Investigation of Anopheles Mosquito Species, Potential Vectors and Susceptibility of Anopheles Mosquitoes in Gold Mine Areas of Shwe Kyin and Bee Lin Townships in Myanmar

Thaung S, Mya MM, Oo SZM, Hein ZM and Maung YNM

Published on: 2022-11-10

Abstract

The study on vector bionomics and susceptibility of Anopheles mosquito vectors for malaria was studied at Gold mine areas of Aukminzaung village in Shwe Kyin Township Bago Region and Kyauklonegyi village Bee Lin Township Mon State in 2018. Anopheles mosquitoes were collected from fixed mosquito catching stations using animal baited Kanda net(K-net), Light traps indoor and outdoor collection. Mosquitoes were caught with WHO sucking tube from 18:00hour to 06:00 hour of the next morning. Larval survey was conducted in and around three Kilo meters from the study village. Susceptibility of collected mosquitoes was measured using WHO test kits. Vector incrimination study was done by ELISA test for conforming potential vectors. Malaria prevalence rate was found 38(10.35%) in Aukminzaung village and 29(8.82%) in Kyauklonegyi village and P. falciparum infection was high in both areas (i.e. 21(55.26%) and 18(62.07%).  A total of 928 and 329 Anopheles mosquitoes belonging to 7 and 6 species were collected from both Aukminzaung and Kyauklonegyi villages. The major vector An. minimus A (n=84 and 30) and secondary vectors An. maculatus (n=106 and 52), An. vagus (n=153 and 66), An. philippinensis (152 and 20), An. jamesii (n=131 and 83) were collected in both villages. Main vector An. minimus was collected higher (n=4.5 and 1.5/ person) respectively in 21:00-22:00 hour in biting outdoor collection in both villages. All total of 169 and 167 water holding places (water wells, rice fields, water pools, creeks and footprint etc.) were searched for larval positivity and found that 105(62.13%) and 94(56.29%) of the water holding places were positive with Anopheles larvae in both areas. High number of An. minimus larvae were collected in water pools, water wells rice field and An. vagus larvae were collected in foot prints in the villages. Anopheles minimus and An. maculatus mosquitoes were found sporozoites positive and susceptible to WHO recommended Deltamethrin 0.05%, Permethrin 0.75%, and Cyfuthrin 0.15% impregnated paper. Proper treatment of malaria patients and effective vector control measure is needed to eliminate the malaria endemicity in both areas.     

Keywords

Goldmine; Anopheles; Potential; Vector, Endemicity; An. Minimus; Susceptibility; Test kits

Introduction

Previously malaria is priority public health problem in Myanmar. But now malaria is remarkably failed down. In Myanmar, the malaria burden remains high despite a significant reduction of cases during the last decade, in the context of the disease elimination by 2030. Now the malaria is reduced in most of the areas, morbidity and mortality of malaria is reducing from 4% to 0.0001% by 100000 populations 2011 to 2018 despite, malaria remains in remote, gold mine and border areas [1].  The Ministry of Health has planned the elimination of the diseases by 2030 [2]. In the last decade in the Greater Mekong Sub-region, a continued decline of malaria cases (by 74%) and deaths (by 94%), although malaria remained as a major public health problem in a big population of people particularly in hard to reach areas or remote areas and mining and along international borders [3-5]. In countries like Ghana, the overall malaria prevalence was 22.8% in 2006/2007, with ~98% in mining areas that were predominantly Plasmodium falciparum infections [6], meanwhile in PNG (Lihir Island) Plasmodium vivax was more prevalent (57%) in 2006-2011, with a small number of Plasmodium malariae cases (< 3%) [7]. Most malaria cases in Brazil come from rural areas related to gold-mining in the Amazon Region, where 52% of the cases are caused by P. vivax, 30% by P. falciparum, and the rest are mix infections and P. malariae [8]. The malaria is endemic in mining areas in the world including Myanmar. In Myanmar malaria cases were high in Nanmatu and Bawtwin lead Mine [9]. In Myanmar P. vivax is gradually rising in many parts of the country [10,11].   Mining has historically played an important role in the expansion and creation of many productive human settlements and to the national economy of mineral rich countries, but simultaneously it has led to an increase the malaria transmission in mining areas [12]. African and Asian countries, such as Ghana, South Africa, and Papua New Guinea (PNG), report an important percentage of malaria cases originating in gold-mining areas. Myanmar is a mineral rich country and many mine areas as gold, metal, lead mines etc. are available indifferent parts of Myanmar.  African and Asian countries, such as Ghana, South Africa, and Papua New Guinea (PNG), report an important percentage of malaria cases originating in gold-mining areas. Likewise in the American continent, Brazil, Colombia, Venezuela, Suriname, and Peru are countries with significant gold extraction associated with high malaria prevalence [6,13,14], which presents mainly as asymptomatic cases and in age groups involved in mining [15,16]. Small-scale gold mining in remote areas of the Brazilian Amazon rainforest results in mosquito breeding grounds which in turn leads to surges in malaria transmission (www.globalhealth.stanford.edu/programs/ alpha). Ordinarily, rainforests are biologically diverse ecosystems, maintaining mosquito populations at low levels with natural competition and relatively few breeding grounds. Mining results in a completely transformed landscape, with many pools of standing water and a totally altered ecosystem. In these environments, mosquitos thrive. Gold mining in the tropics is often associated with malaria. Alluvial mining and the informal encampments occupied by migrant workers create an environment that is conducive to mosquito breeding and parasite transmission. In the late 2010’s, Costa Rica experienced a malaria resurgence caused by illegal gold mining. In 2018: locally-transmitted malaria cases increase to 70. Upon further investigation, researchers find that transmission is stemming primarily from migrant workers associated with an illegal open-pit gold mining region near the northern border [17].  Migrant miners live in informal settlements without mosquito protection like sealed living spaces or mosquito nets. This sudden influx of people exposed to the malaria vectors in the forest can result in malaria outbreaks. When miners return to their home cities or town, they can unknowingly be infected with malaria, causing malaria outbreaks far from the mining sites [18,19]. In the Amazon regions of Peru, Guyana, and Suriname (Guyana Shield) there has been an increase in malaria transmission mainly in informal mining camps due to the lack of opportune diagnosis, availability, and poor quality of antimalarials [14,20]. In Suriname, 66% of the miner population are Brazilian immigrants [21]. In Myanmar, Gold and other mines are mostly belonging to government. The mines are developed quickly during the past decade because government policies which have favoured foreign investment in mining, but also because there has been a rapid growth of illegal mines and they created big mosquito breeding sites due to big open water pools. There is no evidence of an association between malaria and the legal mining activities, it could be presumed that legal mining is more environment-friendly with less artificially man-made mosquito breeding sites. Although illegal mining has big open water pools created high density of different species of mosquito’s larvae and adults. In Myanmar There are 37 species of Anopheles mosquitoes, 10 species as malaria vectors. of this An. dirus and An. minimus are primary vectors of malaria other as An. annularis and An. sundiacus are local vector of malaria and An. aconitus, An. culicifacies, An. philippinensis, An. hyricanus, An. maculatus, An. jeyporiensis based on their relation to malaria [22,23]. Most of the Gold miners are come from different parts of Myanmar 80% of the gold miners are migrant and they have not malaria immunity, they are highly risk person in mining areas. Migrants are frequently induced by infected mosquitoes and Anopheles mosquitoes breed in manmade temporary and permanent habitats in open sky gold mining at a great variety of different conditions and adapt to local environmental condition. There is ill information about Anopheles mosquitoes, larval habitats, potential vectors and malaria transmission in gold mining areas in Myanmar. Therefore, attempt has been investigated the prevalence of malaria, Anopheles mosquito species, breeding habitats, potential vectors, sibling species and susceptibility of Anopheles mosquitoes in Gold mine areas of Shwe Kyin Township Bago Region and Bee Lin Townships Mon State in Myanmar.

Materials and Methods

Study Areas

 Study was conducted in Aukminzaung village in Shwe Kyin Township Bago Region and Kyauklonegyi village Bee Lin Township Mon State. About 1000 people reside in 225 houses in Aukminzaung village and 600 people reside in 130houses in Kyauklonegyi village during the study period. Malaria is prevalence in both areas.  Both areas are Gold mining areas. Plenty of legal and illegal gold mine are available in both areas. Over 30 gold mines are available in Aukminzaung village Shwe Kyin and 20 to 25 gold mines are available in Kyauklonegyi village, Bee Lin Township Mon State. All the mines are digging in forested hilly and foothill areas. Some are digging in paddy fields. Over 50 workers are working in a gold mining and most of the mining workers are come from different parts of Myanmar. Ninety Percent of the local villagers are farmers working in paddy fields, some are gold miners, bamboo cutter, charcoal maker, hunters, School teachers, health workers and government staff. One primary school are situated in both villages. One monastery and pagoda are available in both villages.

Study Period: Study period was 1year from March 2020 to February 2021.

Study Design: laboratory and field based descriptive study was conducted in both areas.

Blood Sample Collection: Finger prick blood was collected in both gold mining sites. Blood was put on a grease free glass slide. Thick and thin blood films were made on a glass slide and name and number was labeled on the left site of the slides and all the blood sample were dried in room temperature.

Staining Procedure: Thin films were fixed with absolute Alcohol, dried it in room temperature. All dried blood slides were stained with 10 Giemsa’s stain for 10 minutes. After 10 minutes’ blood slides were washed with buffer water and dried in room temperature. Thick and thin film were examined under high power oil immersion lens (x100), Olympus microscope. Malaria parasites were counted against 300WBC according to WHO methods.

Mosquito Collection

 Anopheles mosquitoes were collected using cattle bait big mosquito trap nets called Kanda net (K-net=330x330x180cm) and indoor and outdoor adult mosquitoes were collected by using CDC light traps from 18:00 to 6:00 hours of next day. Hourly catch was done from 18:00 to 24:00 hours using WHO sucking tube. Indoor and outdoor CDC light trap collection was done in ten selected households from 18:00 to 6:00 hours for 5days.

Larval Surveys

Larval surveys for identification of breeding sites were conducted in and around three kilo-meters away from the study sites. For larval detection water pools, domestic wells, stream, creeks, sand pools and all different types of water holding places as water pockets, coconut shells, discarded tins and utensils bamboo stumps including foot print of animals were examined by 3 Dips/water holding place [24]. The captured larvae and pupae were put in labeled plastic bags and brought back to the laboratory for species identification and colonization.

Mosquito Species Identification

Collected Anopheles mosquitoes and adult emerged from larval survey were identified according to different identification keys [25-27].  

Vector Incrimination

For vector incrimination study head and thorax were dissected from vector mosquitoes. Enzyme Linked Immunosorbent assay (ELISA) test for circumsporozoite antigen detection was supplemented according to Wirtz et al., [28].

 Insecticide Susceptibility Test

 Insecticide susceptibility tests (WHO test kit): Collected adult female Anopheles mosquitoes from the entomological survey were tested for measurement of insecticide susceptibility level using WHO test kits and standard procedures [29]. The efficacy of insecticides which are commonly used for vector control in malaria endemic areas namely as Permethrin 0.75%, Cyfluthrin 0.15% and Deltamethrin 0.05% impregnated paper with WHO test kits were provided.

 Procedure

 20 to30 fields collected Anopheles mosquitoes were introduced in WHO insecticide impregnated paper attached plastic tube (WHO test kit) by sucking tube and exposed for 1 hour. After one hour of exposing the mosquitoes were then removed from the plastic tubes and placed in clean plastic tubes without paper with 10% glucose soaked cotton and moisture were maintained by water soak dump towel. Percentage of knockdown was measured after 60 minutes’ exposure and effective mortality was assessed after 24 hours’ exposure. Two replicate testing were done to confirm the susceptibility of mosquitoes. If the quantity of collected mosquitoes was not more than 10 while we used pool mosquito samples to test insecticides susceptibility. Susceptibility of mosquitoes were determined according to WHO [29].

Data Analysis: Data entry and collected monthly malaria and mosquito data were analyzed by using Microsoft Excel software. Mosquito density, main vector of indoor and outdoor light traps, mosquito susceptibility and mortality were calculated in percent.

Results

Microscopic Examination of Malaria Parasite

The result of malaria prevalence in Shwe Kyin Township Bago Region and Bee Lin Township Mon State were shown in Table 1.

Table 1: Malaria parasite positive rate by microscopic examination in villages of Shwe Kyin and Bee Lin.

Study location

Total Examined

Total   positive

Malaria parasite species

Gameto cyte positive

Parasite density index

P.f

P.v

Mixed

Aukminzaung village, Shwe Kyin

367

38

(10.35%)

21

 (55.26%)

15 (39.47%)

2

(5.26%)

5

(1.36%)

1.93

Kyauklonegyi

village, Bee Lin

329

    29

 (8.82%)

     18

(62.07%)

       10

(34.48%)

1 (3.45%)

3(0.91%)

1.81

 

Total population

696

67

 (9.63%)

39

 (58.21%)

25

(37.31%)

3 (4.48%)

8

(1.15%)

1.87

Table 1 shows that malaria parasite positive rate in Aukminzaung village, Shwe Kyin Township Bago Region and Kyauklonegyi village, Bee Lin Township Mon State and found that in Aukminzaung village malaria positive rate in collected population was 38(10.35%) and parasite species positivity rate was found P. falciparum 21(55.26%), P. vivax positive rate was 15(39.47%) and Mixed infection rate was 2(5.26%). Gametocyte positive rate and Parasite density index were 1.36% and 1.93 respectively.  In Kyauklonegyi village, Bee Lin township, the malaria situation was found 29 (8.82%) positivity rate in recruited population of this P. falciparum positivity was 18(62.07%), P. vivax positive rate was 10(34.48%), mix infection was 3(4.48%), gametocyte positive rate was 3(0.91%) and Parasite density index 1.81 respectively. Overall positive rate was found 67(9.63%) highest positive rate was found P. falciparum 39(58.21%) followed by P. vivax 25(37.31%) and lowest was found mix (P.f + P.v) infection 3(4.48%) gametocyte positive rate was found 8(1.15%) and Parasite Density Index was 1.87. In comparison of positivity in both gold mine areas, malaria positivity rate was higher in Aukminzaung village 38(10.35%) than Kyauklonegyi village 29(8.82%). Plasmodium falciparum positive rate was higher in Kyauklonegyi village 62.07% than Aukminzaung village 55.26%. Plasmodium vivax, Mixed, gametocyte and Parasite Density Index were found higher in Aukminzaung village15(39.47%), 2(5.26%),6(1.36%) and 1.93 than Kyauklonegyi village10 (34.48%),1 (3.45%), 3(0.91%) and 1.81 respectively.

Table 2: Total collected Anopheles mosquitoes by different collection methods in Aukminzaung village Shwe Kyin Township.

Sr.No.

Species

CB

LT Indoor

LT Outdoor

Human bait

Total

Density (%)

1

An. minimus

46

22

10

6

84

9.05

2

An. vagus

106

19

20

8

153

16.49

3

An. philippinensis

108

8

24

12

152

16.38

4

An. jamesii

116

3

8

4

131

14.12

5

An. kawari

104

0

14

6

124

13.36

6

An. maculatus

68

6

18

14

106

11.42

7

An. barbirostris

118

18

30

12

178

19.18

 

Total

666

76

124

62

928

100

 

%

71.77

8.19

13.36

6.68

100

 

Table 2 shows that detail of mosquito collection by different methods in Aukminzaung village, Shwe Kyin Township and found that a total of 928 Anopheles mosquitoes were collected. Of this the highest density of Anopheles mosquitoes were collected by Cattle bait catching method 666 (71.77%) followed by Light trap outdoor 124 (13.36%) and lowest was collected by Human bait catching method 62(6.68%). In overall density, An. barbirostris was collected in highest density (19.18%) followed by An. vagus (16.49%) and An. philippinensis (16.38%) and lowest density was observed main vector An. minimus (9.05%). In cattle bait catching method the highest number of An. barbirostris was collected n=118 followed by An. jamesii n=116 and lowest number of An. minimus was collected (n=46). In Light trap indoor collection method, the highest number of An. minimus (n=22) was collected followed by An. vagus (n=19) and lowest number of An. jamesii (n=3) was collected. In light traps outdoor collection method, the highest number of An. barbirostris (n=30) was collected followed by An. philippinensis (n=24) and lowest number of An. jamesii (n=8) was collected. In human bait collection method, the highest number of An. maculatus (n=14) was collected followed by An. philippinensis and An. barbirostris (n=12 each) and lowest number of An. jamesii (n=4) on human landing and biting catch.

Table 3: Total collected Anopheles mosquitoes by different collection methods in Kyauklonegyi village Bee Lin Township Mon State.

Sr.No.

Species

CB

LT Indoor

LT Outdoor

Human bait

Total

Density (%)

1

An. minimus

17

7

4

2

30

9.12

2

An. vagus

36

10

14

6

66

20.06

3

An. philippinensis

16

0

2

2

20

6.08

4

An. jamesii

57

7

10

9

83

25.23

5

An. kawari

0

0

0

0

0

0

6

An. maculatus

42

2

6

2

52

15.81

7

An. barbirostris

60

4

10

4

78

23.71

 

Total

228

30

46

25

329

100

 

%

69.3

9.12

13.98

7.6

100

 

Table 4 Shows that detail of mosquito collection by different methods in Kyauklonegyi village, Bee Lin Township Mon State and found that a total of 329 Anopheles mosquitoes were collected. Of this the highest density of Anopheles mosquitoes were collected by Cattle bait catching method 228 (69.30%) followed by Light trap outdoor 46 (13.98%) and lowest density was collected by Human bait catching method 25(7.60%).

In overall Anopheles mosquito density, An. barbirostris was collected in highest density (23.71%) followed by An. jamesii (25.23%) and third highest was An. vagus (20.06% and lowest density was observed secondary vector An. philippinensis (6.08%). Low density of main vector An. minimus was collected 9.12%. An. kawari was absent in Bee Lin areas.

In cattle bait collection method, the highest number of An. barbirostris was collected (n=60) followed by An. jamesii (n=57) and lowest number of An. philippinensis was collected (n=16). Main vector of An. minimus was collected (n=17). In Light trap indoor method, the highest number of An. vagus was collected (n=10) followed by An. jamesii and An. minimus (n= 7 each) and lowest number of An. maculatus was collected (n=2). In Light trap outdoor catching method, the highest number of An. vagus was collected (n=14) followed by An. barbirostris and An. jamesii (n=10 each) and lowest number of An. philippinensis was collected (n=2). In human bait collection method, the highest number of An. jamesii was collected (n=9) followed by An. vagus (n=6) and lowest number of An. minimus, An. philippinensis, and An. maculatus were collected (n=2)

Table 4: Larval survey in different breeding sources in Aukminzaung village Shwe Kyin Township and Kyauklonegyi village Bee Lin Township of gold mine areas.

Aukminzaung village Shwe Kyin Township

Kyauklonegyi village Bee Lin Township

Breeding sources

No. of search

sites

No. of +ve sites  

 +ve sites

Species of larvae

Density

No. of search

No. of 

 +ve  sites

Species of larvae

Density

Water wells

7

6

53 An. minimus

83(9.82%)

11

9

29 An. minimus

45(7.14%)

 

 

 

14 An. philippinensis

 

 

 

7 An. jamesii

 

 

 

 

16 An. maculatus

 

 

 

9 An. maculatus

 

Creek(Back)

41

21

10 An. minimus

98 (11.60%)

34

12

17 An. minimus

91(14.44%)

 

 

 

32 An. jamesii

 

 

 

19An. jamesii

 

 

 

 

34 An. maculatus

 

 

 

21 An. maculatus

 

 

 

 

22 An. barbirostris

 

 

 

34 An. barbirostris

 

Valley

7

4

11 An. minimus

98(11.60%)

6

4

13 An. minimus

62(9.84%)

 

 

 

16 An .jamesii

 

 

 

15 An. jamesii

 

 

 

 

31 An. barbirostris

 

 

 

21 An. barbirostris

 

 

 

 

23 An. kawari

 

 

 

0 An. kawari

 

 

 

 

17 An. maculatus

 

 

 

13An. maculatus

 

Rice field

56

41

14 An. minimus

162(19.17%)

54

38

11 An.  minimus

145(23.02%)

 

 

 

24 An. maculatus

 

 

 

22 An. maculatus

 

 

 

 

53 An. barbirostris

 

 

 

61 An. barbirostris

 

 

 

 

23 An. kawari

 

 

 

0 An. kawari

 

 

 

 

11 An. philippinensis

 

 

 

8 An. philippinensis

 

 

 

 

37 An. vagus

 

 

 

43 An. vagus

 

Sand pools

7

5

25 An. minimus

48(5.68%)

11

5

17 An. minimus

24 (3.81%)

 

 

 

16 An. maculatus

 

 

 

7  An. maculatus

 

 

 

 

7 An. philippinensis

 

 

 

 

 

Water pools

16

13

63 An. minimus

265(31.36%)

13

9

21 An. minimus

167(26.51%)

 

 

 

39 An. maculatus

 

 

 

25 An. maculatus

 

 

 

 

76 An. barbirostris

 

 

 

58 An. barbirostris

 

 

 

 

38 An. philippinensis

 

 

 

28 An. philippinensis

 

 

 

 

49  An. vagus

 

 

 

35 An. vagus

 

Foot prints

35

15

57An.vagus

91(10.77%)

38

17

68 An. vagus

96(15.24%)

 

 

 

34An. barbirostris

 

 

 

28 An. barbirostris

 

 

169

105(62.13%)

 

845(100%)

167

94(56.29%)

 

630(100%)

Aukminzaung and Kyauklonegyi villages were found out of 169 and 167 breeding sources were detected for larval breeding condition and found that 105(62.13%) and 94(56.29%) breeding sources were Anopheles larvae positive. The highest density of Anopheles larvae was collected from water pools in both villages i.e. 265(31.36%) and 167(26.51%) followed by Rice fields 162(19.17% and 145(23.02%) and lowest density of Anopheles larvae were collected from Sand pools 48(5.68%) and 24(3.81%). In Aukminzaung village the highest number of An. barbirostris larvae was collected from Water pools (n=76) followed by An. minimus larvae from (n=63) from Water pools and lowest number of An. philippinensis was collected from Sand pools (n=7). In Kyauklonegyi village the highest number of An. vagus was collected from foot print (n=68) followed by An. barbirostris from Water pools (n=58) and lowest number of An. jamesii and An. maculatus from Sand pools.  Main vector An. minimus was collected in highest number from Water pools water n= 63 followed by water wells (n=53) and lowest number was collected from creeks (n=10) in Aukminzaung village. In Kyauklonegyi village the highest number of An. minimus was collected from water wells (n=29) followed by water pools (n=21) and lowest number of An. minimus was collected from Rice field (n=11). 

Table 5: Plasmodium sporozoite positivity in salivary gland of Anopheles mosquitoes collected from Aukminzaung and Kyauklonegyi villages by ELISA test.

Sr. No.

 

Aukminzaung village

Kyauklonegyi village

 

Species

Total collected

Sporozoite positivity

Total collected

Sporozoite positivity

1

An. minimus

84

1(1.19%)

30

0

2

An. vagus

153

0

66

0

3

An. philippinensis

152

0

20

0

4

An. jamesii

131

0

83

0

5

An. kawari

124

0

0

0

6

An. maculatus

106

1(0.94%)

52

0

7

An. barbirostris

178

0

78

0

 

Total

928

2(0.22%)

329

0

Table 5 shows that in out of all total of 928 wild caught Anopheles mosquitoes from Aukminzaung village Shwe Kyin Township were detected for sporozoite positivity and found that 2 Anopheles mosquitoes were found sporozoite positive 2/928(0.22%). Of this one An. minimus was detected for Pf sporozoite 1/84(1.19%) and another one An. maculatus was found Pv210 sporozoite positive 1/106(0.94%) and no sporozoite positivity was detected in Anopheles mosquitoes collected from Kyauklonegyi villages Bee Lin Township.

Table 6: Susceptibility status of Anopheles mosquito from Aukminzaung and Kyauklonegyi village using WHO Test kits method.

Sr.

No.

Anopheles Species

No. of tested mosquitoes

WHO insecticides impregnated paper

(WHO 1992)

Susceptibility status

Aukminzaung

Kyauklonegyi

1

An. minimus

30

30

 

Deltamethrin 0.05%,

Pernethrin 0.75%

Cyfuthrin 0.15%

 

 

 

 

 

Susceptible

 

 

 

 

2

 

An. vagus

30

30

3

An. philippinensis

30

20

4

An. jamesii

30

30

5

An. kawari

30

0

6

An. maculatus

30

30

7

An. barbirostris

30

30

Table 6 shows that all collected main vector An. minimus and secondary vector An. philippinensis, An. maculatus and other Anopheles species as An. vagus, An. jamesii, An. kawari and An. barbirostris were found susceptible to WHO recommended Deltamethrin 0.05%, Permethrin 0.75% and Cyfuthrin 0.15% insecticides impregnated paper.

Discussion

Malaria is a deadly diseases and high risk in hard to reach areas, border areas, insurgent areas and mining areas. Gold mine areas is a very high risk area due to most of nonimmune migrant population come from different part of the country. In Myanmar is endemic because tropical climate and plenty of breeding sources are available year round and main vector of An. dirus and An. minimus as well as secondary vectors are found in all season in different parts of hilly and plain areas [23,30-35].

In the present study malaria situation, Anopheles mosquitoes, potential vector, susceptibility and breeding sources in Gold mine areas of Aukminzaung and Kyauklonegyi villages were measured to conform the risk areas in Myanmar. Malaria situation in both gold mine areas were found high risk areas and P. falciparum was high in both areas followed by P. vivax and very low amount of mixed infection were found. Same high prevalence of P. falciparum was found in oil well digging areas in Kazuma village Sake Phyu Township Magway Region and water plant area in Taikky areas in Yangon Region [36,37]. In Katinehtit, Kamamaung Township Kayin State, P. falciparum infection was higher than P. vivax and year round malaria positive cases were found in both areas [38]. Although other researchers revealed that P. vivax is gradually increased in other parts of the country [10,39]. Sanchez, and his party revealed that in gold mining areas in Brazil, Migrant workers live in informal settlements, these conditions result in malaria outbreaks in gold mining regions in the Amazon Brazilian, results in an additional 59 cases of P. vivax malaria, and for each square kilometer of forest cleared, roughly 6.4 additional malaria cases are observed [18].  Douine and associates observed that during the investigation in an illegal gold mine in French Guiana, two malaria episodes were diagnosed among 46 persons. Twenty-six of them were from Sophie, where PCR-Plasmodium prevalence was estimated at 60% (15/26). This result was concordant with previous studies revealing high malaria endemicity in the gold miner population [40]. P. vivax infections, which comprise 24-40% of the total of cases of malaria in mining areas are transmitted by An. darlingi, An. albimanus, An. calderoni, and An. nuñeztovari and this has been observed in the Pacific Coast and South Pacific mining districts and the departments of Nariño, Cauca, and Valle del Cauca [41-43].

Most of the gold miner are migrants who are come from different malaria endemic and non-endemic areas were accumulated in one site and also they have not prevented the mosquito bite when they sleep. In the present study main vector An. minimus was higher in Aukminzaung village than Kyauklonegyi village and secondary vector as An. philippinensis, and An. maculatus were also higher in Aukminzaung village. Same result of main vector An. minimus and An. dirus has been found in Thabwewa village Oktwin Township Bago Region and Katinehtit village Kamamaung village Kayin State [31,44]. Other researcher mention that An. dirus density was higher than An. minimus in Ohnpinquin village Ye Phyu Township Taninthayi Region [32]. Although in Gonminsoe village Bago Region found that secondary vector An. culicifacies was abundantly collected followed by An. minimus was agreed with the present study. An. culicifacies are main vector of malaria in India, Pakistan, Srilankar and Iran and have 5 sibling species as An. culicifacies, A, B, C, D and E. Of this A, C, D and E are main vector of malaria [45-47]. The highest density of An. barbirostris was collected in Aukminzaung and An. jamesii was collected in Kyauklonegyi villages followed An. philippinesis and An. vagus in Aukminzaung and An. barbirostris in Kyauklonegyi. Other researchers revealed that Anopheles sinensis are main vector of malaria in China [48] and the most extensively occurring species of Anopheline mosquitoes in Indonesia are An. sundaicus, An. subpictus, An. barbirostris, An. maculatus, An. aconitus, and An. balabacensis, and all are implicated in malaria transmission [49, 50]. Gould et al., [51] revealed that Anopheles aconitus transmitted malaria in the central plain of Thailand.    

Although 4 different mosquito collection methods, the highest number of main vectors An. minimus was collected by Cattle bait collection method in both villages, followed by light trap outdoor collection method than Indoor light trap and Human bait outdoor methods in all villages. Although other researcher mentioned that main vectors are more likely to human blood and they were interred in households, may be both An. minimus and An. dirus mosquitoes are indoor vectors behavior [31]. Other researchers revealed that highest number of An. dirus was collected in outdoor and An. minimus was higher in indoor human bait collection method [31,32] the result was agreed with the behavior of An. minimus in the present study due to second most higher caught by light trap indoor method. Although in the present study An. minimus was collected in highest number by cattle bait method, it may be due to some behavior changes occurred in An. minimus group because there are 5 sibling species. Maung Maung Mya and his party observed that main vector An. minimus was abundantly collected by animal bait collection method in Ye Township Mon State is agreed with the behavior of An. minimus in the present study [52]. Secondary vector An. philippinensis, An. maculatus and non-vector An. barbirostris were collected in highest number by Cattle bait collection method followed by outdoor light trap catching method and lowest was observed by human bait collection. Same result has been found in Thabwewa village Oktwin and Paukkhaung Townships, Bago Region, Ye Phyu Township in Taninthayi Region, Pyin Oo Lwin Mandalay Region, Kamamaung Township, Kayin State and Ye Township Mon State. [31-34, 52,53].  An. annularis are local vector of malaria in Raphine State [23], An. dirus, An. minimus, An. philippinensis and An. maculatus are main vector of malaria in India and Thailand [46,54-56], An. culicifacies, An. stephensii and An. fluviatellis [45,46], An. dirus, An. minimus and An. maculatus are main vectors of malaria in Thailand and Cambodia [ 54,57]. An. sinensis are main vector of malaria in China [58].  Malaria transmission in both areas of Gem and Bungoma districts in sub-Saharan Africa, the main malaria vectors are An. gambiae s.s., An. arabiensis and An. funestus [59]. In Rakhaine State, An. annularis is responsible for local transmission. An. sundaicus is responsible vector for malaria transmission in coastal regions [22, 23]. Anopheles sundaicus, An. subpictus, An. barbirostris, An. maculatus, An. aconitus, and An. balabacensis, vector of malaria and extensively occurring species of Anopheline mosquitoes in Indonesia [49,50]. Ndoen and his party observed that the three species as An. subpictus, An. barbirostris, and An. vagus, with sufficient longevity for pathogen development, An. barbirostris was found inside dwellings and had peak activity during the night [60].

In gold mining sites, plenty of manmade water pools were created high density of different species of mosquitoes and transmitted various mosquito borne diseases. In the present study in gold mining areas of Aukminzaung and Kyauklonegyi villages were found out of 169 and 167 breeding sources, 105(62.13%) and 94(56.29%) were found Anopheles larvae positive. The highest density of Anopheles larvae was collected from manmade water pools in both villages followed by Rice fields and lowest density of Anopheles larvae were collected from Sand pools in both gold mining areas. Main vector An. minimus was collected in all water sources of both mining areas, the highest number An. minimus larvae were collected from Water pools water followed by water wells and lowest number was collected from creeks in Aukminzaung village and in Kyauklonegyi village the highest number of An. minimus was collected from water wells followed by water pools and lowest number of An. minimus was collected from Rice field. Other researchers revealed that high density of An. minimus and An. culicifacies larvae were collected from sand pools in Thabwewa creek in Oktwin and Gonminsoe creeks in Paukkhaung Township Bago Region and slowly running water in Watwon village Pyin Oo Lwin Township and Slowly running water in the band of Yunsalin Creeks [31,33,34, 38]. Although in the present study low number of An. minimus larvae were collected in Creeks.  Other researchers revealed that high density of main vector An. dirus larvae were collected from rock pools in Thabwewa village and high density of An. dirus larvae were collected from domestic water wells in Mon, Taninthayi and Kayin State [31,32, 61,62]. Pe Than Htun associates mentioned that high density of An. minimus larvae were collected in rice field in Taungoo Township and Bawbin Dam areas of Thae Chaung Township Bago Region [63,64].          

Small-scale mining operations create unnatural forest-edge environments with clear-cut forest and pools of stagnant water, ideal mosquito breeding habitats. In Aukminzaung village the highest number of An. barbirostris and slightly low number of An. minimus larvae was collected from Water pools and lowest number of An. philippinensis was collected from Sand pools.  In Kyauklonegyi village the highest number of An. vagus was collected from foot print followed by An. barbirostris from Water pools and lowest number of An. jamesii and An. maculatus from Sand pools. In Nant Yin village High density of An. minimus and An. dirus larvae were collected from water pools, wells and creeks in Ohnpinkone and Bwedakone wards [65]. An. darlingi and An. albimanus are commonly in these districts and breed in within manmade water pools “open sky” mining. These two vectors maintain transmission which occurs predominantly in the first part of the night [66]. Anopheles. vagus larvae was abundantly collected from Foot print of both areas was agreed with the result of the larval survey study in Myebintha village Magway Region Bawbin dam areas in Bago Region where high density of An. vagus and An. barbirostris were collected from foot print, ponds sand pools, and water pools and An. cilicifacies was collected in sand pools [64] and the first time report of Maung Maung Mya and associates observed that An. minimus, An. maculatus, Cx. quinquefasciatus and Ae. cogalli larvae were bred together in domestic water wells in Ye Township, Mon State [62]. Seven types of mosquitoes breeding sources were investigated in gold mining sites with plenty of different species of mosquitoes. Manmade water pools in Gold mining create high density of mosquito larvae and transmit different types of mosquitoes borne diseases in mining site and near the environs.  Other researcher mention that Gold mining site create temporary or permanent man-made habitats in open sky gold-mining and these conditions can significantly impact the phenology and population dynamics of mosquito larvae populations and indirectly affect the dynamics of mosquito-borne diseases [67].

Malaria parasite infected Anopheles mosquito transmit malaria infection to human. Takken et al. pointed important factors for mosquitoes to become malaria vectors. These include mosquito density, behavioural factors, and vectorial capacity [50]. Behavioural characteristics include feeding habits, anthropophily or zoophily [68]; house-frequenting resting habits, exophily or endophily [69]; and site of feeding, endophagy or exophagy [68]. Vectorial capacity and the ability to transmit malaria is related to mosquito longevity and susceptibility to Plasmodium infection. An infective mosquito is a prerequisite for malaria transmission. It takes between 8 and 35 days to complete the parasite’s life cycle in the mosquito host, depending on the condition of the environment and the species of malaria parasites [69,70]. Sporozoite positive Anopheles mosquitoes are potential vector of malaria and perfume high risk of malaria transmission. In the present vector incrimination study found that a total of 928 and 239 wild caught Anopheles mosquitoes from Aukminzaung village Shwe Kyin and Kyauklonegyi village Bee Lin Townships were detected for sporozoite positivity and found that only 2 Anopheles mosquitoes were found sporozoite positive 2/928(0.22%) in Aukminzaung village. Of this one was An. minimus was detected for Pf sporozoite 1/84(1.19%) and another one was An. maculatus was found Pv210 sporozoite positive 1/106(0.94%). In and Kyauklonegyi villages Bee Lin Township were found no sporozoit positive in all tested Anopheles mosquitoes. Same sporozoite positivity was observed in An. dirus, An. maculatus, An. minimus and An. kochi in Boukpyin Township Taninthayi Region and An. annularis, An. minimus and An. culicifacies were found sircumsporozoite antigen positive in their salivary gland in Rakhine State [71]. Anopheles minimus and An. maculatus was found sporozoite positive in Yesitkan village Taikkyi Township Yangon Region and An. dirus and An. minimus was sporozoite positive in Katinehtit village Kamamaung Township in Kayin State as well as one An. minimus was sporozoites positive in Ye Township Mon State [34,37,62]. Tun Lin and his party observed that An. dirus and An. vagus in Thabwewa village were found sporozoite positive [31]. The importance of An. vagus requires more in depth study due to earlier findings during adult Anopheles surveys found P. falciparum sporozoite positive in An. vagus Although in the present study, An. vagus was negative for sporozoites by ELISA method.  Sporozoite positive Anopheles mosquitoes are potential vector of malaria and transmit malaria. In malaria endemic communities, a significant percentage (5-15%) of the population usually harbour malaria infections without showing clinical symptoms [72]. These individuals represent a pool of parasites for malaria transmission and thus perpetuate its spread [16,73, 35].

Susceptibility status of collected Anopheles mosquitoes were teste with WHO recommended impregnated paper. All collected main vector An. minimus and secondary vector An. philippinensis, An. maculatus and other Anopheles species as An. vagus, An. jamesii, An. kawari and An. barbirostris were found susceptible to WHO recommended Deltamethrin 0.05%, Permethrin 0.75% and Cyfuthrin 0.15% insecticides impregnated paper. Same results have been found in different areas of Myanmar. Maung Maung Mya and his party revealed that Anopheles mosquitoes from Shan, Bago, Mon, Sagaing, Mandalay, Kayin, Magway were susceptible to WHO recommended Test kit [74].  Although other researchers revealed that main vector of An. dirus and An. minimus in Thailand, An. dirus and An. minimus, An. culicifacies, An. stephensii and An. fluviatellis in India were resistance to Deltamethin0.05%, Permethrin0.75% [75]. Anopheles gambia has been shown to be resistant to pyrethroid insecticides in sub Saharan Africa [59]. Pyrethroid resistance has been spreading rapidly in sub-Saharan Africa and has been documented in 23 countries [76]. This may partly be in response to agricultural application and run-off of insecticides into mosquito breeding sites [77-79], but increasingly in response to selection pressure resulting from the scale up of insecticide-treated nets and indoor residual spraying as malaria prevention tools [80-82]. This subset of 192 individuals represented the most abundant species (≥20 samples per species) in the original molecular typing of species in 844 samples were testing for the presence of known insecticide resistance mutations for ace-1 G119S, rdl A296S, and kdr L1014F and examined insecticide mutation rates in rarer species and found that   the same three mutations typed in 68 individual samples representing 14 species and 2 unknown Anopheline samples [84]. Although in the present study all the collected primary, secondary and non-vectors of Anopheles mosquitoes were susceptible to WHO recommended pyrethroid insecticides Therefore, LLINs nets and RIS can prevent effectively in both Gold mining workers and villagers in both areas.A

Conclusion

Malaria epidemics are a high risk to gold miners and villages of both Aukminzaung and Kyauklonegyi villages throughout the year due to in gold mining most of the migrant workers live in inconspicuous settlements without prevention of mosquito bite, resulting high risk of malaria transmission. Plasmodium falciparum parasite positive rate was high in both areas. Main vector of An. minimus and secondary vectors of An. philippinensis and An. minimus and suspected vectors of An. vagus and An. barbirostris, An. jamesii and An. kawari were abundantly collected in both villages except An. kawari in Kyauklonegyi village and An. minimus and An. maculatus were found sporozoite positive in Aukminzaung samples. Therefore, these two vectors are potential vectors in the study area of Aukminzaung village in Shwe Kyin Township. All collected Anopheles mosquitoes were susceptible to WHO recommended Deltamethrin, Permethrin, and Cyfuthrin insecticides. Mosquito surveillance efforts should be continued to monitor changes in malaria vector abundance and distribution due to habitat modification and other plantations in near future in the region and the effects of global climate change and need to supplied Long Lasting Insecticide nets (LLINs) to all migrant workers and villagers because mosquitoes are susceptible to WHO insecticides. Therefore, LLINs nets can prevent the man vector contact effectively and prevent the malaria transmission in all migrants and villages in both areas and need to health care provider for properly care and treatment the antimalarial drugs in gold mining sites.

Acknowledgements

This study was supported by WHO (Apw) grant. I am thankful to our Director General who allowed me to do the research. And also, I am also highly thanks to TMO and authority concerns from Shwe Kyin and Bee Lin who were permitted and helpful to do the research works in their Townships. I am thankful to the staffs of Medical Entomology Research Division, were helpful to the research works till completion. 

Conflict Of Interest

 The author declared no potential conflicts of interest concerning the research, authorship, and publication of this article.

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