Background: Scrub typhus is an acute febrile illness caused by the obligate intracellular bacterium,Orientia tsutsugamushi. Immunochromatography (ICT) and IgM ELISA are two of the routinely employed antibody based assays for diagnosis of Scrub typhus fever in Nepal, although the recommended gold standard diagnostic test is IgM Immunofluorescence assay (IFA). This study evaluated InBios Scrub Typhus Detect™ Immunoglobulin M (IgM) ELISA and IgM Immunofluorescence assays in single serum sample at the time of admission.Method: Study participants (1585 suspected cases), were enrolled based on acute febrile illness with suspected scrub typhus cases in central Nepal. Blood sample was collected from the suspected patients of scrub typhus, presenting with acute febrile illness. IgM antibody toOrientia tsusugamushiwas detected by using Scrub Typhus Detect™ Kit and an in-house IgM IFA. The IFA assay was performed with the Gilliam, Karp, Kato strains andO. chutoantigens following the ARRL protocol.Result: Statistical analysis of IgM ELISA results when compared to reference test, IgM IFA results demonstrated the following characteristics, sensitivity 84.0% (95%CI: 79.73–87.68%), specificity 94.82% (95% CI: 93.43–95.99%), positive likelihood ratio 16.21% (95% CI: 12.71–20.67%), negative likelihood ratio 0.17% (95% CI: 0.13–0.21%), disease prevalence 22.08% (95% CI: 20.06 -24.21%), positive predictive value 82.12% (95% CI: 78.28–85.42%) and negative predictive value 95.44% (95% CI: 94.27–96.38%) respectively.Conclusion: Although IgM IFA is considered the gold standard test for the diagnosis of scrub typhus cases, it is relatively expensive, requires trained personal and a microscope with fluorescence filters. Scrub typhus IgM ELISA may be the best alternative test and possible viable option for resource limited endemic countries like Nepal.

With few studies conducted to date, very little is known about the epidemiology of rickettsioses in Bhutan. Due to two previous outbreaks and increasing clinical cases, scrub typhus is better recognized than other rickettsial infections and Q fever. A descriptive cross-sectional serosurvey was conducted from January to March 2015 in eight districts of Bhutan. Participants were 864 healthy individuals from an urban (30%) and a rural (70%) sampling unit in each of the eight districts. Serum samples were tested by microimmunofluorescence assay for rickettsial antibodies at the Australian Rickettsial Reference Laboratory. Of the 864 participants, 345 (39.9%) were males and the mean age of participants was 41.1 (range 13–98) years. An overall seroprevalence of 49% against rickettsioses was detected. Seroprevalence was highest against scrub typhus group (STG) (22.6%) followed by spotted fever group (SFG) rickettsia (15.7%), Q fever (QF) (6.9%) and typhus group (TG) rickettsia (3.5%). Evidence of exposure to multiple agents was also noted; the commonest being dual exposure to STG and SFG at 5%. A person’s likelihood of exposure to STG and SFG rickettsia significantly increased with age and farmers were twice as likely to have evidence of STG exposure as other occupations. Trongsa district appeared to be a hotspot for STG exposure while Punakha district had the lowest STG exposure risk. Zhemgang had the lowest exposure risk to SFG rickettsia compared to other districts. People living at altitudes above 2000 meters were relatively protected from STG infections but this was not observed for SFG, TG or QF exposure. This seroprevalence study highlights the endemicity of STG and SFG rickettsia in Bhutan. The high seroprevalence warrants appropriate public health interventions, such as diagnostic improvements and clinical treatment guidelines. Future studies should focus on vector profiles, geospatial, bio-social and environmental risk assessment and preventive and control strategies. Rickettsial infections including scrub typhus and Q fever are not widely recognised in Bhutan although the country is situated in an endemic Asian region. With two recorded outbreaks, scrub typhus is known to occur but other rickettsial infections and Q fever have not been recorded. In this first seroprevalence study of rickettsial infections, an overall seroprevalence of 49% was detected against rickettsioses amongst 864 participants. Seroprevalence was highest against scrub typhus group (STG) (22.6%) followed by spotted fever group (SFG) rickettsia (15.7%), Q fever (QF) (6.9%) and typhus group (TG) rickettsia (3.5%). Evidence of exposure to multiple agents were also noted; the commonest being dual exposure to STG and SFG at 5%. A person’s likelihood of exposure to STG and SFG significantly increased with age and farmers were twice as likely to have evidence of STG exposure as other occupations. Trongsa district in central Bhutan appeared to be a hotspot for STG exposure. People living at altitudes above 2000 meters were relatively protected from STG infections but this was not observed for SFG, TG and QF exposure. The findings from this study may direct future research on rickettsioses in Bhutan. These neglected tropical diseases warrant specific public health interventions in Bhutan.

Urine sampling may offer a less invasive solution than cervical sampling to test for human papillomavirus (HPV) for HPV vaccine impact monitoring. Paired samples of urine and exfoliated cervical cells were obtained for 89 women with history of high-risk (HR) HPV-positive normal cytology in Bhutan. Urine sampling protocol included self-collection of first-void urine immediately into a conservation medium and procedures to optimize DNA yield. Colposcopical abnormalities were biopsied. Two HPV assays were used: a multiplex type-specific PCR (E7-MPG) and a less analytically sensitive GP5+/6+ PCR followed by reverse line blot. HPV positivity for 21 types common to both assays was similar in urine and cells by E7-MPG (62.9% and 57.3%, respectively, p = 0.32) but lower in urine by GP5+/6+ (30.3% and 40.4%, p = 0.05). HPV6/11/16/18 positivity did not significantly differ between urine and cells by either assay. Sensitivity of urine (using cells as gold standard) to detect 21 HPV types was 80% and 58% for E7-MPG and GP5+/6+, respectively, with specificity 61% and 89%. HPV type distribution in urine and cells was similar, regardless of assay. The 5 detected CIN3+ were HR-HPV positive in cells by both assays, compared to 4 and 3 by E7-MPG and GP5+/6+, respectively, in urine samples. For the monitoring of vaccine impact, we demonstrate validity of a urine sampling protocol to obtain HPV prevalence data that are broadly comparable to that from cervical cells. However, detection of HPV in urine varies according to assay sensitivity, presumably because low level infections are frequent.

We assessed the level of knowledge, attitudes, and practices on antibiotics through a questionnaire-based cross-sectional survey among the general public in Bhutan. Of the 692 participants, 52.6% (364) were females with a mean age of 34.2 years. More than half of the respondents showed unsatisfactory knowledge varying significantly from 23.1% to 69.6%. Cotrimoxazole (septran) was the least known while amoxicillin was the most known antibiotics assessed. Two-thirds of the responsents (267) knew that inappropriate use of antibiotics could lead to antimicrobial resistance and 89% (319) were aware of the need to complete the antibiotic courses. In bivariate analysis, satisfactory knowledge was associated with the education level of graduate and higher as compared with no education. This study revealed unsatisfactory knowledge and attitude but satisfactory practices on antibiotics use among participants. Efforts are needed to improve public awareness on antibiotics. Enforcement of regulations on sale of antibiotics over the counter needs a revamp.

The global priorities in the field of infectious diseases are constantly changing. While emerging viral infections have regularly dominated public health attention, which has only intensified after the COVID-19 pandemic, numerous bacterial diseases have previously caused, and continue to cause, significant morbidity and mortality—deserving equal attention. Three potentially life-threatening endemic bacterial diseases (leptospirosis, melioidosis, and rickettsioses) are a huge public health concern especially in low- and middle-income countries. Despite their continued threat, these diseases do not receive proportionate attention from global health organizations and are not even included on the WHO list of neglected tropical diseases (NTDs). This, in turn, has led to a vicious circle of neglect with continued, yet conceivably preventable, hospitalizations and deaths each year especially in the vulnerable population. This is a call from a group of multi-institutional experts on the urgent need to directly address the circle of neglect and raise support in terms of funding, research, surveillance, diagnostics, and therapeutics to alleviate the burden of these 3 diseases.