Molecular and Entomological Characterization of 2023 Dengue Outbreak in Dhading District, Central Nepal.

In 2023, Nepal faced its second largest dengue outbreak ever, following a record-breaking number of dengue cases in 2022, characterized by the expansion of infections into areas of higher altitudes. However, the characteristics of the 2023 circulating dengue virus (DENV) and the vector density remain poorly understood. Therefore, we performed DENV serotyping, clinical and laboratory assessment, and entomological analysis of the 2023 outbreak in central Nepal. A total of 396 fever cases in Dhading hospital suspected of being DENV positive were enrolled, and blood samples were collected and tested by different techniques including PCR. Of these, 278 (70.2%) had confirmed DENV infection. Multiple serotypes (DENV-1, -2, and -3) were detected. DENV-2 (97.5%) re-emerged after six years in Dhading while DENV-3 was identified for the first time. Dengue inpatients had significantly higher frequency of anorexia, myalgia, rash, diarrhea, nausea, vomiting, abdominal pain, and thrombocytopenia (p < 0.05). In this area, Aedes mosquitoes largely predominated (90.7%) with the majority being A. aegypti (60.7%). We also found high levels of Aedes index (20.0%) and container index (16.7%). We confirmed multiple DENV serotype circulation with serotype re-emergence and new serotype introduction, and high vector density in 2023. These findings call for the urgent initiation and scaling up of DENV molecular surveillance in human and mosquito populations for dengue control and prevention in Nepal.

An outbreak investigation of scrub typhus in Nepal: confirmation of local transmission.

BACKGROUND: Scrub typhus is a largely ignored tropical disease and a leading cause of undifferentiated febrile illness in the areas of tsutsugamushi triangle caused by Orientia tsutsugamushi. It is frequently diagnosed in South Asian countries, although clear epidemiological information is not available from Nepal. After the 2015 earthquake in Nepal, a sudden upsurge in scrub typhus cases was reported. The objective of this study was to investigate epidemiology of scrub typhus and its causative agents in humans, animals, and chigger mites to understand the ongoing transmission ecology. METHODS: Scrub typhus cases with confirmed diagnosis throughout the country were included in the analysis. Studies were concentrated in the Chitwan district, the site of a major outbreak in 2016. Additional nation-wide data from 2015 to 2017 available from the government database included to analyse the disease distribution by geographical mapping. RESULTS: From 2015 to 2017, 1239 scrub typhus cases were confirmed with the largest outbreak occurring in 2016 with 831 (67.1%) cases. The case fatality rate was 5.7% in 2015 which declined to 1.1% in 2017. A nationwide outbreak of scrub typhus was declared as the cases were detected in 52 out of the 75 districts of Nepal. Seasonal trend was observed with a peak during August and September. In addition to the human cases, the presence of O. tsutsugamushi was also confirmed in animals (rodents) and chigger mites (Leptotrombidium imphalum) from the outbreak areas of southern Nepal. CONCLUSION: The detection of O. tsutsugamushi in humans, animals, and chigger mites from outbreak locations and wide-spread reports of scrub typhus throughout the country consecutively for 3 years confirms the ongoing transmission of O. tsutsugamushi with a firmly established ecology in Nepal. The country's health system needs to be strengthened for systematic surveillance, early outbreak detection, and immediate actions including treatment and preventive measures.

Genetic Diversity of Astrovirus in Children From a Birth Cohort in Nepal.

Objective: This study describes the types of Human astroviruses detected in stool samples collected from a birth cohort of children in Nepal. Methods: Using a commercial kit (ProSpecT), a total of 5,224 diarrheal and non-diarrheal stool samples were screened for Human astrovirus by ELISA. RT-PCR was performed on ELISA positive samples (2.8%) for further confirmation. The primary RT-PCR assay used targets the ORF2 region and detects human astrovirus type 1-8. Samples that were negative in this assay were further analyzed using primers that target the ORF1b region of human astrovirus which detect both classical type (HAstV 1-8) and novel types (MLB1-5, VA 1-5). PCR positive samples were analyzed by Sanger sequencing to determine the genotype. Results: A total of 148 available ELISA positive stool samples were analyzed by RT-PCR and further genotyped. RT-PCR analysis of these samples using the ORF2 and ORF1b assay revealed that 124 (84%) were positive for classical human types (HAstV 1-8). Seven different classical HAstV genotypes based on ORF2 and ORF1a were identified (HAstV 1- HAstV 8) with the greatest prevalence of HAstV 5 genotype (42.2%), followed by HAstV 1 (34.7%), HAstV 2 and HAstV 8 (7.4%), HAstV 4 (4.1%), HAstV 3 (3.3%), and HAstV 6 (0.8%). Non-classical types were not detected in our study. Conclusion: A high diversity of circulating Astrovirus strains were detected in young children, both with and without symptoms of gastroenteritis. HAstV 5 and HAstV 1 were the most common genotypes in young children in Nepal.

Determination of azithromycin heteroresistant Campylobacter jejuni in traveler's diarrhea.

Campylobacter is the most common cause of traveler's diarrhea (TD) and human bacterial gastroenteritis. A heteroresistant Campylobacter jejuni (C. jejuni) isolate, identified by microbiological methods and characterized with molecular techniques, was obtained from a traveler in Nepal suffering TD. The presence of atypical colonies within the clear zone of inhibition was the first evidence of an atypical phenotype, leading to additional characterization of this heteroresistant strain. Antimicrobial susceptibility testing (AST) and population analysis profiling (PAP) demonstrated heteroresistance to azithromycin (AZM), a first-line antibiotic treatment for Campylobacter infections. Molecular analysis indicated a point mutation occurred on the 23S rRNA gene at the A2075G transitions, and the number of mutated gene copies was proportional to AZM resistance. Heteroresistant C. jejuni subpopulations from acute TD are likely underestimated, which may lead to treatment failures, as was the case for this patient. The presence of a heteroresistant strain in a high antibiotic environment may select for additional drug resistance and enable distribution into hospital and local communities.

Astrovirus Infection and Diarrhea in 8 Countries.

BACKGROUND AND OBJECTIVES: Astroviruses are important drivers of viral gastroenteritis but remain understudied in community settings and low- and middle-income countries. We present data from 8 countries with high prevalence of diarrhea and undernutrition to describe astrovirus epidemiology and assess evidence for protective immunity among children 0 to 2 years of age. METHODS: We used 25 898 surveillance stools and 7077 diarrheal stools contributed by 2082 children for enteropathogen testing, and longitudinal statistical analysis to describe incidence, risk factors, and protective immunity. RESULTS: Thirty-five percent of children experienced astrovirus infections. Prevalence in diarrheal stools was 5.6%, and severity exceeded all enteropathogens except rotavirus. Incidence of infection and diarrhea were 2.12 and 0.88 episodes per 100 child-months, respectively. Children with astrovirus infection had 2.30 times the odds of experiencing diarrhea after adjustment for covariates (95% confidence interval [CI], 2.01-2.62; P < .001). Undernutrition was a risk factor: odds of infection and diarrhea were reduced by 10% and 13%, respectively, per increase in length-for-age z score (infection: odds ratio, 0.90 [95% CI, 0.85-0.96]; P < .001; diarrhea: odds ratio, 0.87 [95% CI, 0.79-0.96]; P = .006). Some evidence of protective immunity to infection was detected (hazard ratio, 0.84 [95% CI, 0.71-1.00], P = .052), although this was heterogeneous between sites and significant in India and Peru. CONCLUSIONS: Astrovirus is an overlooked cause of diarrhea among vulnerable children worldwide. With the evidence presented here, we highlight the need for future research as well as the potential for astrovirus to be a target for vaccine development.

Dengue Virus Serotypes 1 and 2 Responsible for Major Dengue Outbreaks in Nepal: Clinical, Laboratory, and Epidemiological Features.

Dengue virus (DENV) is expanding toward previously nonendemic areas. DENV has recently been introduced in Nepal with limited information. We report the clinical features and serotype distribution of DENV in Nepal during the 2010 outbreaks. A total of 1,215 clinical dengue cases at two major hospitals of central and western Nepal were investigated. Demographic, clinical, and laboratory parameters were recorded. Serum specimens were tested for DENV by IgM/IgG enzyme-linked immunosorbent assays (ELISAs) and reverse transcription polymerase chain reaction (RT-PCR). We confirmed DENV infection in 403 (33%) patients from 12 districts with an estimated case fatality rate of 1.5%. DENV infection was more common in adults (87%) and urban settings (74%). We detected all four serotypes but DENV-1 and -2 were mainly responsible for major outbreaks (92%). Overall, 60% of all DENV infections were secondary and 17% were severe dengue; both being more frequent among the DENV-2 infections. Rash, bleeding, abdominal pain, hepatomegaly, elevated liver enzymes, and thrombocytopenia were significantly more common in severe dengue compared with nonsevere infections. We also confirmed the expansion of dengue to hill urban areas (DENV-1 and -2), including the capital Kathmandu (altitude, 1,300 m) though > 90% cases were from southern plains. Differential clinical and laboratory features probably help in clinical decisions. Multiple serotypes circulation and elevated secondary infections pose potential risk of severe outbreaks and deaths in the future. Therefore, a country with recent dengue introduction, like Nepal, urgently requires a systematic surveillance and appropriate control measures in place to respond to any disastrous outbreaks.

Evidence of West Nile virus infection in Nepal.

BACKGROUND: Acute febrile illness is common among those seeking medical care and is frequently treated empirically with the underlying illness remaining undiagnosed in resource-poor countries. A febrile illness study was conducted 2009-2010 to identify known and unknown pathogens circulating in Nepal. METHOD: Study methods included diagnostic testing and preliminary ELISA screening of acute and convalescent samples for diseases both known and unknown to be circulating in Nepal, including West Nile virus (WNV). The molecular assays including Polymerase Chain Reaction (PCR), Sanger sequencing and ultra deep sequencing on MiSeq Illumina Platform were conducted to further confirm the presence of WNV. RESULTS: The study enrolled 2,046 patients presenting undifferentiated febrile illness with unknown etiology. Sera from 14 out of 2,046 patients were tested positive for west nile virus (WNV) by nested Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Only two out of 14 cases were confirmed for the presence of WNV by sequencing and identified as WNV lineage 1 phylogentically. The two patients were adult males with fever and no neurological symptoms from Kathmandu and Bharatpur, Nepal. CONCLUSION: Two out of 2,046 serum samples contained fragments of WNV genome resembling WNV lineage 1, which is evidence of the continued spread of WNV which should be considered a possible illness cause in Nepal.

Bhaktapur, Nepal: the MAL-ED birth cohort study in Nepal.

The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study site in Nepal is located in the Bhaktapur municipality, 15 km east of Kathmandu, the capital city of Nepal. Bhaktapur, an ancient city famous for its traditional temples and buildings, is included on UNESCO's World Heritage List and is a major tourist attraction in Nepal. Nepal is a land-locked country located in South Asia between China and India with an area of 147 181 km(2), ranging from sea-level plains to Mount Everest, the world's highest peak. The total population as of the 2011 census was 26.6 million, with an average annual population growth rate of 1.4. Nepal is one of the world's least developed countries and is ranked 157 of 186 in the 2013 Human Development Report; one-third of the Nepali population lives below the poverty line. The current under-5 mortality rate is 54 per 1000 live births, the infant mortality rate is 46 per 1000 live births, and the neonatal mortality rate is 33 per 1000 live births. Vaccine coverage for all Expanded Program on Immunization vaccines is >80%. Among children, the most common diseases contributing to significant morbidity and mortality are acute respiratory infection and dehydration from severe diarrhea. In this article, we report on the geographic, demographic, and socioeconomic features of the Bhaktapur MAL-ED site and describe the data that informed our cohort recruitment strategy.

Safety and efficacy of a recombinant hepatitis E vaccine.

BACKGROUND: Hepatitis E virus (HEV) is an important cause of viral hepatitis. We evaluated the safety and efficacy of an HEV recombinant protein (rHEV) vaccine in a phase 2, randomized, double-blind, placebo-controlled trial. METHODS: In Nepal, we studied 2000 healthy adults susceptible to HEV infection who were randomly assigned to receive three doses of either the rHEV vaccine or placebo at months 0, 1, and 6. Active (including hospital) surveillance was used to identify acute hepatitis and adverse events. The primary end point was the development of hepatitis E after three vaccine doses. RESULTS: A total of 1794 subjects (898 in the vaccine group and 896 in the placebo group) received three vaccine doses; the total vaccinated cohort was followed for a median of 804 days. After three vaccine doses, hepatitis E developed in 69 subjects, of whom 66 were in the placebo group. The vaccine efficacy was 95.5% (95% confidence interval [CI], 85.6 to 98.6). In an intention-to-treat analysis that included all 87 subjects in whom hepatitis E developed after the first vaccine dose, 9 subjects were in the vaccine group, with a vaccine efficacy of 88.5% (95% CI, 77.1 to 94.2). Among subjects in a subgroup randomly selected for analysis of injection-site findings and general symptoms (reactogenicity subgroup) during the 8-day period after the administration of any dose, the proportion of subjects with adverse events was similar in the two study groups, except that injection-site pain was increased in the vaccine group (P=0.03). CONCLUSIONS: In a high-risk population, the rHEV vaccine was effective in the prevention of hepatitis E. (ClinicalTrials.gov number, NCT00287469 [ClinicalTrials.gov].).

Study of hepatitis B among different categories of health care workers.

OBJECTIVE: To estimate the frequency of hepatitis B virus (HBV) infection among different categories of health care workers (HCWs) so as to identify groups and areas requiring special attention for the prevention of occupational transmission of bloodborne infections. DESIGN: A cross-sectional study. PLACE AND DURATION OF STUDY: The study was conducted in Bir Hospital, the central referral hospital of Nepal from December 2001 to February 2002. PATIENTS AND METHODS: A total of 145 HCWs were selected by random allocation and blood samples were tested for HBV surface antigen (HBsAg), surface antibody (anti-HBs) and core antibody (anti-HBc). Multivariate analysis was done, including calculation of odd ratios for HBV infection by each variable of interest, like age, sex, HCW category, history of blood transfusion and vaccination status. RESULTS: Anti-HBc was positive in 14.5% and HBsAg in 1.4% of HCWs. 20.9% of non-professional staff, 19.2% of nurses, 5.6% of laboratory workers and 3.1% of doctors had evidence of past or present HBV infection. 48.9% of HCWs, with only 16.7% of laboratory workers and 27.9% of non-professional staff, had received a full course of HBV vaccination. The significant risk factors associated with past or present HBV infection were lack of hepatitis B vaccination (p<0.05) and two HCW categories, nurses (p<0.05) and non-professional staff, who clean the used instruments (p<0.05). CONCLUSION: Apart from lack of hepatitis B vaccination, nurses and non-professional staff on their own were found to be significantly more susceptible to HBV infection than others. This highlights the need to implement the internationally recommended measures to protect HCWs from bloodborne infections.