BACKGROUND: Anopheles sawadwongporni Rattanarithikul & Green, Anopheles maculatus Theobald and Anopheles pseudowillmori (Theobald) of the Anopheles maculatus group (Diptera: Culicidae) are recognized as potential malaria vectors in many countries from the Indian subcontinent through Southeast Asia to Taiwan. A number of malaria vectors in malaria hotspot areas along the Thai-Myanmar border belong to this complex. However, the species distribution and dynamic trends remain understudied in this malaria endemic region. METHODS: Mosquitoes of the Maculatus group were collected using CDC light traps every other week from four villages in Tha Song Yang District, Tak Province, Thailand from January to December 2015. Adult female mosquitoes were morphologically identified on site using taxonomic keys. Molecular species identification was performed by multiplex PCR based on the internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA) and sequencing of the cox1 gene at a DNA barcoding region in a subset of 29 specimens. RESULTS: A total of 1328 An. maculatus (sensu lato) female mosquitoes were captured with An. maculatus, An. sawadwongporni and An. pseudowilmori accounting for 75.2, 22.1 and 2.7% respectively. The field captured mosquitoes of the Maculatus group were most abundant in the wet season and had a preferred distribution in villages at higher elevations. The phylogenetic relationships of 29 cox1 sequences showed a clear-cut separation of the three member species of the Maculatus group, with the An. pseudowillmori cluster being separated from An. sawadwongporni and An. maculatus. CONCLUSIONS: This study provides updated information for the species composition, seasonal dynamics and microgeographical distribution of the Maculatus group in malaria-endemic areas of western Thailand. This information can be used to guide the planning and implementation of mosquito control measures in the pursuance of malaria transmission.

BACKGROUND: The malaria vector Anopheles minimus has been influenced by external stresses affecting the survival rate and vectorial capacity of the population. Since An. minimus habitats have continuously undergone ecological changes, this study aimed to determine the population genetic structure and the potential gene flow among the An. minimus populations in Thailand. METHODS: Anopheles minimus was collected from five malaria transmission areas in Thailand using Centers for Disease Control and Prevention (CDC) light traps. Seventy-nine females from those populations were used as representative samples. The partial mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and cytochrome b (Cytb) gene sequences were amplified and analyzed to identify species and determine the current population genetic structure. For the past population, we determined the population genetic structure from the 60 deposited COII sequences in GenBank of An. minimus collected from Thailand 20 years ago. RESULTS: The current populations of An. minimus were genetically divided into two lineages, A and B. Lineage A has high haplotype diversity under gene flow similar to the population in the past. Neutrality tests suggested population expansion of An. minimus, with the detection of abundant rare mutations in all populations, which tend to arise from negative selection. CONCLUSIONS: This study revealed that the population genetic structure of An. minimus lineage A was similar between the past and present populations, indicating high adaptability of the species. There was substantial gene flow between the eastern and western An. minimus populations without detection of significant gene flow barriers.

Background

Malaria is heterogeneously distributed across landscapes. Human population movement (HPM) could link sub-regions with varying levels of transmission, leading to the persistence of disease even in very low transmission settings. Malaria along the Thai-Myanmar border has been decreasing, but remains heterogeneous. This study aimed to measure HPM, associated predictors of travel, and HPM correlates of self-reported malaria among people living within malaria hotspots.

Methods

526 individuals from 279 households in two malaria hotspot areas were included in a prospective observational study. A baseline cross-sectional study was conducted at the beginning, recording both individual- and household-level characteristics. Individual movement and travel patterns were repeatedly observed over one dry season month (March) and one wet season month (May). Descriptive statistics, random effects logistic regressions, and logistic regressions were used to describe and determine associations between HPM patterns, individual-, household-factors, and self-reported malaria.

Results

Trips were more common in the dry season. Malaria risk was related to the number of days doing outdoor activities in the dry season, especially trips to Myanmar, to forest areas, and overnight trips. Trips to visit forest areas were more common among participants aged 20-39, males, individuals with low income, low education, and especially among individuals with forest-related occupations. Overnight trips were more common among males, and individual with forest-related occupations. Forty-five participants reported having confirmed malaria infection within the last year. The main place of malaria blood examination and treatment was malaria post and malaria clinic, with participants usually waiting for 2-3 days from onset fever to seeking diagnosis. Individuals using bed nets, living in houses with elevated floors, and houses that received indoor residual spraying in the last year were less likely to report malaria infection.

Conclusion

An understanding of HPM and concurrent malaria dynamics is important for consideration of targeted public health interventions. Furthermore, diagnosis and treatment centres must be capable of quickly diagnosing and treating infections regardless of HPM. Coverage of diagnosis and treatment centres should be broad, maintained in areas bordering malaria hotspots, and available to all febrile individuals.

Background

Centers for Disease Control and Prevention miniature light traps (CDC-LT) baited with CO2 are a routine tool for adult mosquito sampling used in entomological surveys, and for monitoring and surveillance of disease vectors. The present study was aimed at evaluating the performance of baited and unbaited CDC-LT for indoor and outdoor trapping of endemic mosquito species in northwestern Thailand.

Methods

CDC-LT (n = 112) with and without dry ice baits were set both indoors and outdoors in 88 selected houses for stretches of 5 consecutive nights per month in 7 villages in Tha Song Yang district, Tak province between January 2011 and March 2013. Individual traps were repeatedly placed in the same location for a median of 6 (range 1-10) times. Mosquitoes were identified by morphological characteristics and classified into blood-fed, empty, male/female and gravid. Absolute mosquito numbers were converted to capture rates (i.e., mosquitoes per trap and year). Capture rates were compared using multilevel negative binomial regression to account for multiple trap placements and adjust for regional and seasonal differences.

Results

A total of 6,668 mosquitoes from 9 genera were collected from 576 individual CDC-LT placements. Culex was the predominant captured genus (46%), followed by anopheline mosquitoes (45%). Overall, CO2 baited traps captured significantly more Culex (especially Culex vishnui Theobald) and Anopheles mosquitoes per unit time (adjusted capture rate ratio (aCRR) 1.64 and 1.38, respectively). Armigeres spp. mosquitoes were trapped in outdoor traps with significantly higher frequency (aCRR: 1.50), whereas Aedes albopictus (Skuse) had a tendency to be trapped more frequently indoors (aCRR: 1.89, p = 0.07). Furthermore, capture rate ratios between CO2 baited and non-baited CDC-LT were significantly influenced by seasonality and indoor vs. outdoor trap placement.

Conclusion

The present study shows that CDC-LT with CO2 baiting capture significantly more Culex and Anopheles mosquitoes, some of which (e.g., Cx. vishnui, Cx. quinquefasciatus Say, An. minimus s.l. Theobald, An. maculatus s.l. Theobald) represent important disease vectors in Thailand. This study also shows significant differences in the capture efficiency of CDC-LT when placed indoors or outdoors and in different seasons. Our study thus provides important guidelines for more targeted future vector trapping studies on the Thai-Myanmar border, which is an important cross-border malaria transmission region in Thailand.

Background

The Thai-Myanmar border is a remaining hotspot for malaria transmission. Malaria transmission in this region continues year-round, with a major peak season in July-August, and a minor peak in October-November. Malaria elimination requires better knowledge of the mosquito community structure, dynamics and vectorial status to support effective vector control.

Methods

Adult Anopheles mosquitoes were collected using CDC light traps and cow bait in 7 villages along the Thai-Myanmar border in January 2011 - March 2013. Mosquitoes were determined to species by morphological characters. Plasmodium-positivity was determined by circumsporozoite protein ELISA.

Results

The 2986 Anopheles mosquitoes collected were assigned to 26 species, with Anopheles minimus sensu lato (s.l.) (40.32%), An. maculatus s.l. (21.43%), An. annularis s.l. (14.43%), An. kochi (5.39%), An. tessellatus (5.26%), and An. barbirostris s.l. (3.52%) being the top six most abundant species. Plasmodium-infected mosquitoes were found in 22 positive samples from 2906 pooled samples of abdomens and heads/thoraxes. Four mosquito species were found infected with Plasmodium: An. minimus s.l., An. maculatus s.l., An. annularis s.l. and An. barbirostris s.l. The infectivity rates of these mosquitoes were 0.76, 0.37, 0.72, and 1.74%, respectively. Consistent with a change in malaria epidemiology to the predominance of P. vivax in this area, 20 of the 22 infected mosquito samples were P. vivax-positive. The four potential vector species all displayed apparent seasonality in relative abundance. While An. minimus s.l. was collected through the entire year, its abundance peaked in the season immediately after the wet season. In comparison, An. maculatus s.l. numbers showed a major peak during the wet season. The two potential vector species, An. annularis s.l. and An. barbirostris s.l., both showed peak abundance during the transition from wet to dry season. Moreover, An. minimus s.l. was more abundant in indoor collections, whereas An. annularis s.l. and An. barbirostris s.l. were more abundant in outdoor collections, suggesting their potential role in outdoor malaria transmission.

Conclusions

This survey confirmed the major vector status of An. minimus s.l. and An. maculatus s.l. and identified An. annularis s.l. and An. barbirostris s.l. as additional vectors with potential importance in malaria transmission after the wet season.

Malaria infections remain an important public health problem for the Thai-Myanmar border population, despite a plan for the elimination by the end of 2026 (Thailand) and 2030 (Myanmar). This study aimed to explore spatiotemporal patterns in Plasmodium falciparum and Plasmodium vivax incidence along the Thai-Myanmar border. Malaria cases among Thai citizens in 161 sub-districts in Thailand's Kanchanaburi and Tak Provinces (2012-2017) were analyzed to assess the cluster areas and temporal trends. Based on reported incidence, 65.22% and 40.99% of the areas studied were seen to be at elimination levels for P. falciparum and P. vivax already, respectively. There were two clear clusters of malaria in the region: One in the northern part (Cluster I), and the other in the central part (Cluster II). In Cluster I, the malaria season exhibited two peaks, while there was only one peak seen for Cluster II. Malaria incidence decreased at a faster rate in Cluster I, with 5% and 4% reductions compared with 4% and 3% reductions in P. falciparum and P. vivax incidence per month, respectively, in Cluster II. The decreasing trends reflect the achievements of malaria control efforts on both sides of the Thai-Myanmar border. However, these clusters could act as reservoirs. Perhaps one of the main challenges facing elimination programs in this low transmission setting is maintaining a strong system for early diagnosis and treatment, even when malaria cases are very close to zero, whilst preventing re-importation of cases.

The occurrence and spread of insecticide resistance has had a negative effect on the efficacy of insecticide-based tools and is distributed worldwide, including the Greater Mekong Subregion (GMS). This study aims to determine the insecticide susceptibility of malaria and dengue vectors in malaria and dengue hotspots on the Thai-Myanmar border. Mosquito larvae and pupae were obtained from water sources from December 2019 to April 2020 in Tha Song Yang District, Tak province, western Thailand. WHO bioassay susceptibility tests were conducted with three classes of insecticides to evaluate the knockdown and mortality rates of Anopheles and Aedes aegypti female adults. V1016G and F1534C kdr mutations in the voltage-gated sodium channel of Ae. aegypti were identified using a multiplex PCR. A total of 5764 female mosquitoes were bioassayed in this study, including Anopheles spp. (92.63%) and F1 Ae. aegypti (7.37%). After 24 h of observation, An. minimus s.l. (n = 3885) and An. maculatus s.l. (n = 1138) in Suan Oi (SO) and Tala Oka (TO) were susceptible to pyrethroids, organophosphates and carbamates (except bendiocarb) with 98-100% mortality (MR). Resistance to bendiocarb was detected with a mortality rate of 88.80%, 88.77%, and 89.92% for An. minimus s.l. (n = 125, 125) and An. maculatus s.l. (n = 66), respectively. The first generation of Ae. aegypti adult females were suspected of resistance to deltamethrin (n = 225, MR = 96.89%) and confirmed resistance to permethrin (n = 200, MR = 20.00%). V1016G and F1534C mutations were detected in three genotypes, heterozygote and homozygote forms. The correlation between the kdr alleles and deltamethrin resistance was significant. In conclusion, bendiocarb resistance was found in primary malaria vectors, An. minimus s.l. and An. maculatus s.l. F1 Ae. aegypti population was pyrethroids-resistant, associated with kdr alleles. Therefore, molecular analysis should be conducted to gain insights into the mechanism of insecticide resistance. Routine malaria vector control programmes, such as fogging implementation in hotspot villages to induce Aedes resistance available in peri-domestic sites, are questionable.

Mosquitoes rely mainly on the olfactory system to track hosts. Sensilla contain olfactory neuron receptors that perceive different kinds of odorants and transfer crucial information regarding the surrounding environment. Anopheles maculatus and An. sawadwongporni, members of the Maculatus Group, are regarded as vectors of malaria in Thailand. The fine structure of their sensilla has yet to be identified. Herein, scanning electron microscopy is used to examine the sensilla located on the antennae of adults An. maculatus and An. sawadwongporni, collected from the Thai-Myanmar border. Four major types of antennal sensilla are discovered in both species: chaetica, coeloconica, basiconica (grooved pegs) and trichodea. The antennae of female An. maculatus have longer lengths (μm, mean ± SE) in the long sharp-tipped trichodea (40.62 ± 0.35 > 38.20 ± 0.36), blunt-tipped trichodea (20.39 ± 0.62 > 18.62 ± 0.35), and basiconica (7.84 ± 0.15 > 7.41 ± 0.12) than those of An. sawadwongporni. Using light microscopy, it is found that the mean numbers of large sensilla coeloconica (lco) on both flagella in An. maculatus (left: 32.97 ± 0.48; right: 33.27 ± 0.65) are also greater when compared to An. sawadwongporni (left: 30.40 ± 0.62; right: 29.97 ± 0.49). The mean counts of lco located on flagellomeres 1-3, 6, and 9 in An. maculatus are significantly higher than those of An. sawadwongporni. The data in this study indicate that two closely related Anopheles species exhibit similar morphology of sensilla types, but show variations in length, and likewise in the number of large sensilla coeloconica between them, suggesting they might be causative factors that affect their behaviors driven by the sense of smell.

In the course of malaria elimination in the Greater Mekong Subregion (GMS), malaria epidemiology has experienced drastic spatiotemporal changes with residual transmission concentrated along international borders and the rising predominance of Plasmodium vivax. The emergence of Plasmodium falciparum parasites resistant to artemisinin and partner drugs renders artemisinin-based combination therapies less effective while the potential spread of multidrug-resistant parasites elicits concern. Vector behavioral changes and insecticide resistance have reduced the effectiveness of core vector control measures. In recognition of these problems, the Southeast Asian International Center of Excellence for Malaria Research (ICEMR) has been conducting multidisciplinary research to determine how human migration, antimalarial drug resistance, vector behavior, and insecticide resistance sustain malaria transmission at international borders. These efforts allow us to comprehensively understand the ecology of border malaria transmission and develop population genomics tools to identify and track parasite introduction. In addition to employing in vivo, in vitro, and molecular approaches to monitor the emergence and spread of drug-resistant parasites, we also use genomic and genetic methods to reveal novel mechanisms of antimalarial drug resistance of parasites. We also use omics and population genetics approaches to study insecticide resistance in malaria vectors and identify changes in mosquito community structure, vectorial potential, and seasonal dynamics. Collectively, the scientific findings from the ICEMR research activities offer a systematic view of the factors sustaining residual malaria transmission and identify potential solutions to these problems to accelerate malaria elimination in the GMS.

The malaria landscape in the Greater Mekong Subregion has experienced drastic changes with the ramp-up of the control efforts, revealing formidable challenges that slowed down the progress toward malaria elimination. Problems such as border malaria and cross-border malaria introduction, multidrug resistance in Plasmodium falciparum, the persistence of Plasmodium vivax, the asymptomatic parasite reservoirs, and insecticide resistance in primary vectors require integrated strategies tailored for individual nations in the region. In recognition of these challenges and the need for research, the Southeast Asian International Center of Excellence for Malaria Research has established a network of researchers and stakeholders and conducted basic and translational research to identify existing and emerging problems and develop new countermeasures. The installation of a comprehensive disease and vector surveillance system at sentinel sites in border areas with the implementation of passive/active case detection and cross-sectional surveys allowed timely detection and management of malaria cases, provided updated knowledge for effective vector control measures, and facilitated the efficacy studies of antimalarials. Incorporating sensitive molecular diagnosis to expose the significance of asymptomatic parasite reservoirs for sustaining transmission helped establish the necessary evidence to guide targeted control to eliminate residual transmission. In addition, this program has developed point-of-care diagnostics to monitor the quality of artemisinin combination therapies, delivering the needed information to the drug regulatory authorities to take measures against falsified and substandard antimalarials. To accelerate malaria elimination, this program has actively engaged with stakeholders of all levels, fostered vertical and horizontal collaborations, and enabled the effective dissemination of research findings.