1. 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

2. 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

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)
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%)

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

7. 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

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

9. 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)

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 environmental factors 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. 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

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 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

20. Results and discussion

21. 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

22. 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.

23. 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

24. 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

25. 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)

26. 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

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. 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.

29. Section 2
False-positivity of circumsporozoite protein (CSP)-ELISA in zoophilic anophelines 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, 10:195 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

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

33. 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

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

35. Results and discussion

36. 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

37. 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

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
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 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

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

46. Results and discussion

47. 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
0-601 mm
64-88%
716
Lama mm
56-88%
2443

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

51. 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

52. 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

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

54. 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

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

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

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

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

59. 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

60. Results and discussion

61. 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
US$
101
50.8
g) Malaria history
Yes

62. 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

63. 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

64. 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

65. 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.

66. 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.

67. Thank you for your kind attention