Kabirul BASHAR JSPS RONPAKU PhD Research Fellow Kanazawa University, Japan &

1. VECTOR INCRIMINATION IN MALARIA ENDEMIC HILLY AREAS OF BANGLADESH Advisor Nobuko TUNOAssociate Professor Graduate School of Natural Science and Technology Kanazawa University, Japan Kabirul BASHARJSPS RONPAKU PhD Research FellowKanazawa University, Japan & Assistant ProfessorJahangirnagar University, Bangladesh

2. General background Malaria is a serious and sometimes fatal disease caused by human Plasmodiums that infects a certain type of Anopheles mosquito which feeds on humans. • Plasmodium falciparum • Plasmodium vivax • Plasmodium ovale • Plasmodium malaria • Plasmodium knowlesi – a species that causes malaria among monkeys

3. Life cycle of malaria parasite Fig. courtesy: CDC

4. Global Malaria Map 2009 Map courtesy: malaria atlas project

5. Estimated malaria incidence and death by region (WHO, 2010)

6. Malaria situation in Bangladesh, 2011 (WHO, 2011)

7. Malaria endemic areas in Bangladesh

8. Dominant malaria vector in the world Map courtesy: malaria atlas project

9. Incriminated malaria vector in Bangladesh Secondary vector An. aconitus An. annularis An. vagus Primary vector An. baimai An. philippinensis An. sundaicus An. minimus (Elias et al., 1982; Maheswary et al., 1992, 1993,1994)

10. Background of the study • Vector incrimination is a prerequisite for understanding of malaria transmission. • Poor information of malaria vector in Bangladesh.

11. Study objectives • Incrimination vector species in malaria endemic areas To study host preference To determine infection rate with Plasmodium species To examine places and hours of blood feeding • To study the relation between environmentalfactors and malaria • To assess the socio- demographic factors related to malaria

12. Outlines of the presentation • Section 1: Blood feeding pattern • Section 2: Plasmodium infection rate • Section 3: Seasonal prevalence • Section 4: KAP study

13. Section 1 Blood-feeding patterns of Anopheles mosquitoes in a malaria-endemic area of Bangladesh

14. Background • Anophelines exhibit a wide range of host preferences • Degree of anthropophily, affect the efficacy of malaria vector

15. Study site

16. Blood Meal ELISA Positive Negative PCR Combined the results

17. Mosquito collection and identification Human bait Light trap Pyrethrum spray Identification

18. Host detection of blood meals in mosquitoes Enzyme-Linked Immuno Sorbent Assay (ELISA) using three kinds of antibodies Human Bovine Goat Polymerase chain reaction (PCR) using primers of 5 animals humans, bovines, dogs, goats/sheep, and pigs Followed by Electrophoresis to know size of amplified products

19. HBI is the proportion of human blood within an entire mosquito blood meal The selection (Bi) index for species iwas calculated as Where Bi is the selection index for mosquito species i,wiis the forage ratio for mosquito species i, and n is the number of different types of blood sources available

20. Results and discussion

21. Anophelesspecies composition as the proportion collected using light traps (LTs), pyrethrum spray (PS), and human bait (HB)

22. Species composition was method specific. Light trap caught most of species. 100% different species assemblies were caught by Pyrethrum spray catch and Human bait catch.

23. Percentage of blood meals identified and their feeding status

24. Human blood index (HBI)

25. Human blood index was higher in the mosquitoes caught at outdoors (light trap) (Chi square test, p<0.01)

26. Host selection index (Bi) of Anopheles species in Kumari, Bangladesh

27. Summary of the results • Species composition differed up to sampling method • HB is effective to collect anthropophilic species • HBI values was higher in outdoor collected mosquitoes • Mosquito host preference depends on the availability of host

28. Discussion • We need to compare multiple sampling methods since respective method cause specific bias. • HBI values were high in An. baimai, An. minimus, An. annularis. An. jamesiiand An. pallidus. • Biting may take place in outdoors=bed nets may be not enough to prevent mosquitoes biting. • Mosquito host preference depends on the availability of host.

29. Section 2 False-positivity of circumsporozoite protein (CSP)-ELISA in zoophilicanophelines in Bangladesh

30. Background • Effective control is only possible if potential vectors are accurately determined • There are three methods to find parasites in mosquitoes, microscopic observation, ELISA, and PCR • CSP-ELISA have been widely used in the world after 1980’s • There have been false-positive of Plasmodium reported in the ELISA method

31. Durnez et al. Malaria Journal 2011, 10:195False positive circumsporozoite protein ELISA:a challenge for the estimation of the entomological inoculation rate of malaria and forvector incrimination They reported that false positive may occur in CSP-ELISA when tested-mosquitoes took blood meals from cattle, pig, or goats. The fault can be detected by two ways • Heat specimens at 100℃ for 10 minutes before application • Reconfirm by PCR

32. Study areas Materials and Methods Lama, Bandarban Srimangal, Moulvibazar Durgapur, Netrokona Dhobaura, Mymensing

33. CSP- ELISA CSP Negative CSP Positive Heat CSP Combined the results and analysis

34. Test of Plasmodium infection status of mosquito • CSP-ELISA Pf PV210 PV 247 • Heat stable CSP-ELISA Pf PV210 PV 247

35. Results and discussion

36. Number of Anopheline with positive reaction by heat stable CSP-ELISA assay

37. Number of false and true positive cases in anophelines tested for Plasmodium

38. Correlation between the human blood index (HBI) and true-positivity ratios in 15 Anopheles species

39. Summary of the results • We detected Plasmodium in 10 species. • Five species of 10 were the firstly reported in Bangladesh • False-positive reactions (40%) occurred in zoophilic species

40. Discussion • Vector species and high malaria endemic areas have changed comparing with our study and previous studies between 1980-1994. • We need to change the standard method of CSP-ELISA to avoid false positivity.

41. Section 3 Seasonal abundance of Anopheles mosquitoes and their association with environmental factors and malaria incidence in Bangladesh

42. Background • Meteorologicalfactors affect on mosquito breeding • The relationship between climate variables and mosquito abundance can provide important information to determine parasite activity levels and, therefore, disease risk • Exact information on the seasonal prevalence of mosquito fauna in a region is essential for the development of efficient vector control programs

44. Study design • We chose 2 sites in malaria endemic area • Collected mosquitoes (January to December) • Collected daily environmental variable data, i.e., Temperature,Rainfall, and Relative Humidity • Collected malaria patient data • Analyse the data with Canoco for windows 4

45. Correlation Malaria cases Mosquito collection CCA Daily Rainfall Temperature Humidity

46. Results and discussion

47. Total mosquito captured and average temperature, rainfall and humidity in the study areas

48. Monthly malaria cases and environmental variables in Kumari

49. Monthly malaria cases and species abundance in Kumari

50. Monthly malaria cases and species abundance in Kumari