Challenges for achieving safe and effective radical cure of Plasmodium vivax: a round table discussion of the APMEN Vivax Working Group.

The delivery of safe and effective radical cure for Plasmodium vivax is one of the greatest challenges for achieving malaria elimination from the Asia-Pacific by 2030. During the annual meeting of the Asia Pacific Malaria Elimination Network Vivax Working Group in October 2016, a round table discussion was held to discuss the programmatic issues hindering the widespread use of primaquine (PQ) radical cure. Participants included 73 representatives from 16 partner countries and 33 institutional partners and other research institutes. In this meeting report, the key discussion points are presented and grouped into five themes: (i) current barriers for glucose-6-phosphate deficiency (G6PD) testing prior to PQ radical cure, (ii) necessary properties of G6PD tests for wide scale deployment, (iii) the promotion of G6PD testing, (iv) improving adherence to PQ regimens and (v) the challenges for future tafenoquine (TQ) roll out. Robust point of care (PoC) G6PD tests are needed, which are suitable and cost-effective for clinical settings with limited infrastructure. An affordable and competitive test price is needed, accompanied by sustainable funding for the product with appropriate training of healthcare staff, and robust quality control and assurance processes. In the absence of quantitative PoC G6PD tests, G6PD status can be gauged with qualitative diagnostics, however none of the available tests is currently sensitive enough to guide TQ treatment. TQ introduction will require overcoming additional challenges including the management of severely and intermediately G6PD deficient individuals. Robust strategies are needed to ensure that effective treatment practices can be deployed widely, and these should ensure that the caveats are outweighed by  the benefits of radical cure for both the patients and the community. Widespread access to quality controlled G6PD testing will be critical.

Development and evaluation of a spatial decision support system for malaria elimination in Bhutan.

BACKGROUND: Bhutan has reduced its malaria incidence significantly in the last 5 years, and is aiming for malaria elimination by 2016. To assist with the management of the Bhutanese malaria elimination programme a spatial decision support system (SDSS) was developed. The current study aims to describe SDSS development and evaluate SDSS utility and acceptability through informant interviews. METHODS: The SDSS was developed based on the open-source Quantum geographical information system (QGIS) and piloted to support the distribution of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) in the two sub-districts of Samdrup Jongkhar District. It was subsequently used to support reactive case detection (RACD) in the two sub-districts of Samdrup Jongkhar and two additional sub-districts in Sarpang District. Interviews were conducted to ascertain perceptions on utility and acceptability of 11 informants using the SDSS, including programme and district managers, and field workers. RESULTS: A total of 1502 households with a population of 7165 were enumerated in the four sub-districts, and a total of 3491 LLINs were distributed with one LLIN per 1.7 persons. A total of 279 households representing 728 residents were involved with RACD. Informants considered that the SDSS was an improvement on previous methods for organizing LLIN distribution, IRS and RACD, and could be easily integrated into routine malaria and other vector-borne disease surveillance systems. Informants identified some challenges at the programme and field level, including the need for more skilled personnel to manage the SDSS, and more training to improve the effectiveness of SDSS implementation and use of hardware. CONCLUSIONS: The SDSS was well accepted and informants expected its use to be extended to other malaria reporting districts and other vector-borne diseases. Challenges associated with efficient SDSS use included adequate skills and knowledge, access to training and support, and availability of hardware including computers and global positioning system receivers.

Targeting populations at higher risk for malaria: a survey of national malaria elimination programmes in the Asia Pacific.

BACKGROUND: Significant progress has been made in reducing the malaria burden in the Asia Pacific region, which is aggressively pursuing a 2030 regional elimination goal. Moving from malaria control to elimination requires National Malaria Control Programmes (NMCPs) to target interventions at populations at higher risk, who are often not reached by health services, highly mobile and difficult to test, treat, and track with routine measures, and if undiagnosed, can maintain parasite reservoirs and contribute to ongoing transmission. METHODS: A qualitative, free-text questionnaire was developed and disseminated among 17 of the 18 partner countries of the Asia Pacific Malaria Elimination Network (APMEN). RESULTS: All 14 countries that responded to the survey identified key populations at higher risk of malaria in their respective countries. Thirteen countries engage in the dissemination of malaria-related Information, Education, and Communication (IEC) materials. Eight countries engage in diagnostic screening, including of mobile and migrant workers, military staff, and/or overseas workers. Ten countries reported distributing or recommending the use of long-lasting insecticide-treated nets (LLINs) among populations at higher risk with fewer countries engaging in other prevention measures such as indoor residual spraying (IRS) (two countries), spatial repellents (four countries), chemoprophylaxis (five countries), and mass drug administration (MDA) (three countries). Though not specifically tailored to populations at higher risk, 11 countries reported using mass blood surveys as a surveillance tool and ten countries map case data. Most NMCPs lack a monitoring and evaluation structure. CONCLUSION: Countries in the Asia Pacific have identified populations at higher risk and targeted interventions to these groups but there is limited information on the effectiveness of these interventions. Platforms like APMEN offer the opportunity for the sharing of protocols and lessons learned related to finding, targeting and successfully clearing malaria from populations at higher risk. The sharing of programme data across borders may further strengthen national and regional efforts to eliminate malaria. This exchange of real-life experience is invaluable to NMCPs when scarce scientific evidence on the topic exists to aid decision-making and can further support NMCPs to develop strategies that will deliver a malaria-free Asia Pacific by 2030.

A new species of the Anopheles barbirostris complex (Diptera: Culicidae) from Bhutan.

Anopheles sarpangensis Somboon, Namgay & Harbach is described as a new species of the Barbirostris Complex of the subgenus Anopheles Meigen. The adults differ from other species of the complex in having wings with a prehumeral pale spot on the costa, foretarsomere 1 with an apical pale band more than half as long as foretarsomere 5, midtarsomeres 1 and 2, and occasionally 3 and 4, with incomplete apical pale bands, and abdominal sterna with many white scales as in An. campestris Reid, An. saeungae Taai & Harbach, and An. wejchoochotei Taai & Harbach. The pupa and larva are indistinguishable from the pupae and larvae of other species of the complex. Phylogenetic analysis revealed that COI sequences form a clade separate from clades consisting of the sequences of other species of the complex. The immature stages were commonly found in fishery ponds in foothills at low to moderately high altitudes in the Sarpang District of Bhutan.

Efforts toward the Elimination of Visceral Leishmaniasis in South Asia: A Review of Progress in Bhutan.

Leishmaniases are a group of diseases under the category of neglected tropical diseases targeted for global elimination. However, they continue to pose major clinical and public health problems, especially among those living in poor socioeconomic conditions. Here, we summarize leishmaniasis elimination efforts in Bhutan. Between 1994 and 2022, Bhutan recorded 54 cases of leishmaniasis across 14 of its 20 districts. There are seven species of Phlebotomus and three species of Sergentomyia sand flies documented in the country. At a subnational level, all endemic districts recorded a visceral leishmaniasis annual incidence <1 per 10,000 population, meeting the regional elimination targets. Serological testing with ELISA and molecular testing with polymerase chain reaction were established at the Royal Center for Disease Control in 2022. A leishmaniasis prevention and management guideline was adopted in 2023 to aid clinicians in diagnosis and management. Active and passive case surveillance was integrated with the national infectious disease early warning and response system. Risk-based entomological surveillance and control have also been prioritized. Climate change may play a major role in rendering districts in the temperate zone favorable for vector proliferation. The country's medical university introduced a diploma course in medical entomology in 2023 to augment the human resources needed for vector surveillance efforts. However, leishmaniasis elimination lacks dedicated programmatic management amid competing priorities for resources against other infectious diseases. Leishmaniasis elimination requires a targeted and programmatic approach in Bhutan, including cross-border collaborative efforts with neighboring Indian states. Bhutan remains highly committed to achieving leishmaniasis elimination targets.

Review of the Mimeticus Subgroup of Culex (Culex) (Diptera: Culicidae), With an Assessment of the Specific Status of Three Nominal Species Described From India.

The Mimeticus Subgroup of the subgenus Culex, genus Culex Linnaeus, is reviewed and revised. Mitochondrial COI sequences of species of the subgroup available in GenBank were analysed to verify the identity of voucher specimens. The specific status of three species described from India, Cx. gaugleri Suman, Cx. katezari Karlekar, Andrew & Deshpande, and Cx. kodaikanalensis Suman, Veer & Chandra, was examined and found to be unsupportable. Culex gaugleri and Cx. kodaikanalensis are synonyms of Cx. jacksoni based on the similarity of DNA sequences and dubious evidence of morphological differentiation. Culex katezari is recognized as a nomen dubium because the morphological description and molecular information provided by the authors are insufficient for its identification. A number of specimens registered in GenBank were found to be misidentified. In addition, available data indicate that the Mimeticus Subgroup may include at least three unrecognized species, referred to informally as Cx. mimeticus form B, Cx. mimulus form X, and Cx. mimulus form Y.

The female and pupa of Uranotaenia (Pseudoficalbia) bhutanensis (Diptera: Culicidae).

Uranotaenia bhutanensis Somboon, Namgay & Harbach was recently described as a new species of the Bimaculata Series of the subgenus Pseudoficalbia Theobald based on larvae collected from a log hole at high altitude in Bhutan. The adult female and pupa are described in this article based on the subsequent discovery of 2 paratype females with associated pupal exuviae.

Malaria control in Bhutan: case study of a country embarking on elimination.

BACKGROUND: Bhutan has achieved a major reduction in malaria incidence amid multiple challenges. This case study seeks to characterize the Bhutan malaria control programme over the last 10 years. METHODS: A review of the malaria epidemiology, control strategies, and elimination strategies employed in Bhutan was carried out through a literature review of peer-reviewed and grey national and international literature with the addition of reviewing the surveillance and vector control records of the Bhutan Vector-Borne Disease Control Programme (VDCP). Data triangulation was used to identify trends in epidemiology and key strategies and interventions through analysis of the VDCP surveillance and programme records and the literature review. Enabling and challenging factors were identified through analysis of socio-economic and health indicators, corroborated through a review of national and international reports and peer-review articles. FINDINGS: Confirmed malaria cases in Bhutan declined by 98.7% from 1994 to 2010. The majority of indigenous cases were due to Plasmodium vivax (59.9%) and adult males are most at-risk of malaria. Imported cases, or those in foreign nationals, varied over the years, reaching 21.8% of all confirmed cases in 2006. Strategies implemented by the VDCP are likely to be related to the decline in cases over the last 10 years. Access to malaria diagnosis in treatment was expanded throughout the country and evidence-based case management, including the introduction of artemisinin-based combination therapy (ACT) for P. falciparum, increasing coverage of high risk areas with Indoor Residual Spraying, insecticide-treated bed nets, and long-lasting insecticidal nets are likely to have contributed to the decline alongside enabling factors such as economic development and increasing access to health services. CONCLUSION: Bhutan has made significant strides towards elimination and has adopted a goal of national elimination. A major challenge in the future will be prevention and management of imported malaria infections from neighbouring Indian states. Bhutan plans to implement screening at border points to prevent importation of malaria and to targeted prevention and surveillance efforts towards at-risk Bhutanese and migrant workers in construction sites.

The Anopheles lindesayi Species Complex (Diptera: Culicidae) in Bhutan.

This paper deals with species A, C, D, and E of the Lindesayi Complex of Anopheles subgenus Anopheles (Diptera: Culicidae) that were recently recorded from Bhutan. Species B is not included due to insufficient data. Species A is Anopheles lindesayi sensu stricto, and species C, D, and E are new species that are formally described and named herein as Anopheles druki Somboon, Namgay & Harbach, Anopheles himalayensis Somboon, Namgay & Harbach and Anopheles thimphuensis Somboon, Namgay & Harbach, respectively. Morphological characteristics of the adults, larval and pupal stages of each species are provided and compared with An. lindesayi from Bhutan.

Uranotaenia (Pseudoficalbia) bhutanensis (Diptera: Culicidae), A New Species From Bhutan.

A new species of the subgenus Pseudoficalbia of the genus Uranotaenia is described from larvae found in a hole in a log at high altitude in montane forest in the Mongar District of Bhutan. Based on morphology, the larvae are sufficiently distinct from other species of the subgenus to be described and formally named here as Uranotaenia bhutanensis Somboon, Namgay & Harbach, n. sp. The species is a member of the Bimaculata Series. The chaetotaxy of the fourth-instar larva is provided.

The Anopheles baileyi species complex (Diptera: Culicidae: Anophelinae) in Bhutan.

This paper deals with three species (informal species A, B and C) of the Baileyi Complex of Anopheles subgenus Anopheles (Diptera: Culicidae) that were recently reported from Bhutan. Species A is An. baileyi sensu stricto, while species B and C are new species which are formally described and named herein as An. monticola Somboon, Namgay & Harbach and An. bhutanensis Somboon, Namgay & Harbach, respectively. Morphological characters of the adults, male genitalia, larval and pupal stages of the two new species, and the chaetotaxy of the immature stages, are provided and compared with An. baileyi from Bhutan. Although the patterns of wing spots of the adults and the pupal setae of the three species are very similar, significant differences in structures of the male genitalia distinguish them. Larvae of An. baileyi and An. monticola are similar, but differ from the larva of An. bhutanensis in the development of setae 3- and 13-T. The first antennal flagellomere of females of An. baileyi and An. bhutanensis has a distinct patch of pale scales, which is absent in An. monticola.

Reinertia, a New Subgenus of the Genus Aedes Meigen and Its Type Species Aedes (Reinertia) suffusus (Diptera: Culicidae), Newly Recorded From Bhutan.

A new subgenus, Reinertia Somboon, Namgay & Harbach, of the genus Aedes Meigen and its type species, Ae. suffusus Edwards, are described from specimens reared from larvae and pupae found in a tree hole in Bhutan. The scutum of the adults is mostly covered with narrow pale falcate scales. The proboscis, maxillary palpus, tibiae, and tarsi are dark-scaled. The gonocoxite of the male genitalia bears a unique setose basomesal sclerite. The larva closely resembles larvae of the subgenus Downsiomyia Vargus in having setae 4-6-C with numerous branches and inserted more or less on level with seta 7-C, abdominal seta 12-I is present and the comb is composed of 6-10 spine-like scales arranged in an irregular row. Surprisingly, Reinertia shares features of the adult habitus, male genitalia, and larva with the Palearctic subgenus Dahliana Reinert, Harbach & Kitching. However, in phylogenetic analyses of the mitochondrial COI gene of species representing 38 subgenera of Aedes and six other genera of the tribe Aedini Neveu-Lemaire, Reinertia was not associated with Dahliana or Downsiomyia. In both maximum likelihood and Bayesian analyses of the data, Ae. suffusus was recovered as the weakly supported sister of a clade composed of five species of the subgenus Protomacleaya Theobald. In the absence of strong support, and because Protomacleaya is an unnatural group of species that resemble each other phenetically by virtue of what they lack, Ae. suffusus cannot be placed in the subgenus Protomacleaya. Thus, the morphological and molecular data attest the uniqueness of Ae. suffusus and its recognition as a monobasic subgeneric lineage.

Culex (Culex) longitubus, A New Species of the Mimeticus Subgroup (Diptera: Culicidae) From Bhutan.

Culex longitubus Somboon, Namgay & Harbach is described as a new species of the Mimeticus Subgroup of the subgenus Culex. The larva is most similar to the larva of Cx. tianpingensis Chen from China, but is distinguished by the length of the siphon and the anal papillae, the form of the comb scales and pecten spines, and the development of setae 7-P, 13-T, 1-X, and 4-X. The adults have wing markings and male genitalia similar to those of species of the Mimeticus Complex. Phylogenetic analysis of COI sequences revealed that the new species is closely related to Cx. murrelli Lien of the Mimulus Complex. The immature stages of the new species were found in stagnant pools and marshes at high altitudes in several districts of Bhutan.

Culex bhutanensis, a new species of the Mimeticus Subgroup of the nominotypical subgenus of the genus Culex Linnaeus (Diptera: Culicidae) from Bhutan.

Culex bhutanensis Somboon, Namgay & Harbach is described as a new species of the Mimeticus Subgroup of the subgenus Culex Linnaeus. Based on morphological distinctions and phylogenetic analysis of COI sequences, the new species is a member of the Mimulus Complex. The adults have wing markings similar to those of Cx. mimulus Edwards and Cx. murrelli Lien, but are distinguished by the presence of a line of pale scales or scattered pale scales along the posterior margin of the proximal portion of the costa. The larva is most similar to the larva of Cx. orientalis Edwards from Japan and South Korea, but is distinguished by the form of the comb scales, pecten spines and seta 2-X. The pupa differs from the pupae of other species of the subgroup in having seta 8-C with more branches. The immature stages of the new species were found in rice paddies, stagnant pools and marshes at high altitudes in several districts of Bhutan.

Description of Aedes (Hulecoeteomyia) bhutanensis n. sp. (Diptera: Culicidae) from Bhutan.

A new species of the subgenus Hulecoeteomyia of the genus Aedes reared from pupae found with two larvae in a small pool on a log in mountainous forest of Bhutan is diagnosed and formally named Aedes bhutanensis Somboon & Harbach, n. sp. The specific status of the species is supported by differential morphological characters of females, larvae and pupae, and sequences of the mitochondrial COI gene. Dichotomous keys are provided for identification of the adult females and larvae of the species of the subgenus.

Molecular and morphological evidence of sibling species in Anopheles baileyi Edwards (Diptera: Culicidae) in Bhutan and Thailand.

This paper reports the results of a molecular and morphological study of Anopheles baileyi in Bhutan and Thailand. Phylogenetic analyses of ribosomal (ITS2) and mitochondrial DNA (COI) sequences revealed the presence of four genetically distinct clades, three in Bhutan (Clades I, II and III) and one in Thailand (Clade IV). Most of the larvae in the Bhutanese clades differed from those in the Thai clade in having seta 4-C branched, whereas it is single in the latter. The adults of each clade showed variation of wing markings and overlapping characters. The combination of characteristics of thoracic setae 1,2-P and abdominal seta 3-I was found to be useful for distinguishing the larvae. Pupae were inseparable. We provisionally recognize mosquitoes of Clades I, II, III and IV as members of a sibling species complex, the Baileyi Complex, denoted as species A, B, C and D, respectively. Species A is most likely the type form of An. baileyi s.s. because it was found adjacent to the type locality (Yatung, Tibet), and the others are unrecognized species.

Molecular and morphological evidence for sibling species within Anopheles (Anopheles) lindesayi Giles (Diptera: Culicidae) in Bhutan.

This paper reports the results of a comparative molecular and morphological study of An. lindesayi collected from various districts of Bhutan and An. l. cameronensis from Thailand, compared with GenBank accessions and publications for An. l. japonicus from Japan, South Korea and China, An. l. pleccau from Taiwan, and An. lindesayi from India. Phylogenetic analyses based on ribosomal (ITS2) and mitochondrial (COI) DNA sequences using the Maximum Likelihood method revealed five genetically distinct clades (A, B, C, D and E) in Bhutan. Specimens in Clade A correspond to the original description of An. lindesayi, particularly in wing markings, the pattern of basal pale scales on the hindfemur and the single seta 4-C of larvae, and their COI sequences were closely related to one Indian sequence. Larvae of Clades B, C, D and E are similar in having seta 4-C branched rather than single. The adults of Clades C, D and E (B not available) are distinguishable from those of Clade A and other subspecies. Specimens of Clade C are unique in having a long pale spot on wing vein R and the subcosta, scattered pale scales on several veins and a dark spot at the tip of vein R2. The adults of Clades D and E are similar in having a dark spot at the tip of vein R2 and no scattered pale scales on all other veins. We provisionally recognize mosquitoes of Clades A, B, C, D and E as species A, B, C, D and E, respectively, of the Lindesayi Complex. Species A is An. lindesayi sensu stricto and the others are unnamed species. Concomitantly, the previous concept of the "Lindesayi Complex", which included An. lindesayi, An. menglangensis, An. nilgiricus and An. wellingtonianus, is now recognized as the Lindesayi Subgroup of the Lindesayi Group (Anopheles Series, subgenus Anopheles) with the five sibling species of An. lindesayi comprising a more apposite Lindesayi Complex within the subgroup.

A Multiplex PCR Based on Mitochondrial COI Sequences for Identification of Members of the Anopheles barbirostris Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region.

A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of COI sequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp for An. barbirostris s.s., 238 bp for An. dissidens, 611 bp for An. saeungae, 502 bp for An. wejchoochotei and 365 bp for An. barbirostris A3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement with COI sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct COI sequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.

A spatio-temporal analysis to identify the drivers of malaria transmission in Bhutan.

At a time when Bhutan is on the verge of malaria elimination, the aim of this study was to identify malaria clusters at high geographical resolution and to determine its association with local environmental characteristics. Malaria cases from 2006-2014 were obtained from the Vector-borne Disease Control Program under the Ministry of Health, Bhutan. A Zero-Inflated Poisson multivariable regression model with a conditional autoregressive (CAR) prior structure was developed. Bayesian Markov chain Monte Carlo (MCMC) simulation with Gibbs sampling was used to estimate posterior parameters. A total of 2,062 Plasmodium falciparum and 2,284 Plasmodium vivax cases were reported during the study period. Both species of malaria showed seasonal peaks with decreasing trend. Gender and age were not associated with the transmission of either species of malaria. P. falciparum increased by 0.7% (95% CrI: 0.3%, 0.9%) for a one mm increase in rainfall, while climatic variables (temperature and rainfall) were not associated with P. vivax. Insecticide treated bed net use and residual indoor insecticide coverage were unaccounted for in this study. Hot spots and clusters of both species were isolated in the central southern part of Bhutan bordering India. There was significant residual spatial clustering after accounting for climate and demographic variables.

Morphological and molecular evidence for a new species of Lutzia (Diptera: Culicidae: Culicini) from Thailand.

We found a species of LutziaTheobald (1903) (Diptera: Culicidae) in Chiang Mai Province and other provinces in northern Thailand which bears morphological and DNA sequence differences from the three species of Lutzia, subgenus Metalutzia Tanaka, previously recorded from Thailand, namely Lt. fuscana (Wiedemann), Lt. halifaxii (Theobald) and Lt. vorax Edwards. The adults of the Chiang Mai form (CM form) have abdominal banding patterns similar to those of Lt. vorax from Japan (which includes the type locality of this species), but differ in having the mediocubital crossvein usually positioned before rather than beyond the radiomedial crossvein. The thoracic and abdominal integument of Lt. vorax larvae is covered with relatively short pointed spicules whereas it is covered by denser, longer and more acutely pointed spicules in the CM form. Some differences are also found in the development of thoracic seta 1-M, and abdominal setae 8-II and 8-III. The pupa of the CM form clearly differs from the pupa of Lt. vorax in having setae 1 and 5 of abdominal segments V and VI branched rather than single. The characters of the wing, larva and pupa of the CM form are similar to those of Lt. fuscana and Lt. halifaxii. However, whereas the phallosome of males of the CM form is similar to males of Lt. vorax and Lt. halifaxii, it is distinct from males of Lt. fuscana. Phylogenetic analysis of mitochondrial cytochrome c oxidase subunits I and II sequences revealed that the CM form falls outside a clade comprised of specimens of Lt. vorax from Japan, Korea, Thailand and Bhutan (Kimura 2-parameter, K2P, genetic distances 3.9-5.6, and 5.1-6.6, respectively). However, the two gene sequences of the CM form are not clearly distinct from clades comprised of sequences from specimens of Lt. fuscana and Lt. halifaxii (K2P 0.2-2.4%). However, based on the combination of morphological and molecular data, the current study provides evidence that the CM form is a previously unrecognized species of the genus Lutzia.