Characterizing antibody responses to mosquito salivary antigens of the Southeast Asian vectors of malaria and dengue with a human challenge model of controlled exposure: a protocol.

Background: Measurement of antibody titers directed against mosquito salivary antigens in blood samples has been proposed as an outcome measure to assess human exposure to vector bites. However, only a handful of antigens have been identified and the specificity and longitudinal dynamics of antibody responses are not well known. We report the protocol of a clinical trial of controlled exposure to mosquito bites that aims to identify and validate biomarkers of exposure to bites of mosquito vector species that transmit malaria and dengue in Southeast Asia and some other parts of the world. Methods: This study is an exploratory factorial randomized control trial of controlled exposure to mosquito bites with 10 arms corresponding to different species ( Aedes aegypti, Ae. albopictus, Anopheles dirus, An. maculatus and An. minimus) and numbers of bites (35 or 305 bites in total over 6 weeks). Blood samples will be collected from study participants before, during and after mosquito biting challenges. Candidate peptides will be identified from published literature with antigen prediction algorithms using mosquito DNA sequence data and with immunoblotting assays carried out using protein extracts of dissected mosquito salivary glands and participants samples. Antibody titers against candidate peptides will be determined in participants samples with high-throughput cutting-edge immuno-assays. Quantification of the antibody response profile over time (including an estimate of the decay rate) and the effect of the number of bites on the antibody response will be determined using linear and logistic mixed-effects models for the continuous and the binary response, respectively. Conclusion: This research is expected to generate important knowledge for vector sero-surveillance and evaluation of vector-control interventions against malaria and dengue in the Greater Mekong Subregion. Registration: This study is registered with clinicaltrials.gov (NCT04478370) on July 20 th, 2020.

STK11 Causative Variants and Copy Number Variations Identified in Thai Patients With Peutz-Jeghers Syndrome.

Introduction Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant inherited disorder caused by germline mutations in the serine-threonine kinase 11 (STK11) tumor suppressor gene. This syndrome is characterized by hamartomatous gastrointestinal polyps, mucocutaneous melanin pigmentation, and a higher risk of developing various cancers. Methods We summarized the clinical and molecular characteristics of five unrelated Thai patients with PJS. Denaturing high-performance liquid chromatography (DHPLC) screening, coupled with direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA), were applied for the molecular analysis of STK11. Results A total of four STK11 pathogenic changeswere identified in the five PJS patients, including two frameshift variants (a novel c.199dup, p.Leu67ProfsTer96 and a known c.834_835del, p.Cys278TrpfsTer6) and two types of copy number variations (CNV), exon 1 deletion and exons 2-3 deletion. Among reported STK11 exonic deletions, exon 1 and exons 2-3 deletions were found to be the two most commonly deleted exons. Conclusion All identified STK11 mutations were null mutations that were associated with more severe PJS phenotypes and cancers. This study broadens the phenotypic and mutational spectrum of STK11 in PJS.

Outdoor residual spraying for malaria vector-control in Kayin (Karen) state, Myanmar: A cluster randomized controlled trial.

Outdoor and early biting by mosquitoes challenge the efficacy of bed nets and indoor residual spraying against malaria in the Greater Mekong Subregion. The objective of this study was to assess the efficacy of outdoor residual spraying (ORS) for malaria vector-control in this region. A cluster randomized controlled trial was conducted between July 2018 and April 2019 in twelve villages in Karen (Kayin) state, Myanmar. Villages were randomly assigned to receive either a single round of ORS with a capsule suspension of lambda-cyhalothrin for two days in October or no intervention (six villages per group). The primary endpoint was the biting rate of malaria mosquitoes assessed with human-landing catch and cow-baited trap collection methods, and was analyzed with a Bayesian multi-level model. In the intervention villages, the proportion of households located within the sprayed area ranged between 42 and 100% and the application rate ranged between 63 and 559 g of active ingredient per hectare. At baseline, the median of Anopheles biting rate estimates in the twelve villages was 2 bites per person per night (inter-quartile range [IQR] 0-5, range 0-48) indoors, 6 bites per person per night (IQR 2-16, range 0-342) outdoors and 206 bites per cow per night (IQR 83-380, range 19-1149) in the cow-baited trap. In intention-to-treat analysis, it was estimated that ORS reduced biting rate by 72% (95% confidence interval [CI] 63-79) from Month 0 to Month 3 and by 79% (95% CI 62-88) from Month 4 to Month 6, considering control villages as the reference. In conclusion, ORS rapidly reduces the biting rates of malaria mosquitoes in a Southeast Asian setting where the vectors bite mostly outdoors and at a time when people are not protected by mosquito bed nets.

Impact of outdoor residual spraying on the biting rate of malaria vectors: A pilot study in four villages in Kayin state, Myanmar.

Outdoor and early mosquito biters challenge the efficacy of bed-nets and indoor residual spraying on the Thailand-Myanmar border. Outdoor residual spraying is proposed for the control of exophilic mosquito species. The objective of this study was to assess the impact of outdoor residual spraying on the biting rate of malaria vectors in Kayin state, Myanmar. Outdoor residual spraying using lambda-cyhalothrin was carried out in two villages in December 2016 (beginning of the dry season) and two villages were used as a control. Malaria mosquitoes were captured at baseline and monthly for four months after the intervention using human-landing catch and cow-baited trap collection methods. The impact of outdoor residual spraying on human-biting rate was estimated with propensity score adjusted generalized linear mixed-effect regressions. At baseline, mean indoor and outdoor human-biting rate estimates ranged between 2.12 and 29.16 bites /person /night, and between 0.20 and 1.72 bites /person /night in the intervention and control villages respectively. Using model output, we estimated that human-biting rate was reduced by 91% (95%CI = 88-96, P <0.0001) immediately after outdoor residual spraying. Human-biting rate remained low in all sprayed villages for 3 months after the intervention. Malaria vector populations rose at month 4 in the intervention villages but not in the controls. This coincided with the expected end of insecticide mist residual effects, thereby suggesting that residual effects are important determinants of intervention outcome. We conclude that outdoor residual spraying with a capsule suspension of lambda-cyhalothrin rapidly reduced the biting rate malaria vectors in this area where pyrethroid resistance has been documented.

Longevity of the insecticidal effect of three pyrethroid formulations applied to outdoor vegetation on a laboratory-adapted colony of the Southeast Asian malaria vector Anopheles dirus.

Outdoor residual spraying is proposed for the control of exophilic mosquitoes. However, the residual effect of insecticide mists applied to outdoor resting habitats of mosquitoes is not well characterized. The objective of this study was to assess the longevity of the residual insecticidal effect of three pyrethroid formulations applied to outdoor vegetation against the Southeast Asian malaria vector Anopheles dirus. Lambda-cyhalothrin capsule suspension, deltamethrin emulsifiable concentrate and bifenthrin wettable powder were sprayed on dense bamboo bushes on the Thailand-Myanmar border during the dry season 2018. The duration and magnitude of the residual insecticidal effect were assessed weekly with a standard cone assay, using freshly collected insecticide-treated bamboo leaves and a laboratory-adapted colony of Anopheles dirus sensu stricto susceptible to pyrethroids. The experiment was repeated during the rainy season to assess the persistence of the lambda-cyhalothrin formulation after natural rains and artificial washings. During the dry season (cumulative rainfall = 28 mm in 111 days), mortality and knockdown (KD) rates were >80% for 60 days with bifenthrin and 90 days with lambda-cyhalothrin and deltamethrin. The 50% knockdown time (TKD50) was <15 min with lambda-cyhalothrin and deltamethrin, and <30 min with bifenthrin. During the rainy season (cumulative rainfall = 465 mm in 51 days), mortality and KD rates were >80% for 42 days and TKD50 was <15 min with lambda-cyhalothrin. Additional artificial washing of the testing material with 10L of tap water before performing the cone tests had no significant effect on the residual insecticidal effect of this formulation. Long-lasting residual insecticidal effect can be obtained when spraying pyrethroid insecticides on the outdoor resting habitats of malaria vectors.

Detection of diverse Wolbachia 16S rRNA sequences at low titers from malaria vectors in Kayin state, Myanmar.

Background : Natural Wolbachia infections in malaria mosquitoes were recently reported in Africa, and negatively correlated with the development of Plasmodium falciparum in the vectors. The occurrence and effects of Wolbachia infections outside Africa have not been described and may have been underestimated. Methods : Mosquitoes were collected by human-landing catch during May and June 2017 in ten villages in Kayin state, Myanmar. Closely related species of malaria vectors were identified with molecular assays. Wolbachia infection rates were assessed by quantitative real-time PCR. Results: Malaria vectors were identified in the Funestus, Maculatus and Leucosphyrus Groups . Wolbachia were detected in 6/6 Anopheles species and in 5/10 villages. Mean prevalence of Wolbachia infection was 2.7% (95%CI= [1.3; 4.9]). The median Wolbachia load was seven orders of magnitude less in naturally infected malaria vectors than in artificially infected laboratory-reared Aedes aegypti. Phylogenetic analysis based on 16S rRNA sequences revealed a high diversity of Wolbachia strains and identified lineages different from those described in Africa. Conclusion: Natural Wolbachia infections are common and widespread in malaria vectors in Kayin state, Myanmar. Their effects on Anopheles mosquitoes and malaria transmission is yet to be determined.

Contribution of Asymptomatic Plasmodium Infections to the Transmission of Malaria in Kayin State, Myanmar.

BACKGROUND: The objective of mass antimalarial drug administration (MDA) is to eliminate malaria rapidly by eliminating the asymptomatic malaria parasite reservoirs and interrupting transmission. In the Greater Mekong Subregion, where artemisinin-resistant Plasmodium falciparum is now widespread, MDA has been proposed as an elimination accelerator, but the contribution of asymptomatic infections to malaria transmission has been questioned. The impact of MDA on entomological indices has not been characterized previously. METHODS: MDA was conducted in 4 villages in Kayin State (Myanmar). Malaria mosquito vectors were captured 3 months before, during, and 3 months after MDA, and their Plasmodium infections were detected by polymerase chain reaction (PCR) analysis. The relationship between the entomological inoculation rate, the malaria prevalence in humans determined by ultrasensitive PCR, and MDA was characterized by generalized estimating equation regression. RESULTS: Asymptomatic P. falciparum and Plasmodium vivax infections were cleared by MDA. The P. vivax entomological inoculation rate was reduced by 12.5-fold (95% confidence interval [CI], 1.6-100-fold), but the reservoir of asymptomatic P. vivax infections was reconstituted within 3 months, presumably because of relapses. This was coincident with a 5.3-fold (95% CI, 4.8-6.0-fold) increase in the vector infection rate. CONCLUSION: Asymptomatic infections are a major source of malaria transmission in Southeast Asia.

Entomological determinants of malaria transmission in Kayin state, Eastern Myanmar: A 24-month longitudinal study in four villages.

Background: The Thailand-Myanmar borderland is an area endemic for malaria where transmission is low, seasonal and unstable. The epidemiology has been described but there is relatively few data on the entomological determinants of malaria transmission. Methods: Entomological investigations were conducted during 24 months in four villages located in Kayin state, on the Myanmar side of the Thailand-Myanmar border. Anopheles mosquitoes were identified by morphology, and molecular assays were used in order to discriminate between closely related sibling species of malaria vectors. Plasmodium infection rate was determined using quantitative real-time PCR. Results: The diversity of Anopheles mosquitoes was very high and multiple species were identified as malaria vectors. The intensity of human-vector contact (mean human-biting rate= 369 bites/person/month) compensates for the low infection rate in naturally infected populations of malaria vectors (mean sporozoite index= 0.04 and 0.17 % for P. falciparum and P. vivax respectively), yielding intermediary level of transmission intensity (mean entomological inoculation rate= 0.13 and 0.64 infective bites/person/month for P. falciparum and P. vivax, respectively). Only 36% of the infected mosquitoes were collected indoors between 09:00 pm and 05:00 am, suggesting that mosquito bed-nets would fail to prevent most of the infective bites in the study area. Conclusion: This study provided a unique opportunity to describe the entomology of malaria in low transmission settings of Southeast Asia. Our data are important in the context of malaria elimination in the Greater Mekong Subregion.