Distribution and Seasonal Variation of Phlebotomus and Sergentomyia Sand Fly Populations in Bhutan.

Leishmaniasis is considered one of the neglected tropical diseases in the world. Although Bhutan is a member of the visceral leishmaniasis elimination consortium in South Asia, not much attention has been accorded to the disease because of its low incidence. The vector that transmits Leishmania remains poorly understood. In this backdrop, sand-fly surveys were regularly conducted at multiple sites where leishmaniasis cases have been reported in Bhutan. Collections were made using CDC light traps and cattle-baited net traps in 15 villages from February 2019 to May 2022. Six species of Phlebotomus and four species of Sergentomyia were identified from these sites that included two Phlebotomus and three Sergentomyia species discovered for the first time in Bhutan. Sand-fly density varied significantly from village to village, and it showed strong seasonality with peak numbers collected from June to October and almost zero from December to February. Overall, sand-fly density was highest in the basements of the houses and were higher outdoors than indoors. Cattle-baited net traps collected few sand flies during the surveillance period. This work constituted the first systematic sand-fly population surveillance conducted in Bhutan and will provide a baseline for future vector ecology and Leishmania epidemiological studies.

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.

Epidemiological Analysis of the 2019 Dengue Epidemic in Bhutan.

Bhutan experienced its largest and first nation-wide dengue epidemic in 2019. The cases in 2019 were greater than the total number of cases in all the previous years. This study aimed to characterize the spatiotemporal patterns and effective reproduction number of this explosive epidemic. Weekly notified dengue cases were extracted from the National Early Warning, Alert, Response and Surveillance (NEWARS) database to describe the spatial and temporal patterns of the epidemic. The time-varying, temperature-adjusted cohort effective reproduction number was estimated over the course of the epidemic. The dengue epidemic occurred between 29 April and 8 December 2019 over 32 weeks, and included 5935 cases. During the epidemic, dengue expanded from six to 44 subdistricts. The effective reproduction number was <3 for most of the epidemic period, except for a ≈1 month period of explosive growth, coinciding with the monsoon season and school vacations, when the effective reproduction number peaked >30 and after which the effective reproduction number declined steadily. Interventions were only initiated 6 weeks after the end of the period of explosive growth. This finding highlights the need to reinforce the national preparedness plan for outbreak response, and to enable the early detection of cases and timely response.

Atypical Presentation of Post-Kala-Azar Dermal Leishmaniasis in Bhutan.

This article describes an atypical case of post-kala-azar dermal leishmaniasis associated with complications due to delayed diagnosis and poor case management. The grave consequences of the prolonged disease process that continued for over 2 decades with eventual healing included facial disfigurement, visual impairment, and mental distress both to the patient and the family. The persistent infection within the skin over a lengthy period with likely increased risk of infection spread in the community highlights its potential negative impact on the ongoing leishmaniasis elimination program in the Indian subcontinent. Bhutan is a member of the leishmaniasis elimination network in Asia, and the government continues to invest in maintenance of the national healthcare system. The case study reveals the gaps in the healthcare system with hardships faced by a patient to access quality healthcare and poor patient outcome used as proxy indicators. It also points to the need to enhance access to healthcare to ensure early diagnosis and effective treatment for leishmaniasis patients including those who live in remote areas, in order to achieve the planned disease elimination targets. It also points towards the key challenges faced by a resource poor nation such as Bhutan in achieving universal health coverage and reaching the set goals for disease elimination. The findings underscore the need for a careful review of the national health care system and to address the deficiencies.

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.

Malaria elimination in Bhutan: asymptomatic malaria cases in the Bhutanese population living in malaria-risk areas and in migrant workers from India.

In 2018, Bhutan reported 54 cases of malaria, of which six were indigenous, 14 introduced and 34 imported. Considering the continuous reduction in the number of indigenous cases, Bhutan plans to eliminate malaria by 2025 under the Bhutan Malaria Elimination Strategy. The study was conducted to assess the presence of asymptomatic plasmodial infection in both, Bhutanese population living in malaria-risk areas and in migrant workers to guide the elimination strategies. A cross-sectional study was conducted from April to May 2016 in 750 Bhutanese people and 473 migrant workers. Plasmodium falciparum and Plasmodium vivax infections were investigated by using a rapid diagnostic test (RDT) and the polymerase chain reaction (PCR). Prevalence of asymptomatic plasmodial infection based on PCR was 0.27% (95% CI: 0.05-1.07%) among Bhutanese people with a mean age of 43 years old. The proportions of males and females were 45% and 55%, respectively. Among migrant workers, the prevalence of asymptomatic plasmodial infection was 0.42% (95% CI: 0.07-1.69%) with a mean age of 30 years old. The majority of migrant workers were from the neighboring Indian State of West Bengal (57.51%), followed by Assam (12.26%). RDT in both study groups did not detect any plasmodial infection. The presence of a low prevalence of asymptomatic plasmodial infection indicates that the current elimination strategies and interventions are effective.

A checklist of the Anopheles mosquito species (Diptera: Culicidae) in Bhutan.

The present paper records, for the first time, the Anopheles fauna of Bhutan, determined from surveys conducted from 2007 to early 2018. Adult mosquitoes were collected mainly on cattle bait and occasionally in human landing catches. Collections of immature stages were performed in various aquatic habitats. Larvae were preserved or reared to adults. Identification was based on morphological characters using available keys. A total of 30 species were identified, including nine species of subgenus Anopheles and 21 species of subgenus Cellia. Distribution and collection data are provided with notes on the locations and habitats of the species. Anopheles pseudowillmori is suspected to be a vector of malarial parasites in the plains and hilly forested areas of the country because it is widely distributed and the most common species collected in human landing catches. Notes also include observed morphological variation observed in An. baileyi and An. lindesayi, which differ from the type forms. Corrections are made for previous reports of Anopheles in Bhutan. The need for further surveys and molecular identification of members of species complexes and morphological variants is emphasized.