Identification of Southeast Asian Anopheles mosquito species using MALDI-TOF mass spectrometry

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

Transmission-blocking activities of artesunate, chloroquine, and methylene blue on Plasmodium vivax gametocytes

Plasmodium vivax is now the main cause of malaria outside Africa. The gametocytocidal effects of antimalarial drugs are important to reduce malaria transmissibility, particularly in low-transmission settings, but they are not well characterized for P. vivax. The transmission-blocking effects of chloroquine, artesunate, and methylene blue on P. vivax gametocytes were assessed. Blood specimens were collected from patients presenting with vivax malaria, incubated with or without the tested drugs, and then fed to mosquitos from a laboratory-adapted colony of Anopheles dirus (a major malaria vector in Southeast Asia). The effects on oocyst and sporozoite development were analyzed under a multi-level Bayesian model accounting for assay variability and the heterogeneity of mosquito Plasmodium infection. Artesunate and methylene blue, but not chloroquine, exhibited potent transmission-blocking effects. Gametocyte exposures to artesunate and methylene blue reduced the mean oocyst count 469-fold (95% CI: 345 to 650) and 1,438-fold (95% CI: 970 to 2,064), respectively. The corresponding estimates for the sporozoite stage were a 148-fold reduction (95% CI: 61 to 470) and a 536-fold reduction (95% CI: 246 to 1,311) in the mean counts, respectively. In contrast, high chloroquine exposures reduced the mean oocyst count only 1.40-fold (95% CI: 1.20 to 1.64) and the mean sporozoite count 1.34-fold (95% CI: 1.12 to 1.66). This suggests that patients with vivax malaria often remain infectious to anopheline mosquitos after treatment with chloroquine. Use of artemisinin combination therapies or immediate initiation of primaquine radical cure should reduce the transmissibility of P. vivax infections

Improved species assignments across the entire Anopheles genus using targeted sequencing

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

Déterminants entomologiques de la transmission du paludisme dans la zone transfrontalière entre la Thaïlande et le Myanmar dans un contexte d'élimination de Plasmodium falciparum

The Thai-Myanmar border is an area of low, seasonal and unstable malaria transmission. Consequently, the infection is symptomatic in all age groups and malaria morbidity and mortality is high especially in pregnant women and children.Antimalarial drug resistances in Plasmodium falciparum constitute the main obstacle to tackle malaria in this area. In the absence of alternative antimalarial drugs, the development of artemisinin resistance in Plasmodium falciparum is a major threat to global public health as it could impair the important yet labile progress made to reduce malaria endemicity (on the TMB, malaria incidence drastically decrease from 2 cases / person / year in 1980 to 0.1 cases / person / year after 2010. The primary objective of this work is to evaluate the impact of antimalarial-based elimination strategy relying on universal coverage with early diagnosis and treatment and elimination of malaria transmission hotspots (villages were the prevalence of asymptomatic infection, usually submicroscopic)with mass drig administration. Secondary objectives aim at describing and understanding the modalities and dynamic of malaria transmission in the studied area.La zone transfrontalière entre la Thaïlande et le Myanmar (ZTTM) est une zone d'endémicité palustre où l'intensité de la transmission est faible, saisonnière et instable. Par conséquent, le paludisme est symptomatique dans toutes les classe d'âge et responsable d'une mobi-mortalité élevée, en particulier chez les femmes enceintes et les enfants. Les résistances de Plasmodium falciparum aux médicaments antipaludiques constituent le principal obstacle à la lutte contre le paludisme dans la région. L'apparition de souches de P. falciparum résistantes aux derniers médicaments encore efficaces (notamment les résistances à l'artémisinine) est particulièrement inquiétante car elle menace les acquis fragiles toutefois très importants en terme de contrôle du paludisme (dans la ZTTM, l'incidence du paludisme a été divisée par 20 au cours des trente dernières années passant 2 cas/personne/an dans le milieu des années 1980 à <0.1 cas/personne/an après 2010). L'objectif principal de cette thèse est de d'évaluer l'impact d'une stratégie d'élimination de P. falciparum basée sur des moyens médicamenteux (accès universel au diagnostic et traitement précoce et élimination du réservoir asymtpomatique, généralement sous-microscopique, par le traitement de masse)sur les indicateurs entomologiques de la transmission du paludisme. Les objectifs secondaires sont multiples et visent à mieux décrire et comprendre les modalités et la dynamique de la transmission du paludisme

Entomological determinants of malaria transmission along the Thai-Myanmar border in the context of Targeted Chemo-Elimination of Plasmodium falciparum

La zone transfrontalière entre la Thaïlande et le Myanmar (ZTTM) est une zone d'endémicité palustre où l'intensité de la transmission est faible, saisonnière et instable. Par conséquent, le paludisme est symptomatique dans toutes les classe d'âge et responsable d'une mobi-mortalité élevée, en particulier chez les femmes enceintes et les enfants. Les résistances de Plasmodium falciparum aux médicaments antipaludiques constituent le principal obstacle à la lutte contre le paludisme dans la région. L'apparition de souches de P. falciparum résistantes aux derniers médicaments encore efficaces (notamment les résistances à l'artémisinine) est particulièrement inquiétante car elle menace les acquis fragiles toutefois très importants en terme de contrôle du paludisme (dans la ZTTM, l'incidence du paludisme a été divisée par 20 au cours des trente dernières années passant 2 cas/personne/an dans le milieu des années 1980 à <0.1 cas/personne/an après 2010). L'objectif principal de cette thèse est de d'évaluer l'impact d'une stratégie d'élimination de P. falciparum basée sur des moyens médicamenteux (accès universel au diagnostic et traitement précoce et élimination du réservoir asymtpomatique, généralement sous-microscopique, par le traitement de masse)sur les indicateurs entomologiques de la transmission du paludisme. Les objectifs secondaires sont multiples et visent à mieux décrire et comprendre les modalités et la dynamique de la transmission du paludisme.The Thai-Myanmar border is an area of low, seasonal and unstable malaria transmission. Consequently, the infection is symptomatic in all age groups and malaria morbidity and mortality is high especially in pregnant women and children.Antimalarial drug resistances in Plasmodium falciparum constitute the main obstacle to tackle malaria in this area. In the absence of alternative antimalarial drugs, the development of artemisinin resistance in Plasmodium falciparum is a major threat to global public health as it could impair the important yet labile progress made to reduce malaria endemicity (on the TMB, malaria incidence drastically decrease from 2 cases / person / year in 1980 to 0.1 cases / person / year after 2010. The primary objective of this work is to evaluate the impact of antimalarial-based elimination strategy relying on universal coverage with early diagnosis and treatment and elimination of malaria transmission hotspots (villages were the prevalence of asymptomatic infection, usually submicroscopic)with mass drig administration. Secondary objectives aim at describing and understanding the modalities and dynamic of malaria transmission in the studied area

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 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

pWCP is a widely distributed and highly conserved Wolbachia plasmid in Culex pipiens and Culex quinquefasciatus mosquitoes worldwide

Abstract Mosquitoes represent the most important pathogen vectors and are responsible for the spread of a wide variety of poorly treatable diseases. Wolbachia are obligate intracellular bacteria that are widely distributed among arthropods and collectively represents one of the most promising solutions for vector control. In particular, Wolbachia has been shown to limit the transmission of pathogens, and to dramatically affect the reproductive behavior of their host through its phage WO. While much research has focused on deciphering and exploring the biocontrol applications of these WO-related phenotypes, the extent and potential impact of the Wolbachia mobilome remain poorly appreciated. Notably, several Wolbachia plasmids, carrying WO-like genes and Insertion Sequences (IS), thus possibly interrelated to other genetic units of the endosymbiont, have been recently discovered. Here we investigated the diversity and biogeography of the first described plasmid of Wolbachia in Culex pipiens (pWCP) in several islands and continental countries around the world—including Cambodia, Guadeloupe, Martinique, Thailand, and Mexico—together with mosquito strains from colonies that evolved for 2 to 30 years in the laboratory. We used PCR and qPCR to determine the presence and copy number of pWCP in individual mosquitoes, and highly accurate Sanger sequencing to evaluate potential variations. Together with earlier observation, our results show that pWCP is omnipresent and strikingly conserved among Wolbachia populations within mosquitoes from distant geographies and environmental conditions. These data suggest a critical role for the plasmid in Wolbachia ecology and evolution, and the potential of a great tool for further genetic dissection and possible manipulation of this endosymbiont

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