Insecticide resistance in malaria vectors along the Thailand-Myanmar border

Abstract

Background: There is a paucity of data about the susceptibility status of malaria vectors to Public Health insecticides along the Thailand-Myanmar border. This lack of data is a limitation to guide malaria vector-control in this region. The aim of this study was to assess the susceptibility status of malaria vectors to deltamethrin, permethrin and DDT and to validate a simple molecular assay for the detection of knock-down resistance (kdr) mutations in the study area.

Methods: Anopheles mosquitoes were collected in four sentinel villages during August and November 2014 and July 2015 using human landing catch and cow bait collection methods. WHO susceptibility tests were carried out to measure the mortality and knock-down rates of female mosquitoes to deltamethrin (0.05%), permethrin (0.75%) and DDT (4%). DNA sequencing of a fragment of the voltage-gated sodium channel gene was carried out to identify knock-down resistance (kdr) mutations at position 1014 in mosquitoes surviving exposure to insecticides.

Results: A total of 6295 Anopheles belonging to ten different species were bioassayed. Resistance or suspected resistance to pyrethroids was detected in An. barbirostris (s.l.) (72 and 84% mortality to deltamethrin (n = 504) and permethrin (n = 493) respectively), An. hyrcanus (s.l.) (33 and 48% mortality to deltamethrin (n = 172) and permethrin (n = 154), respectively), An. jamesii (87% mortality to deltamethrin, n = 111), An. maculatus (s.l.) (85 and 97% mortality to deltamethrin (n = 280) and permethrin (n = 264), respectively), An. minimus (s.l.) (92% mortality, n = 370) and An. vagus (75 and 95% mortality to deltamethrin (n =148) and permethrin (n = 178), respectively). Resistance or suspected resistance to DDT was detected in An. barbirostris (s.l.) (74% mortality, n = 435), An. hyrcanus (s.l.) (57% mortality, n = 91) and An. vagus (97% mortality, n = 133). The L1014S kdr mutation at both heterozygous and homozygous state was detected only in An. peditaeniatus (Hyrcanus Group).

Conclusion: Resistance to pyrethroids is present along the Thailand-Myanmar border, and it represents a threat for malaria vector control. Further investigations are needed to better understand the molecular basis of insecticide resistance in malaria vectors in this area.

Keywords: Anopheles; Insecticide resistance; Malaria; Pyrethroids; Southeast Asia; Thailand-Myanmar border; kdr mutation.

Fig. 1

Map of the study sites. Three entomological surveys were conducted in four villages situated on the Myanmar side of the Thai-Myanmar border (HKT, Htee Kaw Taw; KNH, Ka Nu Hta; TPN, Htoo Pyin Nyar; and TOT, Tar Au Ta). Mosquitoes were collected using indoor and outdoor Human Landing Catch (HLC) in five sites and using Cow Bait Collection (CBC) in one site. Mosquitoes were shipped daily at the Shoklo Malaria Research Unit (SMRU, Mae Sot, Thailand) for identification, rearing and bioassays

Fig. 2

Mortality and knock-down (KD) rate determined following the WHO susceptibility test procedure for insecticide monitoring in malaria vectors. Alive female Anopheles were exposed during 1 h to insecticides (deltamethrin 0.05%, permethrin 0.75% and DDT 4%). KD rate was recorded at the end of the exposition period; the mortality rate was recorded after a 24 exposition period. Abbreviation: NA, not available

Fig. 3

Kinetics of the knock-down (KD) rate during insecticide exposure. KD rate was recorded every five minutes during the exposition period to insecticide. Dash-line indicates a 50% KD rate

Fig. 4

Molecular detection of the 1014 knock-down resistance (kdr) mutation on the voltage-gated sodium channel (VGSG) gene. a DNA sequence alignment of the fragment of VGSC gene encompassing nucleotides corresponding to the codon 1014 in various Anopheles species collected on the TMB. Bold character indicates the coding part of the DNA sequence (exon 20); codon 1014 is figured in red. b Consensus amino-acid sequence of the exon 20 determined for each anopheline taxa. The non-synonymous TCG polymorphism detected in An. hyrcanus (s.l.) is responsible for the L1014S kdr mutation; both heterozygous and homozygous mutations were detected. Other polymorphisms are either synonymous or located on non-coding part of the DNA sequence (GenBank Accession numbers KY677707–KY677716)