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.

Occurrence of Echinococcusgranulosussensulato and Other Taeniids in Bhutan.

The present research shows the results of a national study documenting the occurrence and genetic diversity of Echinococcus and Taenia species across Bhutan. Environmental dog faecal samples (n = 953) were collected from 2016 to 2018 in all 20 Bhutanese districts, mainly in urbanised areas. Cystic echinococcosis cysts were isolated from 13 humans and one mithun (Bos frontalis). Isolation of taeniid eggs from faeces was performed by sieving/flotation technique, followed by DNA isolation, PCR and sequence analyses for species identification (gene target: small subunit of ribosomal RNA). Genetic diversity of E. granulosuss.s. was based on the sequence (1609 bp) of the cox1 gene. A total of 67 out of 953 (7%) dog faecal samples were positive for at least one taeniid species. From the 670 free-roaming dog faecal samples, 40 (5.9%) were positive for taeniid DNA, 22 (3.2%) of them were identified as E. granulosuss.s. and four (0.5%) as E. ortleppi (G5). From the 283 faecal samples originating from yak-grazing areas, 27 (9.5%) were taeniid positive, including eight (2.8%) infected with E. granulosuss.s. and four (1.4%) with E. ortleppi. E. granulosuss.s. was identified in all isolates from human and the cyst from mithun. A haplotype network (cox1 gene) from E. granulosuss.s, including isolates from 12 dogs, two human and one mithun, revealed eight different haplotypes. The most common cox1 haplotype was the globally distributed Eg01, followed by Eg40 and Eg37 (previously described in China). Five new cox1 haplotypes (EgBhu1-5) originated from human, dogs, and a mithun were identified. The study indicated the contamination of urban areas and pastures with Echinococcus eggs in seven districts in Bhutan. The molecular characterisation of E. granulosuss.l. revealed different E. granulosuss.s. haplotypes as well as E. ortleppi. The transmission of T. multiceps was documented only in the western part of the country. Considering the zoonotic feature of E. granulosus s.s. and E. ortleppi and the economic impact of coenurosis caused by T. multiceps (also known as gid) in Bhutan, the findings of this study represent a significant contribution towards an epidemiological baseline for the establishment of a national control programme.

Cerebral cysticercosis in a wild Bengal tiger (Panthera tigris tigris) in Bhutan: A first report in non-domestic felids.

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population.

Further development of a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of foot-and-mouth disease virus and validation in the field with use of an internal positive control.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hooved animals. Global outbreaks have highlighted the significant economic, trade, psychosocial and animal welfare impacts that can arise from the detection of disease in previously 'FMD-free' countries. Rapid and early diagnosis provides significant advantages in disease control and minimization of deleterious consequences. We describe the process of further development and validation of a reverse-transcription loop-mediated isothermal amplification foot-and-mouth disease virus (RT-LAMP-FMDV) test, using a published LAMP primer set, for use in the field. An internal positive control (IPC) was designed and introduced for use with the assay to mitigate any intrinsic interference from the unextracted field samples and avoid false negatives. Further modifications were included to improve the speed and operability of the test, for use by non-laboratory trained staff operating under field conditions, with shelf-stable reaction kits which require a minimum of liquid handling skills. Comparison of the assay performance with an established laboratory-based real-time reverse transcriptase PCR (rRT-PCR) test targeting the 3D region of FMD virus (Tetracore Inc) was investigated. LAMP has the potential to complement current laboratory diagnostics, such as rRT-PCR, as a preliminary tool in the investigation of FMD. We describe a strategic approach to validation of the test for use in the field using extracted RNA samples of various serotypes from Thailand and then finally unextracted field samples collected from FMD-suspected animals (primarily oral lesion swabs) from Bhutan and Australia. The statistical approach to validation was performed by Frequentist and Bayesian latent class methods, which both confirmed this new RT-LAMP-FMDV test as fit-for-purpose as a herd diagnostic tool with diagnostic specificity >99% and sensitivity 79% (95% Bayesian credible interval: 65, 90%) on unextracted field samples (oral swabs).

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.