 Owusu-Agyei, S.1

1. Kintampo Health Research Centre, Ghana 2. Noguchi Memorial Institute for Medical Research

College of Health Sciences, University of Ghana3. Department of Infectious and Tropical Diseases,

London School of Hygiene and Tropical Medicine

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Tuffobi

Kawampe

ChirandaYaara

Nyamebekyere No1Asantekwa

Akora

Kintampo Central

Bawakura

BrediAmpoma

KrutakyiAjina

Sabule

20 0 20 Kilometers

BoundaryRoads

# Towns/Villages

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Towns/Villages in Kintampo District where the survey was conducted

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Kawampe

Chiranda

Nyamebekyere No 1

AkoraNyamebekyereBrechakrom

Attakrom/Attakura 2

Ajina Krutakyi

Ampoma

Bredi junctionBredi

BawakuraNante-zongoSoraa

Sabule

Asantekwa

Yaara GbuongyongaTuffobi

NtarebanTuffobi

Kintampo Central

20 0 20 Kilometers

BoundaryRoads

# Villages

N

Towns/Villages in Kintampo District

BURKINA FASO

TO

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GULF OF GUINEA

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BACKGROUND 1

Current malaria vaccine candidate molecules, as well as new antimalarial combination drugs, at various stages of development, eventually reaching clinical testing stages, will require to be tested in malaria endemic African populations before licensure and deployment.

Thus in the advent of malaria vaccine trials in Africa, and for the fact that this district is a clinical trial site, it was of immense importance to ensure that malaria indices were well characterized prior to any tests; as this could be helpful in monitoring disease-conditions prevailing before, during and after the trials.

BACKGROUND 2

Study was conducted as part of a study which was determining the patterns and characteristics of Plasmodium falciparum (p.f) infections and morbidity in Kintampo as part of the site’s agenda for malaria vaccines and drugs intervention studies.

It was of immense importance, to rely on our strong demographic surveillance-system to facilitate the effective tracing of participants to enable collection of blood samples consistently during this study.

GENERAL OBJECTIVE

To determine the distribution of p.f expressing the polymorphic putative protective antigen: merozoite surface protein 2 (MSP2), in an asymptomatic cohort of different age groups during different seasons of the year.

STUDY DESIGN 1

Using the Kintampo Demographic Surveillance System we

1. Divided the study site into sixteen clusters

2. Identified compounds/residence of all our study participants

3. Followed-up participants on a two-month rotation.

STUDY DESIGN 2 Finger prick blood was collected onto filter

papers on each bi-monthly rotational visit through out the entire study year

The blood-blot filter papers, of 600 randomly selected (100 from each rotation) positive microscopically/asymptomatic participants aged 3 weeks to 78 years, were analysed using a nested polymerase chain (PCR) reaction protocol.

GENOTYPING Parasite genomic material was extracted using the

chelex (Wooden et al, 1993) method Negative controls were generated during

extraction while positive controls were obtained from MR4.

Nested PCR MSP2 genotyping protocol of Cattamanchi et al (2003) was used

Amplified DNA was resolved by a 2% agarose gel electrophoresis using a reference ladder

RESULTS 1-Electrophoregram showing MSP2 polymorphisms:

2.0% ethidium bromide-stained agarose gel

RESULTS 2Age prevalence of Mean Multiplicity of Infection (MOI)

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1

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3

4

5

6

7

8

9

10

0 - 4 years 5 - 9 years 10 - 17 years 18 - 79 years

Age

Me

an

Mu

ltip

lic

ity

Of

Infe

cti

on

s

Mean MOI

Linear (MeanMOI)

MOI = Average of all MSP2 antigenic variants distributed among hosts of a particular group

RESULTS 3

Rainy and dry seasons, key determinants of many infectious diseases in the country, were experienced through-out the year

Seasonal Mean Multiplicity of Infections

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1

2

3

4

5

6

7

8

NOV/DEC 03 JAN/FEB 04 M AR/APR 04 M AY/JUN 04 JUL/AUG 04 SEP/OCT 04

Months/Seasons

Me

an

Mu

ltip

licit

y O

f In

fec

tio

ns

All age groups Under five years

RESULTS 4Seasonal prevalence of MOI and MSP2 antigenic families: FC27 and IC/3D7

Seasons Months Total alleles Mean MOI Range Antigen Freq %

DRY SEASON (NOV/DEC)

131 1.8 0-4 F 39 29.8

I 92 70.2

DRY SEASON

(JAN/FEB)

230 2.7 0-6 F 72 31.3

I 158 68.7

RAINY SEASON (MAR/APR)

279 3.0 0-7 F 102 36.6

I 177 63.4

DRY/RAIN SEASON

(MAY/JUN)

131 2.0 0-5 F 25 19.1

I 106 80.9

RAINY SEASON (JUL/AUG)

193 2.4 0-6 F 48 24.9

I 145 75.1

RAINY SEASON (SEP/OCT)

51 1.3 0-4 F 10 19.6

I 41 78.4

RESULTS SUMMARISED

In March/April of the study year, children less than 5 years old had a mean MOI of 7, while all age groups had mean MOI of 3.

In May/June a mean MOI of 2 was observed in children under 5 years while a mean MOI of 1 was observed in all age groups.

Mean MOI of 7 was most frequent between January and

April, while the least and most frequent MOI of 1 was observed in November/December.

During March/April, IC/3D7 out-numbered FC27 variants by a ratio of 2:1. However, in the other seasons, the proportion was wider: 4:1 respectively. Both antigenic variants peaked during March/April, and were at their lowest numbers during September/October.

DISCUSSION OF RESULTS Lower acquired immunity of under-fives, therefore higher

probability to habour more MSP2 variants

Younger individuals lack experience to recognize and treat malaise conditions

Trends observed thought to be as a result of:1. Rains Start (Mar/Apr) - High MOI2. Rains subside (May/Jun) – MOI drops3. 2nd Rainy Season (Jul/Aug) – High MOI4. Dry Season begins (Sep/Oct) – Lowest MOI5. Dry Season (Nov – Feb) – Steady rise in MOI

IC/3D7 the less virulent variant

CONCLUSIONS

Younger and older participants were asymptomatic, but younger participants carried relatively higher number of p.f variants.

IC/3D7 strains of P.f is the most frequent strain to occur in the asymptomatic population given any time of year.

Potential of this asymptomatic population to harbour and transmit both susceptible and resistant P.f parasites is highest between January and April.

Thus, not only should public health and environmental practices be strictly adhered to during these months, but also, potential visitors to this site during these months should make prophylaxis a priority.

ACKNOWLEDGEMENT Chiefs and community members of the

district

Participants (guardians and children) in the study

Staff at KHRC/NMIMR/LSHTM especially

staff who participated in the study

Funding from the Bill and Melinda Gates Foundation and GHS/MOH of Ghana

THANK YOU