Demographic risk factors for extra-pulmonary tuberculosis: a rising public health threat in Bhutan.

BACKGROUND: Recently, extra-pulmonary tuberculosis (EPTB) has been increasingly reported in Bhutan, rising from 26% in 2001 to 39% in 2010. In 2016, almost half (49%) of all reported TB cases were classified as EPTB. Thus, this study was conducted to understand the epidemiology and identify risk factors contributing to increasing notification of EPTB in Bhutan. METHODS: A case-control study was conducted by recruiting all 110 cases of nationally notified EPTB (Extrapulmonary bacteriologically confirmed/EPBC) as cases and 235 Pulmonary TB (Pulmonary bacteriologically confirmed/PBC) as controls. Data were collected on socio-demography, clinical and diagnostic, behavioral and lifestyle and environmental exposures using a structured questionnaire between April and September, 2018. RESULTS: The median age of the cases was 25 years, ranged 9-79 years. Lymphatic TB was predominant (n = 78; 70.91%) followed by genitourinary TB (n = 4 (3.64%). The likelihood of EPTB decreased with increase in age (p = 0.023). The odds of EPTB in females was 1.65 times higher than the males (p = 0.038). Increase in Body Mass Index (adjusted odds ratio (AOR): 1.1; 95% confidence interval (CI): 1.052-1.200) and urban residency were (AOR 1.6; 95% CI 1.016-2.805) were found to have higher odds of developing EPTB. CONCLUSION: Females, urban residents, and those with higher BMI are at increased risk of developing EPTB. These at-risk groups can be used to target limited public health resources to control EPTB in Bhutan.

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.

Human rabies encephalomyelitis in the background of rabies outbreak in animals in Gelephu, Bhutan, 2023: a case report.

BACKGROUND: Rabies continues to pose significant public health challenges in many developing countries including Bhutan. A probable case of rabies was admitted to our hospital and its reporting led to the uncovering of an outbreak in domestic and wild animals. We discuss the challenges in the diagnosis and management of rabies in a resource-limited setting. CASE PRESENTATION: A 35-year-old male presented with intermittent fever, bilateral lower limb weakness that was rapidly progressive, urinary incontinence with episodes of palpitations and sweating. He had sustained a Category III bite on the right lower thigh with four bite marks, inflicted by a stray dog. He had received post-exposure prophylaxis with intra-dermal anti-rabies vaccine. On initial examination, the patient was in distress but cooperative for the interview. He had pulse rate ranging from 60 to 100/min with episodes of diaphoresis and palpitations, but with normal capillary blood glucose. In the lower limb, the muscle power was zero with absent tendon reflexes in the lower limb and impaired abdominal reflex below T10 level. He had hyperaesthesia below T8, hydrophobia, aerophobia and photophobia. He had multiple spontaneous fasciculations in both the thighs and right deltoid and these later involved the intercostal muscles, neck and face muscles. He had altered sensorium and desaturation for which he required mechanical ventilation. Polymerase chain reaction for rabies virus was negative in cerebrospinal fluid and saliva. Rabies virus neutralizing antibody was negative in cerebrospinal fluid but had high titres in the serum. He received Human Rabies Immunoglobulin after admission. He was managed in the intensive care unit and died 23 days later. After this case was notified, a rapid response team was deployed in the field, and uncovered rabies outbreak in animals in the locality. CONCLUSIONS: This case called for a serious evaluation of the country's efforts in achieving zero rabies deaths by 2030. The management of this case identified several critical areas of context-specific interventions in Bhutan. There is also an urgent need to improve diagnostic capabilities at the national reference laboratory and enhance the technical competencies of healthcare workers in the management of dog bite cases.

Environmental, Occupational, and Demographic Risk Factors for Clinical Scrub Typhus, Bhutan.

Underdiagnosis and underreporting of scrub typhus has increasingly affected public health in Bhutan since its initial detection in 2008. Identifying scrub typhus risk factors would support early diagnosis and treatment for this nonspecific febrile disease, reducing the incidence of potentially fatal complications. We conducted a hospital-based, case‒control study during October‒December 2015 in 11 scrub typhus‒prone districts. We identified harvesting cardamom as the major risk factor (odds ratio 1,519; p<0.001); other factors were traditional housing, largely caused by an outside toilet location, as well as owning a goat and frequently sitting on grass. Harvesting vegetables, herding cattle in the forest, and female sex were protective. Age had a nonlinear effect; children and the elderly were more likely to seek treatment for clinical scrub typhus. This study has informed public health policies and awareness programs for healthcare workers through development of National Guidelines for Prevention, Treatment and Control of Scrub Typhus in Bhutan.

High quality social environment buffers infants' cognitive development from poor maternal mental health: Evidence from a study in Bhutan.

Poor maternal mental health negatively impacts cognitive development from infancy to childhood, affecting both behavior and brain architecture. In a non-western context (Thimphu, Bhutan), we demonstrate that culturally-moderated factors such as family, community social support, and enrichment may buffer and scaffold the development of infant cognition when maternal mental health is poor. We used eye-tracking to measure early building blocks of cognition: attention regulation and social perception, in 9-month-old Bhutanese infants (N = 121). The cognitive development of Bhutanese infants in richer social environments was buffered from poor maternal mental health, while for infants in environments with lower rates of protective social environment factors, worse maternal mental health significantly predicted greater costs for infant attention, a fundamental building block cognition. International policies and interventions geared to improve maternal mental health and child health outcomes should incorporate each regions' unique family, cultural, and community support structures.

Antibiotic Prescription in Veterinary Consultations in Bhutan: A Retrospective Cross-Sectional Study.

The veterinary prescription of antibiotics in low- and middle-income countries (LMIC) remains largely undocumented. In Bhutan, however, the national veterinary service keeps records of their activities and prescriptions, which offer an opportunity to establish a benchmark to assess the use of these agents in this and other LMIC. A cross-sectional retrospective study was designed and 2,266 handwritten veterinary records from 2017 were sampled from 23 animal health premises (AHPs) to estimate individual and an overall proportion of consultations that resulted in an antibiotic prescription. The frequency of antibiotic prescription per species, type of AHP, and according to WHO's AWaRe index and OIE list of priority antimicrobials were also explored. It was estimated that 31% (95% confidence interval: 29-33%; intracluster correlation: 0.03) of the veterinary consultations resulted in an antibiotic prescription. The incidence of antibiotic prescription was highest in consultations of poultry across AHP. Across species, diarrhea and wounds were frequently treated with broad-spectrum antibiotics including sulfonamides, tetracyclines, trimethoprim + sulfa, and penicillin. Between 45% and 70% antibiotics prescribed correspond to AWaRe's access group and up to 25% to AWaRe's watch group. Over 70% of antibiotics dispensed in veterinary consultations for any species correspond to the OIE's veterinary critically important antimicrobial agents. Overall, the study demonstrated positive features of veterinary antimicrobial stewardship in Bhutan, given the conservative proportion of consultation that results in this type of prescription and the type of antibiotic prescribed. Although the veterinary service closely follows the Bhutanese Standard Treatment Guidelines, the prescription of antibiotics to key species should be closely monitored. Our study suggests that further improvements of antibiotic stewardship can be achieved through standardisation of antibiotic prescription to some species, a revision of the guidelines toward reducing the prescription of antibiotics of high relevance for human medicine, and by including details of clinical investigation, use of tests, and treatment outcomes in veterinary consultation records.

Identification, Distribution, and Habitat Suitability Models of Ixodid Tick Species in Cattle in Eastern Bhutan.

Tick infestation is the most reported parasitological problem in cattle in Bhutan. In May and June 2019, we collected ticks from 240 cattle in two districts of Eastern Bhutan. Tick presence, diversity, and infestation prevalence were examined by morphological identification of 3600 live adult ticks. The relationships between cattle, geographic factors, and infestation prevalence were assessed using logistic regression analyses. Habitat suitability for the tick species identified was determined using MaxEnt. Four genera and six species of ticks were found. These were Rhipicephalus microplus (Canestrini) (70.2% (95% confidence interval (CI): 68.7-71.7)), Rhipicephalus haemaphysaloides Supino (18.8% (95% CI: 17.5-20.1)), Haemaphysalis bispinosa Neumann (8.2% (95% CI: 7.3-9.1)), Haemaphysalis spinigera Neumann (2.5% (95% CI: 2-3)), Amblyomma testudinarium Koch (0.19% (95% CI: 0.07-0.4)), and a single unidentified Ixodes sp. Logistic regression indicated that the variables associated with infestation were: longitude and cattle age for R. microplus; latitude for R. haemaphysaloides; and altitude and cattle breed for H. bispinosa and H. spinigera. MaxEnt models showed land cover to be an important predictor for the occurrence of all tick species examined. These findings provide information that can be used to initiate and plan enhanced tick surveillance and subsequent prevention and control programs for ticks and tick-borne diseases in cattle in Bhutan.

A knowledge, attitudes, and practices study on ticks and tick-borne diseases in cattle among farmers in a selected area of eastern Bhutan.

Livestock farming plays an important role in supporting the livelihood of resource-poor subsistence farmers in Bhutan. However, ticks and tick-borne diseases (TBDs) are one of the major constraints to livestock farming due to their negative effect on health and production. To date, no study has been conducted in Bhutan to assess farmers' knowledge, attitude, and practices (KAP) about ticks and TBDs in cattle, although such information is essential in ensuring the development and adoption of effective prevention and control measures. Therefore, a KAP survey was conducted among 246 cattle owners in the Samkhar sub-district of eastern Bhutan in June 2019, using a structured questionnaire. Based on our scoring criteria, 52% [95%CI: 45.5-58.4] had adequate knowledge about ticks as potential vectors of diseases. Logistic regression analysis showed that the individuals who practiced a stall-feeding system of cattle rearing were 2.8 times [OR = 2.8 (95%CI: 1.66-4.78)] more likely to have adequate knowledge than others. Sixty-eight percent [95%CI: 62.5-74.4] had a favorable attitude toward tick prevention and control programs. Men were 1.95 times [OR = 1.95 (95%CI: 1.09-3.55)] more likely to have a favorable attitude than women, and the individuals who practiced a stall-feeding system were 2.59 times [OR = 2.59 95%CI: 1.45-4.78)] more likely to have a favorable attitude than others, after adjusting for the effect of other variables in the model. Overall, only 38% [95%CI 32.5-45] of the respondents reported tick infestation as one of the most important animal health problems, but 100% reported using acaricides to control ticks in cattle. Despite a high level of acaricide usage, the level of knowledge was low among the farmers interviewed. Findings from this study underline the importance of considering identified knowledge gaps and initiating education efforts to improve the adoption of effective tick prevention and control measures among farmers.

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.

Epidemiological and histopathological characteristics of head and neck cancers in Bhutan from 2011 to 2017: a retrospective descriptive study.

BACKGROUND: Head and neck cancers are among the commonest cancers in the developing world. Personal habits, such as the use of tobacco, betel nut and alcohol are strongly associated with the development of head and neck cancers at certain sites. Therefore, they are among the preventable cancers. In Bhutan, there has not yet been a study conducted on head and neck cancers. OBJECTIVE: To describe baseline epidemiological and histopathological characteristics of head and neck cancers in Bhutan. METHODS: This is a 7-year descriptive study of all cases of head and neck cancers presented at the Jigme Dorji Wangchuk National Referral Hospital from 2011 to 2017. The data were collected from the hospital's medical records section, histopathology records, patient referral unit and some treatment centres in India. Prior approval was sought from the Research and Ethics Board for Health, the Ministry of Health and the hospital management. RESULTS: There were a total of 515 cases of head and neck cancers from 2011 to 2017. The crude incidence rate was 10 per 100,000 and the overall age adjusted rate was 12.3 (95% CI 9.5-15.1) per 100,000 population. The prevalence during this 7-year period was 69.1 per 100,000 population. The commonest cancers are thyroid, oral cavity, hypopharyngeal, laryngeal and nasopharyngeal cancer in decreasing order. Head and neck cancers are more common in males than females in the majority of sites except thyroid, salivary gland and sinonasal malignancies. Thyroid cancers and nasopharyngeal cancers are found to affect younger age groups. Tashigang (48) followed by Paro (43) recorded the highest number of cases. Squamous cell carcinoma is the commonest histopathology type in almost all the cases, while papillary carcinoma is the commonest among thyroid cancers. Personal habits, such as smoking, chewing tobacco, betel nut and alcohol consumption, were found to be more common among patients suffering from oral cavity, laryngeal, hypopharyngeal and oropharyngeal cancers. CONCLUSION: Head and neck cancers are the third most common cancer in Bhutan after stomach cancer and cervical cancer. Thyroid, oral cavity and hypopharynx are the top three anatomical sites for head and neck cancers in Bhutan. The current epidemiological and histopathological profile of head and neck cancers will form a baseline of information and basis for further research on head and neck cancers in Bhutan.

Evaluation of post-exposure prophylaxis practices to improve the cost-effectiveness of rabies control in human cases potentially exposed to rabies in southern Bhutan.

BACKGROUND: Rabies is endemic in southern Bhutan, associated with 1-2 human deaths and high post exposure prophylaxis (PEP) costs annually. Evaluation of clinicians' management of human cases potentially exposed to rabies could contribute to improving PEP prescribing practices to both reduce unnecessary costs associated with PEP and reach the target of zero human deaths due to rabies by 2023. METHODS: A cross-sectional survey of 50 clinicians' management of human cases potentially exposed to rabies was conducted in 13 health centers in high-rabies-risk areas of Bhutan during February-March 2016. RESULTS: Data were collected on clinicians' management of 273 human cases potentially exposed to rabies. The 50 clinicians comprised health assistants or clinical officers (55%) and medical doctors (45%) with a respective median of 19, 21 and 2 years' experience. There was poor agreement between clinicians' rabies risk assessment compared with an independent assessment for each case based on criteria in the National Rabies Management Guidelines (NRMG). Of the 194 cases for which clinicians recorded a rabies risk category, only 53% were correctly classified when compared with the NRMG. Clinicians were more likely to underestimate the risk of exposure to rabies and appeared to prescribe PEP independently of their risk classification.. Male health assistants performed the most accurate risk assessments while female health assistants performed the least accurate. Clinicians in Basic Health Units performed less accurate risk assessments compared with those in hospitals. CONCLUSIONS: This study highlights important discrepancies between clinicians' management of human cases potentially exposed to rabies and recommendations in the NRMG. In particular, clinicians were not accurately assessing rabies risk in potentially exposed cases and were not basing their PEP treatment on the basis of their risk assessment. This has significant implications for achieving the national goal of eliminating dog-mediated human rabies by 2030 and may result in unnecessary costs associated with PEP. Recommendations to improve clinician's management of human cases potentially exposed to rabies include: reviewing and updating the NRMG, providing clinicians with regular and appropriately targeted training about rabies risk assessment and PEP prescription, and regularly reviewing clinicians' practices.

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.

Clinical and Epidemiological Patterns of Scrub Typhus, an Emerging Disease in Bhutan.

Scrub typhus (ST) is a vector-borne rickettsial infection causing acute febrile illness. The re-emergence of ST in the Asia-Pacific region represents a serious public health threat. ST was first detected in Bhutan in 2008. However, the disease is likely to be under-diagnosed and under-reported, and the true impact is difficult to estimate. At the end of 2014, the SD Bioline Tsutsugamushi TestTM rapid diagnostic test (RDT) kits became available in all hospitals to assist clinicians in diagnosing ST. We conducted a retrospective descriptive study, reviewing records from all hospitals of Bhutan to identify all RDT-positive clinical cases of ST in Bhutan in 2015. The aim was to evaluate the burden of ST in Bhutan, describe the demographic, spatial and temporal patterns of disease, and identify the typical clinical presentations. The annual incidence of RDT-positive cases of ST reporting to Bhutanese hospitals in 2015 was estimated to be 62 per 100,000 population at risk. The incidence of disease was highest in the southern districts with a subtropical climate and a high level of agricultural production. The highest proportion of cases (87%) was rural residents, with farmers being the main occupational category. The disease was strongly seasonal, with 97% of cases occurring between June and November, coinciding with the monsoon and agricultural production seasons. Common ST symptoms were not specific, and an eschar was noted by clinicians in only 7.4% of cases, which is likely to contribute to an under-diagnosis of ST. ST represents an important and neglected burden, especially in rural communities in Bhutan. The outcomes of this study will inform public health measures such as timely-awareness programmes for clinicians and the public in high-risk areas, to improve the diagnosis, treatment and clinical outcomes of this disease.

Survey and Phylogenetic Analysis of Rodents and Important Rodent-Borne Zoonotic Pathogens in Gedu, Bhutan.

Rodents are well-known reservoirs and vectors of many emerging and re-emerging infectious diseases, but little is known about their role in zoonotic disease transmission in Bhutan. In this study, a cross-sectional investigation of zoonotic disease pathogens in rodents was performed in Chukha district, Bhutan, where a high incidence of scrub typhus and cases of acute undifferentiated febrile illness had been reported in people during the preceding 4-6 months. Twelve rodents were trapped alive using wire-mesh traps. Following euthanasia, liver and kidney tissues were removed and tested using PCR for Orientia tsutsugamushi and other bacterial and rickettsial pathogens causing bartonellosis, borreliosis, human monocytic ehrlichiosis, human granulocytic anaplasmosis, leptospirosis, and rickettsiosis. A phylogenetic analysis was performed on all rodent species captured and pathogens detected. Four out of the 12 rodents (33.3%) tested positive by PCR for zoonotic pathogens. Anaplasma phagocytophilum, Bartonella grahamii, and B. queenslandensis were identified for the first time in Bhutan. Leptospira interrogans was also detected for the first time from rodents in Bhutan. The findings demonstrate the presence of these zoonotic pathogens in rodents in Bhutan, which may pose a risk of disease transmission to humans.

One Health research and training and government support for One Health in South Asia.

INTRODUCTION: Considerable advocacy, funding, training, and technical support have been provided to South Asian countries to strengthen One Health (OH) collaborative approaches for controlling diseases with global human pandemic potential since the early 2000s. It is essential that the OH approach continues to be strengthened given South Asia is a hot spot for emerging and endemic zoonotic diseases. The objectives of this article are to describe OH research and training and capacity building activities and the important developments in government support for OH in these countries to identify current achievements and gaps. MATERIALS AND METHODS: A landscape analysis of OH research, training, and government support in South Asia was generated by searching peer-reviewed and grey literature for OH research publications and reports, a questionnaire survey of people potentially engaged in OH research in South Asia and the authors' professional networks. RESULTS: Only a small proportion of zoonotic disease research conducted in South Asia can be described as truly OH, with a significant lack of OH policy-relevant research. A small number of multisectoral OH research and OH capacity building programmes were conducted in the region. The governments of Bangladesh and Bhutan have established operational OH strategies, with variable progress institutionalising OH in other countries. Identified gaps were a lack of useful scientific information and of a collaborative culture for formulating and implementing integrated zoonotic disease control policies and the need for ongoing support for transdisciplinary OH research and policy-relevant capacity building programmes. DISCUSSION: Overall we found a very small number of truly OH research and capacity building programmes in South Asia. Even though significant progress has been made in institutionalising OH in some South Asian countries, further behavioural, attitudinal, and institutional changes are required to strengthen OH research and training and implementation of sustainably effective integrated zoonotic disease control policies.

Comparison of mark-resight methods to estimate abundance and rabies vaccination coverage of free-roaming dogs in two urban areas of south Bhutan.

In Bhutan, Capture-Neuter-Vaccinate-Release (CNVR) programs have been implemented to manage the dog population and control rabies, but no detailed evaluation has been done to assess their coverage and impact. We compared estimates of the dog population using three analytical methods: Lincoln-Petersen index, the Chapman estimate, and the logit-normal mixed effects model, and a varying number of count periods at different times of the day to recommend a protocol for applying the mark-resight framework to estimate free-roaming dog population abundance. We assessed the coverage of the CNVR program by estimating the proportion of dogs that were ear-notched and visually scored the health and skin condition of free-roaming dogs in Gelephu and Phuentsholing towns in south Bhutan, bordering India, in September-October 2012. The estimated free-roaming dog population in Gelephu using the Lincoln-Petersen index and Chapman estimates ranged from 612 to 672 and 614 to 671, respectively, while the logit-normal mixed effects model estimate based on the combined two count events was 641 (95% CI: 603-682). In Phuentsholing the Lincoln-Petersen index and Chapman estimates ranged from 525 to 583 and 524 to 582, respectively, while the logit-normal mixed effects model estimate based on the combined four count events was 555 (95% CI: 526-587). The total number of dogs counted was significantly associated with the time of day (AM versus PM; P=0.007), with a 17% improvement in dog sightings during the morning counting events. We recommend to conduct a morning marking followed by one count event the next morning and estimate population size by applying the Lincoln-Peterson corrected Chapman method or conduct two morning count events and apply the logit-normal mixed model to estimate population size. The estimated proportion of vaccinated free-roaming dogs was 56% (95% CI: 52-61%) and 58% (95% CI: 53-62%) in Gelephu and Phuentsholing, respectively. Given coverage in many neighbourhoods was below the recommended threshold of 70%, we recommend conducting an annual "mass dog vaccination only" campaign in southern Bhutan to create an immune buffer in this high rabies-risk area. The male-to-female dog ratio was 1.34:1 in Gelephu and 1.27:1 in Pheuntsholing. Population size estimates using mark-resight surveys has provided useful baseline data for understanding the population dynamics of dogs at the study sites. Mark-resight surveys provide useful information for designing and managing the logistics of dog vaccination or CNVR programs, assessing vaccination coverage, and for evaluating the impact of neutering programs on the size and structure of dog populations over time.