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.

Investigation of Relative Development and Reproductivity Fitness Cost in Three Insecticide-Resistant Strains of Aedes aegypti from Thailand.

Knockdown resistance (kdr) and detoxification enzymes are major resistance mechanisms in insecticide-resistant Aedes aegypti throughout the world. Persistence of the resistance phenotype is associated with high fitness of resistance alleles in the absence of insecticide pressure. This study determined the relative fitness cost of three insecticide-resistant strains of Aedes aegypti-PMD, PMD-R, and UPK-R-and a hybrid under similar laboratory conditions in the absence of insecticide. The PMD strain is resistant to DDT with no kdr alleles; the PMD-R is resistant to DDT and permethrin with 1534C homozygous kdr alleles; and UPK-R is resistant to DDT, permethrin, and deltamethrin with 989P + 1016G homozygous alleles. The DDT-resistant PMD strain had the highest fitness compared with the two DDT/pyrethroid-resistant strains (PMD-R and UPK-R) and hybrid. Consistent fitness costs were observed in the DDT/pyrethroid-resistant strains and hybrid, including shorter wing length, reduced egg hatchability, shorter female lifespan, and shorter viability of eggs after storage, whereas no effect was observed on blood feeding rate. In addition, reduced egg production was observed in the PMD-R strain and prolonged developmental time was seen in the UPK-R strain. The corresponding hybrid that is heterozygous for kdr alleles was fitter than either of the homozygous mutant genotypes. This is in accordance with the high frequency of heterozygous genotypes observed in natural populations of Ae. aegypti in Chiang Mai city.

Pyriproxyfen-Treated Polypropylene Sheets and Resting Boxes for Controlling Mosquitoes in Livestock Operations.

Many insect vector species of medical and veterinary importance are found abundantly in areas where animals are held. In these areas, they often rest for a period of time on objects around the animals both before and after blood feeding. However, the use of neurotoxic insecticides for vector control is not advised for use in such shelters as these chemicals can pose hazards to animals. The present study evaluated the efficacy of pyriproxyfen (PPF), an insect growth regulator, applied to polypropylene sheets and resting boxes on the reproductivity of mosquitoes found in animal shelters in Chiang Mai, Thailand. The sheets sprayed with 666 mg PPF/m² were set on the inner wall of a cowshed and kept in place for 3 h (6.00 to 9.00 pm). During this time, fully blood-fed female mosquitoes that landed and remained continuously on the sheets for 5, 10, and 20 min were collected. The results, involving Anopheles subpictus, An. vagus, Culex gelidus, Cx. tritaeniorhynchus, and Cx. vishnui, revealed significant reductions in oviposition rates, egg hatchability, pupation, and adult emergence in the PPF-treated groups compared to the control groups. Adult emergence rates were reduced to 85.6⁻94.9% and 95.5⁻100% in those exposed for 10 and 20 min, respectively. The sheets retained their effectiveness for three months. The PPF-treated (666 mg/m²) resting boxes (35 × 35 × 55 cm) were placed overnight at a chicken farm where Cx. quinquefasciatus predominated. Blood-fed mosquitoes were collected in the morning and reared in the laboratory. Oviposition rates were reduced by 71.7% and adult emergence was reduced by 97.8% compared to the controls. PPF residual spray on surface materials in animal sheds is a potential method for controlling mosquitoes. Further studies are needed to evaluate the impact of PPF-treated materials on wild populations.

Serologic characteristics of hepatitis B virus among hill-tribe children in Omkoi district, Chiangmai province, Thailand.

INTRODUCTION: Thailand has integrated hepatitis B (HB) vaccination of newborns into the national Expanded Program on Immunization (EPI) in 1992. This has led to a dramatic decrease of HBsAg prevalence in children. However, HB vaccine coverage in remote areas is not well-known. This study aimed to investigate serologic characteristics of hepatitis B virus (HBV) among hill-tribe children in Omkoi District, Chiangmai Province, Thailand. METHODOLOGY: This cross-sectional study was conducted on stored samples collected from hill-tribe children attending the primary/secondary school in Omkoi District in December 2014. Sera were tested for HBsAg, anti-HBs and anti-HBc using enzyme immunoassays (MUREX, DiaSorin, Italy). Samples with anti-HBc positive were further assessed for HBV DNA using an in-house HBV DNA semi-nested polymerase chain reaction (PCR) assay. RESULTS: Of 210 children evaluated, 4 (1.9%:95% CI 0.5-4.8) were HBsAg-positive. Of the 206 children HBsAg negative, 17 were anti-HBc and anti-HBs positive, 15 anti-HBc positive only, 26 anti-HBs positive only and 148 negative for both anti-HBc and anti-HBs. None of the children with anti-HBc were positive for HBV DNA. CONCLUSIONS: A high percentage of children had no markers of HBV protection suggesting that HB vaccine coverage was not optimal in this area. Our results warrant HBV serologic investigations in other remote areas to assess whether HB vaccine coverage needs to be improved and to identify children who should be vaccinated.

Effect of Relaxation of Deltamethrin Pressure on Metabolic Resistance in a Pyrethroid-Resistant Aedes aegypti (Diptera: Culicidae) Strain Harboring Fixed P989P and G1016G kdr Alleles.

Mutation of the voltage-gated sodium channel genes or knockdown resistance (kdr) and metabolic resistance in Aedes aegypti (L.) (Diptera: Culicidae) are important resistance mechanisms against pyrethroids. The present study investigated the effect of relaxation of deltamethrin selection pressure on the level of mixed-function oxidases (MFO), when the allele frequency of S989P+V1016G mutations is fixed in a resistant Ae. aegypti strain (UPK-R) from Chiang Mai, Thailand. The mosquitoes were divided into two groups, exposure and nonexposure groups, and maintained for 12 generations in an insectary room. Adults of the exposure group (F3 to F12) were treated with 0.05% deltamethrin-impregnated papers. The median lethal concentrations (LC50) of deltamethrin of larvae were determined by World Health Organization (WHO) bioassay. MFO activity was determined in F0 and F12. The results revealed that there was a decreasing trend of adult mortality rates in the exposure group over time. The larval LC50 values of the exposure group were gradually increased, whereas those of the nonexposure group were gradually decreased. The level of MFO activity in the nonexposure group (F12) was lower than the parent and exposure groups (F12) by 1.5 and 4-fold in the larvae, respectively, and 1.5 and 2.5-fold in the adult females, respectively. However, the frequency of P989+G1016 alleles in both groups was 100% up to F12 when the experiment ended. This study indicates that there was a significant but small reduction in the activity levels of MFOs when pyrethroid selection pressure is relaxed in this kdr strain of Ae. aegypti.

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.

A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

BACKGROUND: Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale. METHODS: A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR). RESULTS: The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory. CONCLUSIONS: Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

Excessive fluoride consumption increases haematological alteration in subjects with iron deficiency, thalassaemia, and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency.

Excessive fluoride consumption leads to accelerated red blood cell death and anaemia. Whether that increases the haematological alteration in subjects with haematological disorders (iron deficiency, thalassaemia, and G-6-PD deficiency) is still unclear. The fluoride in serum and urine and haematological parameters of students at Mae Tuen School (fluoride endemic area) were analysed and compared to those of students at Baan Yang Poa and Baan Mai Schools (control areas). Iron deficiency, thalassaemia, and G-6-PD deficiency were also diagnosed in these students. The students at Mae Tuen School had significantly (P < 0.001) higher levels of mean fluoride in the serum and urine than those in control areas. In both control and fluoride endemic areas, students with haematological disorders had significantly lower levels of Hb, Hct, MCV, MCH, and MCHC than those without haematological disorders. Moreover, the lowest levels of Hb, MCH, and MCHC were observed in the students with haematological disorders who live in the fluoride endemic area. Thus, the excessive fluoride consumption increased haematological alteration in subjects with iron deficiency, thalassaemia, and G-6-PD deficiency and that may increase the risk of anaemia in these subjects.

Additive effect of knockdown resistance mutations, S989P, V1016G and F1534C, in a heterozygous genotype conferring pyrethroid resistance in Aedes aegypti in Thailand.

BACKGROUND: Mutation in the voltage-gated sodium channel gene that results in knockdown resistance (kdr), is a major mechanism of pyrethroid resistance in several mosquito species. In Aedes aegypti, V1016G (occurring with and without S989P) and F1534C mutations are common and widely distributed throughout Asia. The G1016 allele is known to be associated with resistance to type I and II pyrethroids. The C1534 allele is primarily associated with resistance to type I pyrethroids and is known to be a recessive allele in conferring kdr. METHODS: We performed crossing experiments using a P989 + G1016 homozygous mutant strain (UPK-R), a C1534 homozygous mutant strain (PMD-R) and a pyrethroid susceptible strain (PMD) to determine the insecticide susceptibility of different genotypic hybrids. Allele-specific PCR methods were used to confirm the genotypes. Metabolic resistance caused by oxidative enzymes and esterase enzymes was ruled out by the addition of piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate, BNPP), respectively. RESULTS: The median lethal concentration (LC50) of deltamethrin susceptibility of a S/P989 + V/G1016 + F/F1534 double heterozygous hybrid from the UPK-R × PMD cross was 0.57 (95 % CI: 0.51-0.63) µgl(-1), which was about 12-fold lower than for UPK-R, 6.98 (6.10-8.04) µgl(-1), and only about 4-fold greater than the susceptible PMD, 0.13 (0.12-0.15) µgl(-1). This resistance returned to 0.08 (0.07-0.09) µgl(-1) on the addition of PBO suggesting that the P989 + G1016 kdr alleles are recessive. The LC50 of the S/P989 + V/G1016 + F/C1534 triple heterozygous hybrid was 3.58 (3.21-3.95) µgl(-1), which was intermediate between that of the homozygous mutant genotypes, being 2-fold higher than the C1534 homozygote and 2-fold lower than the P989 + G1016 homozygote. These minor differences and the high LC50 values of the triple mutated heterozygote indicate there is some degree of functional equivalence of the P989 + G1016 and C1534 alleles in the heterozygote. Addition of PBO decreased the LC50 values by 2-fold, from 3.58 (3.21-3.95) to 1.52 (1.35-1.73) µgl(-1), suggesting that oxidase enzymes play a partial role in resistance. The results are consistent with the median lethal time (LT50) of the triple mutated heterozygote against 0.05 % deltamethrin paper. An adult susceptibility test also revealed that the triple mutated heterozygote was resistant to deltamethrin and permethrin. CONCLUSIONS: The combination of the three kdr alleles in the triple mutated heterozygote, S/P989 + V/G1016 + F/C1534, confers high resistance to pyrethroids. This heterozygous form is common in Ae. aegypti populations throughout Thailand and may have an adverse effect on the efficacy of a mosquito control program using insecticide-based approaches.

Temporal frequency of knockdown resistance mutations, F1534C and V1016G, in Aedes aegypti in Chiang Mai city, Thailand and the impact of the mutations on the efficiency of thermal fogging spray with pyrethroids.

In Thailand, control of dengue outbreaks is currently attained by the use of space sprays, particularly thermal fogging using pyrethroids, with the aim of killing infected Aedes mosquito vectors in epidemic areas. However, the principal dengue vector, Aedes aegypti, is resistant to pyrethroids conferred mainly by mutations in the voltage-gated sodium channel gene, F1534C and V1016G, termed knockdown resistance (kdr). The objectives of this study were to determine the temporal frequencies of F1534C and V1016G in Ae. aegypti populations in relation to pyrethroid resistance in Chiang Mai city, and to evaluate the impact of the mutations on the efficacy of thermal fogging with the pyrethroid deltamethrin. Larvae and pupae were collected from several areas around Chiang Mai city during 2011-2015 and reared to adulthood for bioassays for deltamethrin susceptibility. These revealed no trend of increasing deltamethrin resistance during the study period (mortality 58.0-69.5%, average 62.8%). This corresponded to no overall change in the frequencies of the C1534 allele (0.55-0.66, average 0.62) and G1016 allele (0.34-0.45, average 0.38), determined using allele specific amplification. Only three genotypes of kdr mutations were detected: C1534 homozygous (VV/CC); G1016/C1534 double heterozygous (VG/FC); and G1016 homozygous (GG/FF) indicating that the F1534C and V1016G mutations occurred on separate haplotypic backgrounds and a lack of recombination between them to date. The F1 progeny females were used to evaluate the efficacy of thermal fogging spray with Damthrin-SP(®) (deltamethrin+S-bioallethrin+piperonyl butoxide) using a caged mosquito bioassay. The thermal fogging spray killed 100% and 61.3% of caged mosquito bioassay placed indoors and outdoors, respectively. The outdoor spray had greater killing effect on C1534 homozygous and had partially effect on double heterozygous mosquitoes, but did not kill any G1016 homozygous mutants living outdoors. As this selection pressure would be expected to have led to an increase in frequency of the G1016 allele, it is likely that the relatively stable kdr mutation allele frequencies observed here result from balancing selection, in the form of overdominance for VG/FC genotypes and/or the effects of fluctuating environments that vary in insecticide exposure.

Red Cell Indices and Formulas Used in Differentiation of ß-Thalassemia Trait from Iron Deficiency in Thai Adults.

ß-Thalassemia (ß-thal) and iron deficiency cause most microcytic anemias. Red cell indices and formulas have been established as simple, fast, and inexpensive in discrimination between these two hematological disorders in school children. However, whether these formulas could be applied to diagnose ß-thal trait and iron deficiency in adult Thai subjects is unclear. The aim of this study was to examine the diagnostic accuracy of five red cell indices [red blood cell (RBC) counts, mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW)] and nine formulas (RDW/RBC, RDW Index, Sirdah, Green and King, Mentzer, England and Fraser, Ehsani, Srivastava and Shine and Lal). Their sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV), efficiency, and Youden's Index were analyzed in 102 ß-thal trait and 64 iron deficiency adult Thai subjects. The RDW/RBC formula proved to be the most reliable index as they had 100.0% specificity and PPV and the highest efficiency (94.58%) and Youden's Index (91.18%), as well as high sensitivity (91.18%) and NPV (87.67%). Therefore, this formula could be used in initial discrimination of ß-thal trait from iron deficiency in adult Thai subjects.

Insecticides resistance in the Culex quinquefasciatus populations from northern Thailand and possible resistance mechanisms.

The mosquito vector Culex quinquefasciatus is known to be resistant to insecticides worldwide, including Thailand. This study was the first investigation of the insecticide resistance mechanisms, involving metabolic detoxification and target site insensitivity in C. quinquefasciatus from Thailand. Adult females reared from field-caught larvae from six provinces of northern Thailand were determined for resistant status by exposing to 0.05% deltamethrin, 0.75% permethrin and 5% malathion papers using the standard WHO susceptibility test. The overall mortality rates were 45.8%, 11.4% and 80.2%, respectively. A fragment of voltage-gated sodium channel gene was amplified and sequenced to identify the knock down resistance (kdr) mutation. The ace-1 gene mutation was determined by using PCR-RFLP. The L1014F kdr mutation was observed in all populations, but the homozygous mutant F/F1014 genotype was found only in two of the six provinces where the kdr mutation was significantly correlated with deltamethrin resistance. However, none of mosquitoes had the G119S mutation in the ace-1 gene. A laboratory deltamethrin resistant strain, Cq_CM_R, has been established showing a highly resistant level after selection for a few generations. The mutant F1014 allele frequency was significantly increased after one generation of selection. A synergist assay was performed to assess the metabolic detoxifying enzymes. Addition of bis(4-nitrophenyl)-phosphate (BNPP) and diethyl maleate (DEM), inhibitors of esterases and glutathione S-transferases (GST), respectively, into the larval bioassay of the Cq_CM strain with deltamethrin showed no significant reduction. By contrast, addition of piperonyl butoxide (PBO), an inhibitor of cytochrome P450 monooxygenases, showed a 9-fold reduction of resistance. Resistance to pyrethroids in C. quinquefasciatus is widely distributed in northern Thailand. This study reports for the first time for the detection of the L1014F kdr mutation in wild populations of C. quinquefasciatus in Thailand. At least two major mechanisms, kdr and cytochrome P450 monooxygenases, confer resistance to deltamethrin in Thai C. quinquefasciatus populations.

Prevalence of anemia, iron deficiency, thalassemia and glucose-6-phosphate dehydrogenase deficiency among hill-tribe school children in Omkoi District, Chiang Mai Province, Thailand.

The prevalaence of anemia, iron deficiency, thalassemia and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency were examined among 265 hill-tribe school children, 8-14 years of age, from Omkoi District, Chiang Mai Province, Thailand. Anemia was observed in 20 school children, of whom 3 had iron deficiency anemia. The prevalence of G-6-PD deficiency and ß-thalassemia trait [codon 17 (A>T), IVSI-nt1 (G>T) and codons 71/72 (+A) mutations] was 4% and 8%, respectively. There was one Hb E trait, and no α-thalassemia-1 SEA or Thai type deletion. Furthermore, anemia was found to be associated with ß-thalassemia trait in 11 children. These data can be useful for providing appropriate prevention and control of anemia in this region of Thailand.

Red cell indices and formulas used in differentiation of ß-thalassemia trait from iron deficiency in Thai school children.

Red cell indices and formulas have been established as simple, fast, and inexpensive means for discrimination between the ß-thalassemia (ß-thal) trait and iron deficiency. However, there were no reports of the diagnostic reliability of different red cell indices and formulas in discrimination of ß-thal trait from iron deficiency in the Thai population. The aim of this study was to examine the diagnostic accuracy of five red cell indices [red blood cell (RBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (Hb) (MCH), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW)] and eight formulas (Sirdah, Green & King, RDW Index, Menzler, England & Fraser, Ehsani, Srivastava, and Shine & Lal). Their sensitivity, specificity, positive and negative prognostic value and efficiency, were analyzed in 77 Thai school children, 21 with the ß-thal trait and 56 with iron deficiency. The Sirdah and Srivastava formulas proved to be the most reliable indexes as they had 100.0% sensitivity and negative predictive value, the highest efficiency (97.4%), and the highest Youden's Index value (96.4%). Therefore, these formulas could be used in initial discrimination of the ß-thal trait from iron deficiency in Thai school children.

Detection of the V1016G mutation in the voltage-gated sodium channel gene of Aedes aegypti (Diptera: Culicidae) by allele-specific PCR assay, and its distribution and effect on deltamethrin resistance in Thailand.

BACKGROUND: Resistance to pyrethroid insecticides is widespread among populations of Aedes aegypti, the main vector for the dengue virus. Several different point mutations within the voltage-gated sodium channel (VGSC) gene contribute to such resistance. A mutation at position 1016 in domain II, segment 6 of the VGSC gene in Ae. aegypti leads to a valine to glycine substitution (V1016G) that confers resistance to deltamethrin. METHODS: This study developed and utilized an allele-specific PCR (AS-PCR) assay that could be used to detect the V1016G mutation. The assay was validated against a number of sequenced DNA samples of known genotype and was determined to be in complete agreement. Larvae and pupae were collected from various localities throughout Thailand. Samples were reared to adulthood and their resistance status against deltamethrin was determined by standard WHO susceptibility bioassays. Deltamethrin-resistant and susceptible insects were then genotyped for the V1016G mutation. Additionally, some samples were genotyped for a second mutation at position 1534 in domain III (F1534C) which is also known to confer pyrethroid resistance. RESULTS: The bioassay results revealed an overall mortality of 77.6%. Homozygous 1016G individuals survived at higher rates than either heterozygous or wild-type (1016 V) mosquitoes. The 1016G mutation was significantly and positively associated with deltamethrin resistance and was widely distributed throughout Thailand. Interestingly, wild-type 1016 V mosquitoes tested were homozygous for the 1534C mutation, and all heterozygous mosquitoes were also heterozygous for 1534C. Mutant homozygous (G/G) mosquitoes expressed the wild-type (F/F) at position 1534. However, the presence of the 1534C mutation was not associated with deltamethrin resistance. CONCLUSIONS: Our bioassay results indicate that all populations sampled display some degree of resistance to deltamethrin. Homozygous 1016G mosquitoes were far likelier to survive such exposure. However, resistance in some populations cannot be explained due to kdr mutations and indicates that other resistance mechanisms are operating. The presence of this mutation alone does not fully explain the resistance phenotype we see among Thai Ae. aegypti populations.

Enzymes-based resistant mechanism in pyrethroid resistant and susceptible Aedes aegypti strains from northern Thailand.

Previous studies have shown that permethrin resistance in our selected PMD-R strain of Aedes aegypti from Chiang Mai, Thailand, was associated with a homozygous mutation in the knockdown resistance (kdr) gene and other mechanisms. In this study, we investigated the metabolic mechanism of resistance of this strain compared to the PMD strain which is susceptible to permethrin. The permethrin susceptibility of larvae was determined by a dose-response bioassay. Two synergists, namely piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate (BNPP), were also added to determine if the resistance is conferred by oxidase or esterase enzymes, respectively. The LC(50) value for PMD-R (25.42 ppb) was ∼25-fold higher than for PMD (1.02 ppb). The LC(50) was reduced 3.03-fold in PMD-R and 2.27-fold in PMD when the oxidase inhibitor (PBO) was added, but little or no reduction was observed in the presence of BNPP, indicating that oxidative enzymes play an important role in resistance. However, the LC(50) previously observed in the heterozygous mutation form was reduced ∼eightfold, indicating that metabolic resistance is inferior to kdr. The levels of cytochrome P450 (P450) extracted from fourth instar larvae were similar in both strains and were about 2.3-fold greater in microsomal fractions than in crude supernatant and cytosol fractions. Microsome oxidase activities were determined by incubation with each of three substrates, i.e., permethrin, phenoxybenzyl alcohol (PBOH), and phenoxybenzaldehyde (PBCHO), in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD(+)), PBO, and BNPP. It is known that hydrolysis of permethrin produces PBOH which is further oxidized to PBCHO by alcohol dehydrogenase (ADH) and then to phenoxybenzoic acid (PBCOOH) by aldehyde dehydrogenase (ALDH). When incubated with permethrin, a small amount of PBCOOH was detected in both strains (about 1.1-1.2 nmol/min/mg protein), regardless of the addition of NADPH. The addition of PBO resulted in about 70% and 50% reduction of PBCOOH in PMD and PMD-R, respectively. The addition of BNPP reduced PBCOOH about 50% and 35% in PMD and PMD-R, respectively. Using PBOH as substrate increased PBCOOH ∼16-fold and ∼40-fold in PMD and PMD-R, respectively. Using PBCHO as substrate increased PBCOOH ∼26-fold and ∼50-fold in PMD and PMD-R, respectively. The addition of NADPH, and particularly NAD(+), increased the level of PBCOOH. Together, the results have indicated the presence of a metabolic metabolism involving P450, ADHs, and ALDHs in both PMD and PMD-R strains, with greater enzyme activity in the latter.

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.