Novel real-time PCR assay detects widespread distribution of knock down resistance (kdr) mutations associated with pyrethroid resistance in the mosquito, Culex quinquefasciatus, in Thailand.

Susceptibility to pyrethroids in the mosquito Culex quinquefasciatus, the major vector of lymphatic filariasis, is being seriously threatened worldwide. Knockdown resistance (kdr), caused by mutations in the voltage gated sodium channel (VGSC) gene, particularly the L1014F mutation, is an important resistance mechanism. Our aim was to develop a real-time PCR with melt curve analysis to evaluate the distribution of the L1014F mutation in Cx. quinquefasciatus throughout Thailand and to determine the polymorphism pattern of a VGSC gene fragment spanning the L1014F mutation. A total of 3760 females from 18 localities across five regions of Thailand were bio-assayed by exposure to 0.05% deltamethrin WHO papers, showing mortality rates ranging from 2.4% to 83.0%. Genotyping of 753 dead and surviving mosquitoes using our novel real-time PCR assay with melt curve analysis and tetra-primer allele-specific PCR revealed the mutant F1014 allele is closely associated with the deltamethrin resistance phenotype. The L1014F mutation was found at high frequency throughout Thailand, particularly in the North. However, some survivors were homozygous for wild type L1014 allele, which were further sequenced for the IIP-IIS6 region of VGSC gene. The haplotype network of phenotypically characterized individuals indicated the presence of other possible kdr alleles/resistance mechanisms at play including two novel mutations, V978E and D992E. The finding of new putative kdr alleles and widespread distribution of the F1014 allele emphasizes the significant role of kdr mutations in pyrethroid resistance in Thai Cx. quinquefasciatus populations. Monitoring kdr variations and phenotypic resistance is critical for managing resistance in Cx. quinquefasciatus.

Efficacy of five commercial household insecticide aerosol sprays against pyrethroid resistant Aedes aegypti and Culex quinquefasciatus mosquitoes in Thailand.

Commercial insecticide aerosol sprays are widely used in households for controlling Aedes aegypti and Culex quinquefasciatus, the primary vectors of dengue virus and filarial worm, respectively. In Thailand, however, both mosquitoes are resistant to pyrethroids conferred by knockdown resistance (kdr) mutations, V1016G and F1534C in Ae. aegypti and L1014F in Cx. quinquefasciatus. This study evaluated the efficacy of five sprays (coded as AS1-AS5) with different formulations of pyrethroids against wild mosquitoes by using a cage bioassay in a furnished bedroom of a house. Five cages containing wild mosquitoes and five cages containing a pyrethroid susceptible strain of Ae. aegypti (25 females each), as a bio-indicator, were allocated in the room and spraying was operated for 15 s. Survivors and dead mosquitoes were genotyped individually for the kdr mutations using allele-specific PCR methods. Both mosquito species showed a high resistance to permethrin and deltamethrin with 12.5-58.0% mortality rates. For controlling Ae. aegypti, the spray AS4 showed the highest efficacy (mortality rates 76.0-100.0%, mean 95.2%), followed by AS2 (73.0-100.0%, mean 93.8%). For controlling Cx. quinquefasciatus, the best result was obtained from AS4 (66.0-98.0% mortality, mean 89.8%), followed by AS2 (73.0-97.0%, mean 84.5%). The sprays (AS4 and AS2) containing both type I and type II pyrethroids were more effective than those containing only type I pyrethroids or pyrethrum with the synergist piperonyl butoxide. The mutant G1016 and F1014 allele frequencies were significantly higher in the survivor groups than the dead groups of Ae. aegypti and Cx. quinquefasciatus, respectively, (P < 0.05). The efficacy of the sprays varied depending on the mosquito species, formulations, nozzles and locations of caged mosquitoes. The V1016G and L1014F mutations are associated with the reduced efficacy of sprays used in households for controlling resistant Ae. aegypti and Cx. quinquefasciatus mosquitoes, respectively.

Associations of childhood experiences and methamphetamine use among Akha and Lahu hill tribe youths in northern Thailand: A cross-sectional study.

BACKGROUND: Methamphetamine (MA) is a commonly used substance among youths, particularly those who are living in poor economic conditions with low levels of education and who have had bad childhood experiences. The Akha and Lahu hill tribe youths living on the Thailand-Myanmar-Laos border are identified as the group most vulnerable to MA use in Thailand. The study aimed to estimate the prevalence of MA use and determine its associations with childhood experiences among Akha and Lahu youths aged 15-24 years in northern Thailand. METHODS: A cross-sectional study was performed. Validated and sealed questionnaires were used to gather information from participants after obtaining the informed consent form. Questionnaires were completed by participants and their parents at home. Logistic regression was used to identify the associations between variables at the α = 0.05 level. RESULTS: A total of 710 participants participated in the study: 54.2% were Akha, 52.5% were females, 50.6% were aged 15-17 years, and 11.4% did not have Thai identification card (ID) cards. The overall prevalence of MA use at least once among Akha and Lahu youths was 14.5%. After controlling for all potential confounding factors, 8 variables were found to be associated with MA use. Males had a greater chance of MA use than females (AOR = 4.75; 95% CI = 2.27-9.95). Participants aged 21-24 years had a greater chance of MA use than those aged 15-17 years (AOR = 2.51; 95% CI = 1.11-5.71). Those who had a family member who used MA had a greater chance of MA use than those who did not (AOR = 5.04; 95% CI = 1.66-15.32). Those who had been physically assaulted by a family member while aged 0-5 years had a greater chance of MA use than those who had not (AOR = 2.29; 95% CI = 1.02-5.12). Those who had been physically assaulted by a family member while aged 6-14 years had a greater chance of MA use than those who had not (AOR = 3.15; 95% CI = 1.32-7.54). Those who had a close friend who used alcohol had a greater chance of MA use than those who did not (AOR = 2.24; 95% CI = 1.24-4.72). Those who had a highly confident personality had a greater chance of MA use than those who did not (AOR = 2.35; 95% CI = 1.17-4.69), and those who smoked had a greater chance of MA use than those who did not (AOR = 8.27; 95% CI = 4.42-15.46). CONCLUSIONS: All relevant government and nongovernment agencies together with the Ministry of Public Health Thailand should address MA use among Akha and Lahu youths by properly developing a community health intervention that lowers risk of MA use by addressing family relationships, male youth behaviors, and focused on those individuals with a highly confident personality.

Current prevalence of intestinal parasitic infections and their impact on hematological and nutritional status among Karen hill tribe children in Omkoi District, Chiang Mai Province, Thailand.

Intestinal parasitic infection represents a substantial problem for children living in rural or limited resources areas and significantly relates to anemia and nutritional status. This study aimed to determine the current prevalence of intestinal parasitic infections among school-age children of Karen hill tribe population in Omkoi District, Chiang Mai Province, Thailand and assess the impact of intestinal parasitic infection on hematological and nutritional status in those children. A total of 375 Karen hill tribe children, 6-14 years of age, in Omkoi District were randomly selected to participate in this study. Stool samples were collected and examined for intestinal parasitic infection through formalin-ether concentration method. Blood samples were collected for hematological and iron analysis. The overall prevalence of intestinal parasitic infection was 47.7% (179/375), with single infections (29.3%) and polyparatism (18.4%). The most common pathogenic parasite was Trichuris trichiura (16.0%), followed by Ascaris lumbricoides (13%) and Giardia lamblia (3.5%). In addition, non-pathogenic amoeba, Entamoeba coli was observed with a high prevalence rate (31.2%). Anemia and eosinophilia prevalence were 6.40% (24/375) and 74.7% (280/375), respectively. Eosinophilia was significantly more prevalent in children with intestinal parasitic infection compared to uninfected children. Among 249 children, 13.7% were iron deficiency, 9.6% were thalassemia and hemoglobinophathy and 8% were G-6-PD deficiency. A high prevalence infection rate was significantly associated with eosinophilia, but independently related to anemia and iron deficiency. Intestinal parasitic infections are endemic in school-age children of Karen hill tribe population in Omkoi District. These data highlight the need for an integrated approach to control transmission of intestinal parasites and improve the health and sanitation status of Karen hill tribe children in Thailand.

High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand.

OBJECTIVES: To develop rapid monitoring tools to detect the F1534C permethrin-resistance mutation in domain IIIS6 of the Aedes aegypti voltage-gated sodium channel gene and determine the frequency and distribution of this mutation in Thailand. METHODS: A TaqMan SNP genotyping and an allele specific PCR (AS-PCR) assay were developed and validated by comparison with DNA sequencing of homozygous susceptible and homozygous resistant laboratory strains, their reciprocal-cross progenies, and field-caught mosquitoes. To determine the resistance phenotype of wild-caught A. aegypti, mosquitoes were exposed to 0.75% permethrin paper. The AS-PCR assay was used to screen 619 individuals from 20 localities throughout Thailand. RESULTS: Overall, both assays gave results consistent with DNA sequencing for laboratory strains of known genotype and for wild-caught A. aegypti. The only slight discrepancy was for the AS-PCR method, which overestimated the mutant allele frequency by 1.8% in wild-caught samples. AS-PCR assays of permethrin-exposed samples show that the mutant C1534 allele is very closely associated with the resistant phenotype. However, 19 permethrin-resistant individuals were homozygous for the wild-type F1534 allele. DNA sequencing revealed all these individuals were homozygous for two other mutations in domain II, V1016G and S989P, which are known to confer resistance (Srisawat et al. 2010). The F1534C mutation is widespread in Thailand with mutant allele frequencies varying among populations from 0.20 to 1.00. CONCLUSIONS: These assays can be used for the rapid detection of the F1534C resistance mutation in A. aegypti populations. The F1534C, and other, mutations underlie an extremely high prevalence of pyrethroid resistance in Thailand.

Expression and characterization of three new glutathione transferases, an epsilon (AcGSTE2-2), omega (AcGSTO1-1), and theta (AcGSTT1-1) from Anopheles cracens (Diptera: Culicidae), a major Thai malaria vector.

Glutathione transferases (GSTs) (E.C.2.5.1.18) are multifunctional enzymes involved in the detoxification of many exogenous and endogenous compounds. This study aimed to characterize several new GSTs from Anopheles cracens, a major Thai malaria vector formerly known as Anopheles dirus. The three recombinant enzymes obtained were from the epsilon, theta and omega classes. They showed 80-93% identity to orthologous An. gambiae GSTs. AcGSTE2-2 possessed peroxidase activity that cannot be detected for the An. gambiae AgGSTE2-2. AcGSTT1-1 had high activity toward several substrates that are specific for mammalian theta class. The AcGSTO1-1 can use 1-chloro-2,4-dinitrobenzene, dichloroacetic acid, and hydroxyethyl disulfide substrates. The enzymes bound but did not metabolize the organophosphate temephos. The epsilon AcGSTE2-2 functioned as a peroxidase and DDT metabolizing enzyme. The theta AcGSTT1-1 functioned not only as peroxidase but also acted as a binding protein for organophosphates. The omega GST had thiol transferase activity suggesting a role in oxidative stress response.