VECTOR INCRIMINATION IN MALARIA ENDEMIC HILLY AREAS OF BANGLADESH Kabirul BASHAR JSPS RONPAKU PhD Research Fellow Kanazawa University, Japan & Assistant Professor Jahangirnagar University, Bangladesh Advisor Nobuko TUNO Associate Professor Graduate School of Natural Science and Technology Kanazawa University, Japan

General background Plasmodium falciparum Plasmodium vivax 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

Life cycle of malaria parasite Fig. courtesy: CDC

Global Malaria Map 2009 Map courtesy: malaria atlas project

Estimated malaria incidence and death by region (WHO, 2010) WHO region (endemic countries) No. of cases, 2009 No. of deaths, 2009 Global Total (106) 225 million (100%) 781 thousand (100%) Africa (43) 176 million (78%) 709 thousand (91%) South-East Asia (10) 34 million (15%) 49 thousand (6%) Eastern Mediterranean (12) 12 million (5%) 16 thousand (2%) Western Pacific (10) 2 million (1%) 5.3 thousand (1%) Americas (23) 1 million (<1%) 1.3 thousand (<1%) Europe (8) 0 (0%)

Malaria situation in Bangladesh, 2011 (WHO, 2011) Total population 162.2 million Population in malarious areas 55.16 million Number of confirmed malaria cases 55,830 P. falciparum proportion (Including RDT Positives) 93% Number of deaths due to malaria 37 (Report)

Malaria endemic areas in Bangladesh Name of District No. of Sub district 1. Kurigram 2 2. Sherpur 3 Mymensingh 4 Netrokona 5 Sunamgonj 6 Sylhet 7 Moulvibazar 8 Hobigonj 9 Chittagong 13 10 Khagrachari 11 Rangamati 12 Bandarban Cox's Bazar

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

Incriminated malaria vector in Bangladesh Primary vector An. baimai An. philippinensis An. sundaicus An. minimus Secondary vector An. aconitus An. annularis An. vagus This species was identified as a vector but now a days Anopheles baimai are not found in many places but there have malaria. (Elias et al., 1982; Maheswary et al., 1992, 1993,1994)

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

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 environmental factors and malaria To assess the socio- demographic factors related to malaria

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

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

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

Study site

Abdomen Positive Negative Collection of Mosquitoes using LT, PSC and HLC Species Identification and Preservation Bisect the mosquitoes Abdomen Thorax +Head Blood Meal ELISA Positive Negative PCR Combined the results

Mosquito collection and identification Human bait Light trap Pyrethrum spray Identification

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

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

Results and discussion

Anopheles species composition as the proportion collected using light traps (LTs), pyrethrum spray (PS), and human bait (HB)   Species LT PS HB N Indoors Outdoors An. vagus 879 0.182 0.212 0.936 An. philippinensis 655 0.338 0.335 0.759 An. minimus s.l. 142 0.081 0.065 0.069 An. peditaeniatus 139 0.094 0.045 0.002 An. barbirostris 130 0.059 0.083 An. karwari 128 0.073 0.061 An. umbrosus 0.032 0.119 An. hyrcanus group 52 0.031 0.023 An. nigerrimus 0.004 An. subpictus 40 0.005 0.056 An. pallidus 24 0.011 0.015 An. baimai 23 0.103 An. maculatus s.l. 22 0.008 An. annularis 18 0.013 An. jeyporiensis 7 0.003 An. kochi 5 An. aitkenii 4 0.001 An. jamesii 3 An. tessellatus 2 An. varuna An. fluviatiles 1 Sum 2456 1094 784 549 29

Sampling methods No. of species Light trap 31 Pyrethrum spray 6 Human bait 4 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.

Percentage of blood meals identified and their feeding status Blood Meal Identified (%) Blood-fed 96.79 Gravid 87.93 Half-gravid 95.60 Unfed 10.67 Total 53.66

Human blood index (HBI) Species Indoor Outdoor Overall HBI % N HBI % An. baimai 3 66.67 2 100 80 An. minimus s.l. 34 47.06 21 38.1 43.64 An. annularis 4 50 33.33 37.5 An. jamesii   An. pallidus 8 An. maculatus s.l. 10 40 1 26.67 An. philippinensis 134 19.4 109 19.27 18.43 An. subpictus 27 14.81 12.12 An. vagus 550 10.55 55 25.45 10.29 An. peditaeniatus 63 7.94 6.41 An. barbirostris 31 3.23 6.45 4.62 An. umbrosus 24 81 6.17 4.55 An. hyrcanus group 16 6.25 7 4.35 An. karwari 41 2.44 3.39 An. aitkenii An. kochi An. nigerrimus 28

Indoor Collections Outdoor collections N HBI 977 6.96 341 11.73 Human blood index was higher in the mosquitoes caught at outdoors (light trap) (Chi square test, p<0.01)

Host selection index (Bi) of Anopheles species in Kumari, Bangladesh p-value Human Bovine Goat Others An. annularis 0.33* 0.39* 0.28* 0.11 An. baimai 0.77* 0.23* 0.05 An. barbirostris 0.04 0.89* 0.07 <0.01 An. maculatus s.l. 0.34* 0.37* 0.06 An. minimus s.l. 0.54* An. pallidus 0.29* 0.49* 0.15 0.02 An. philippinensis 0.16 An. vagus 0.09 0.79* 0.08

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

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. jamesii and 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.

Section 2 False-positivity of circumsporozoite protein (CSP)-ELISA in zoophilic anophelines in Bangladesh

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

Durnez et al. Malaria Journal 2011, 10:195 False positive circumsporozoite protein ELISA:　a challenge for the estimation of the entomological inoculation rate of malaria and for vector 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

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

Collection of Mosquitoes using LT, PSC and HLC Species Identification and Preservation Bisect the mosquitoes body Abdomen Thorax +Head CSP- ELISA CSP Negative CSP Positive Heat CSP Combined the results and analysis

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

Results and discussion

Number of Anopheline with positive reaction by heat stable CSP-ELISA assay Species N Number of positive Pf Pv210 Pv247 Mixed Total An. annularis 37 1 An. baimai 25 2 4 An. barbirostris 140 An. jeyporiensis 149 An. karwari 244 11 An. kochi 44 An. minimus s.l. 211 10 13 An. peditaeniatus 238 3 An. philippinensis 1169 9 An. vagus 1978 8

Species N + True+ % of False+ An. annularis 19 1 0.00 An. baimai 24 5 Number of false and true positive cases in anophelines tested for Plasmodium Species N + True+ % of False+ An. annularis 19 1 0.00 An. baimai 24 5 4 20.00 An. barbirostris 140 3 66.67 An. jeyporiensis 142 An. karwari 244 17 11 35.29 An. kochi 44 2 50.00 An. minimus s.l. 211 16 13 18.75 An. peditaeniatus 139 6 An. philippinensis 1169 38 25 34.21 An. vagus 1978 53 52.83

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

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

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.

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

Background Meteorological factors 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

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

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

Results and discussion

No. of anophelines collected Total mosquito captured and average temperature, rainfall and humidity in the study areas Place Temperature Monthly Rainfall Humidity No. of anophelines collected Sreemangal 17-29.3 0-601 mm 64-88% 716 Lama 21-32.5 0-1191 mm 56-88% 2443

Monthly malaria cases and environmental variables in Kumari

Monthly malaria cases and species abundance in Kumari

Monthly malaria cases and species abundance in Kumari

Pearson’s correlation coefficient (R) of most abundance species with malaria cases p-value An. baimai 0.312 0.323 An. jeyporiensis -0.224 0.484 An. karwari -0.325 0.303 An. minimus s.l. -0.347 0.270 An. peditaeniatus -0.181 0.574 An. philippinensis -0.199 0.535 An. vagus -0.188 0.559 An. willmori 0.486 0.110

Environmental variables Significant variables to affect anopheline species composition and abundance (Canonical correspondence analysis) Environmental variables Kumari Sreemangal F p-value 15 days lag Rainfall 2.78 0.016* 2.08 0.008* Relative Humidity 1.76 0.102 1.86 0.092 Temperature 3.62 0.006* 1.0 0.38

Ordination diagram of most abundance anophelines species and environmental variables of Kumari

Concluding remarks Major peaks of anophelines species in March Smaller peaks during September An. baimai and An. willmori strongly associated with rainfall Temperature has significant effect on mosquito density Incidences of malaria all year round in Bandarban

Section 4 Analysis of some socio-demographic factors related to malaria in Bangladesh

Background Socio-demographic factors affect malaria incidence Little documented evidence exists on the awareness and practices of the adult population regarding malaria.

Materials and Methods Knowledge Attitude Demographic Condition Association with malaria Villagers (interview) Practice

Knowledge, Attitudes, and Practices (KAP) survey Interview with standard pre-tested questionnaires Observe socio-demographic condition

Headlines of the KAP survey Information on household members Economic status Status of the living room Regular practices Malaria awareness Information on bet net and use of bed net Use of insecticide About domestic animal

Results and discussion

Socio demographic characteristics of the respondents Number % a) Gender Female 155 77.9 b) Educational level No formal education 76 38.19 c) Occupation Farmer 41 20.6 House work 49 24.6 d) Tribe No 191 96 e) No. of family member <6 110 55.3 f) Monthly income 70-140 US$ 101 50.8 g) Malaria history Yes

Awareness and practices of the respondents regarding malaria and its transmission Characteristics Number % a) Malaria transmitted by Bitten any mosquito 132 66.3 b) Symptoms of malaria Fever with rigor 149 74.9 Intermittent fever 17 8.5 c) Malaria could be prevented by Limiting mosquito breeding 25 12.6 Bed net 98 49.2 d) Malaria treatment provided by Govt. Hospital 34 17.1 Private health center 96 48.2 e) Enter the house previous night <6 pm 115 57.8 6 pm - 9 pm 81 40.7 f) Spent time last afternoon (6-9PM) Inside 118 59.3 Outside 79 39.7

Association of malaria with the socio demographic conditions and behavior of the respondents Variables Response Malaria cases p-value N % Roof of the house Straw/ thatch 20 10.05 <0.05 (df=2) Tin 80 40.20 Concrete/Cement 1 0.50 Partition of the house Jute stick/bamboo 2.51 NS (df=3) Concrete/cement Mud 37.69 Domestic animals No 18 9.05 (df=1) Yes 83 41.71 Use of Bed net 3 1.51 98 49.25 Use of insecticides 68 34.17 33 16.58

Concluding remarks The respondents did not have adequate knowledge on vector and their biting habits ITN were not found very effective in malaria prevention in Bangladesh People who spent time at outside have more risk to get malaria Malaria risk in endemic areas is larger in residents of poorly built houses

Overall recommendation We confirmed, human-bait is the best method to catch anthropophilic species. LT sampling is taken to as neutral sampling methods to reflect real fauna. Standard method of CSP-ELISA should be modified. People should take preventive measure when they stay outside.

Overall recommendation Mosquito breeding place management must be included in malaria control program. Health education is essential in improving the knowledge of the rural community. Longitudinal entomological survey covering several consecutive years is recommended.

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