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Accurate species identification of the mosquitoes in the genus Anopheles is of crucial importance to implement malaria control measures and monitor their effectiveness. We use a previously developed amplicon panel (ANOSPP) that retrieves sequence data from multiple short nuclear loci for any species in the genus. Species assignment is based on comparison of samples to a reference index using k-mer distance. Here, we provide a protocol to generate version controlled updates of the reference index and present its latest release, NNv2, which contains 91 species, compared to 56 species represented in its predecessor NNv1. With the updated reference index, we are able to assign samples to species level that previously could not be assigned. We discuss what happens if a species is not represented in the reference index and how this can be addressed in a future update. To demonstrate the increased power of NNv2, we showcase the assignments of 1789 wild-caught mosquitoes from Madagascar and demonstrate that we can detect within species population structure from the amplicon sequencing data. [ABSTRACT FROM AUTHOR]

Malaria elimination in Southeast Asia remains a challenge, underscoring the importance of accurately identifying malaria mosquitoes to understand transmission dynamics and improve vector control. Traditional methods such as morphological identification require extensive training and cannot distinguish between sibling species, while molecular approaches are costly for extensive screening. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and cost-effective tool for Anopheles species identification, yet its current use is limited to few specialized laboratories. This study aimed to develop and validate an online reference database for MALDI-TOF MS identification of Southeast Asian Anopheles species. The database, constructed using the in-house data analysis pipeline MSI2 (Sorbonne University), comprised 2046 head mass spectra from 209 specimens collected at the Thailand-Myanmar border. Molecular identification via COI and ITS2 DNA barcodes enabled the identification of 20 sensu stricto species and 5 sibling species complexes. The high quality of the mass spectra was demonstrated by a MSI2 median score (min-max) of 61.62 (15.94–77.55) for correct answers, using the best result of four technical replicates of a test panel. Applying an identification threshold of 45, 93.9% (201/214) of the specimens were identified, with 98.5% (198/201) consistency with the molecular taxonomic assignment. In conclusion, MALDI-TOF MS holds promise for malaria mosquito identification and can be scaled up for entomological surveillance in Southeast Asia. The free online sharing of our database on the MSI2 platform (https://msi.happy-dev.fr/) represents an important step towards the broader use of MALDI-TOF MS in malaria vector surveillance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Advanced cell culture methods for modeling organ-level structure have been demonstrated to replicate in vivo conditions more accurately than traditional in vitro cell culture. Given that the liver is particularly important to human health, several advanced culture methods have been developed to experiment with liver disease states, including infection with Plasmodium parasites, the causative agent of malaria. These models have demonstrated that intrahepatic parasites require functionally stable hepatocytes to thrive and robust characterization of the parasite populations' response to investigational therapies is dependent on high-content and high-resolution imaging (HC/RI). We previously reported abiotic confinement extends the functional longevity of primary hepatocytes in a microfluidic platform and set out to instill confinement in a microtiter plate platform while maintaining optical accessibility for HC/RI; with an end-goal of producing an improved P. vivax liver stage culture model. We developed a novel fabrication process in which a PDMS soft mold embosses hepatocyte-confining microfeatures into polystyrene, resulting in microfeature-based hepatocyte confinement (μHEP) slides and plates. Our process was optimized to form both microfeatures and culture wells in a single embossing step, resulting in a 100 μm-thick bottom ideal for HC/RI, and was found inexpensively amendable to microfeature design changes. Microfeatures improved intrahepatic parasite infection rates and μHEP systems were used to reconfirm the activity of reference antimalarials in phenotypic dose–response assays. RNAseq of hepatocytes in μHEP systems demonstrated microfeatures sustain hepatic differentiation and function, suggesting broader utility for preclinical hepatic assays; while our tailorable embossing process could be repurposed for developing additional organ models. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]