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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. 2. 3. Kintampo Health Research Centre, Ghana Noguchi Memorial Institute for Medical Research College of Health Sciences, University of Ghana Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine
Towns/Villages in Kintampo District N BURKINA FASO # Yaara Gbuongyonga # ## # Tuffobi Ntareban # Kawampe Chiranda Villages Roads Boundary # Tfoi ub # Asantekwa # # # Sabule # Soraa COTE D’IVOIRE Nyamebekyere No 1 Kintampo Central # Akora Nante-zongo # Nyamebekyere # # Brechakrom Bawakura Attakrom/Attakura 2 Ampoma # # Bredi junction TOGO # Ajina # Krutakyi 20 0 20 Kilometers North and South Towns/Villages in Kintampo District where the survey was conducted N # # # Yaara Kawampe Chiranda Tf ob u i # # Nyamebekyere No 1 Asantekwa # Kintampo Central # Sabule # # Bawakura Akora Towns/Villages Roads Boundary # # # Ajina Ampoma # Bredi # Krutakyi 20 GULF OF GUINEA 0 20 Kilometers
BACKGROUND 1 n n 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 n n 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 n To determine the distribution of p. f expressing the polymorphic putative protective antigen: merozoite surface protein 2 (MSP 2), in an asymptomatic cohort of different age groups during different seasons of the year.
STUDY DESIGN 1 n 1. 2. 3. Using the Kintampo Demographic Surveillance System we Divided the study site into sixteen clusters Identified compounds/residence of all our study participants Followed-up participants on a two-month rotation.
STUDY DESIGN 2 § n 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 n n 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 MR 4. Nested PCR MSP 2 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 MSP 2 polymorphisms: 2. 0% ethidium bromide-stained agarose gel
RESULTS 2 MOI = Average of all MSP 2 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
RESULTS 4 Seasonal prevalence of MOI and MSP 2 antigenic families: FC 27 and IC/3 D 7 Seasons Months DRY SEASON (NOV/DEC) DRY SEASON (JAN/FEB) RAINY SEASON (MAR/APR) DRY/RAIN SEASON (MAY/JUN) RAINY SEASON (JUL/AUG) RAINY SEASON (SEP/OCT) Total alleles Mean MOI 131 1. 8 Range Freq % 51 2. 4 1. 3 0 -5 0 -6 0 -4 70. 2 F 72 31. 3 158 68. 7 F 102 36. 6 177 63. 4 F 25 19. 1 106 80. 9 F 48 24. 9 I 193 2. 0 0 -7 92 I 131 3. 0 29. 8 I 279 0 -6 39 I 2. 7 F I 230 0 -4 Antigen 145 75. 1 F 10 19. 6 I 41 78. 4
RESULTS SUMMARISED n 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. n 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. n n 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/3 D 7 out-numbered FC 27 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 n Lower acquired immunity of under-fives, therefore higher probability to habour more MSP 2 variants n Younger individuals lack experience to recognize and treat malaise conditions 5. Trends observed thought to be as a result of: Rains Start (Mar/Apr) - High MOI Rains subside (May/Jun) – MOI drops 2 nd Rainy Season (Jul/Aug) – High MOI Dry Season begins (Sep/Oct) – Lowest MOI Dry Season (Nov – Feb) – Steady rise in MOI n IC/3 D 7 the less virulent variant n 1. 2. 3. 4.
CONCLUSIONS n Younger and older participants were asymptomatic, but younger participants carried relatively higher number of p. f variants. n IC/3 D 7 strains of P. f is the most frequent strain to occur in the asymptomatic population given any time of year. n n 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 n n 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