Circulating serotypes and genotypes of dengue virus during the 2023 outbreak in Eastern Nepal.

Dengue virus (DENV) is one of the most significant mosquito-borne diseases in Nepal. In 2023, DENV outbreaks began in Eastern Nepal, near the border with India, and rapidly spread nationwide. The study aims to describe the outbreak's epidemiological pattern, laboratory characteristics, DENV serotypes, and genotypes. A hospital-based cross-sectional study was conducted in four hospitals in Jhapa, Eastern Nepal, in 2023. Acute serum samples were obtained from dengue suspected patients within 7 days of illness and subjected to virus isolation, conventional and real-time polymerase chain reaction (RT-PCR), and phylogenetic analysis. Out of 60 samples, 42 (70 %), 11 (18.3 %) and 7 (11.7 %) were primary, secondary and non-dengue infection, respectively. Among 53 dengue confirmed patients, 46 (86.7 %) were positive for NS1 and 12 (22.6 %) were positive for both NS1 and IgM. Out of 42 dengue isolates, a new clade of the cosmopolitan genotype of DENV-2 was the most prevalent (28, 66.7 %), followed by genotype III of DENV-3 (11, 26.2 %) and genotype V of DENV-1 (3, 7.1 %). Genotype III of DENV-3 was first introduced in 2022-2023 in Nepal. Phylogenetic analysis of the E gene revealed the DENV-2 isolates from Nepal had 98 % homologous nucleotide similarity with the strains from India and Bangladesh. To our knowledge, this is the first report of circulating serotypes and genotypes of DENV in Jhapa. Integrating molecular findings into the dengue control plan can enhance surveillance efforts, monitor disease trends, and implement proactive measures to reduce the burden of dengue and prevent fatalities in future outbreaks.

Survey of Entamoeba infections in schoolchildren and macaques in Kathmandu, Nepal, and analysis of genetic polymorphisms of Entamoeba nuttalli and Entamoeba dispar isolates.

Entamoeba species infect humans and non-human primates, raising concerns associated with potential zoonotic transmission. Therefore, the prevalence of human Entamoeba infections is crucial for its management in areas, where macaques exhibit high infection rates. Previously, we demonstrated prevalent E. nuttalli infections in rhesus macaques in Kathmandu, Nepal. In this study, we surveyed Entamoeba infection among 185 schoolchildren from two schools visited by wild rhesus macaques to assess the risk of transmission. PCR-based screening for Entamoeba species identified E. coli in 13 % and E. dispar in 0.5 % of the human stool samples. However, E. nuttalli and E. chattoni infections, prevalent in macaques, were not detected in human samples. This suggests that Entamoeba spp. are not transmitted through macaques in the school environment. We surveyed the rhesus macaques living in the temple near schools as well as the rhesus and Assam macaques inhabiting Shivapri Nagarjun National Park, Kathmandu. Among the 49 macaque stool samples, E. chattoni, E. coli, E. nuttalli, and E. dispar were detected in 92 %, 86 %, 41 %, and 18 % of the samples, respectively. Notably, E. dispar infections in macaques were mostly prevalent in the temple. A sample isolated from Nagarujun showed an identical genotype at two tRNA-linked short tandem repeat loci to that of E. dispar isolated from humans, suggesting potential transmission from humans to macaques. Genotypic analysis of cultured E. nuttalli strains obtained from the macaques colonizing three locations demonstrated that the geographical distance rather than differences in macaque species played a crucial role in the genetic diversity of the parasites. The phylogenetic tree of E. nuttalli strains, including the previously isolated strains, reflected the geographical distribution of the isolation sites. This study sheds light on the intricate dynamics of Entamoeba transmission and genetic diversity in macaques and humans.

The Development of New Primer Sets for the Amplification and Sequencing of the Envelope Gene of All Dengue Virus Serotypes.

Dengue virus (DENV) poses a significant threat to global health, infecting approximately 390 million people annually. This virus comprises four serotypes capable of causing severe disease. Genetic analyses are crucial for understanding the epidemiology, evolution, and spread of DENV. Although previous studies have focused on the envelope protein-coding (E) gene, only a few primers can efficiently detect and amplify the viral genes from multiple endemic countries simultaneously. In this study, we designed degenerate primer pairs for each DENV serotype to amplify and sequence the entire E gene, using globally representative sequences for each serotype. These primers were validated using DENV isolates from various Asian countries and demonstrated broad-spectrum detection capabilities and high-quality sequences. The primers provide effective tools for genetic analysis in the regions affected by dengue, aiding strain identification and epidemiological studies during outbreaks.

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.

Evaluating active leprosy case identification methods in six districts of Nepal.

BACKGROUND: Nepal has achieved and sustained the elimination of leprosy as a public health problem since 2009, but 17 districts and 3 provinces with 41% (10,907,128) of Nepal's population have yet to eliminate the disease. Pediatric cases and grade-2 disabilities (G2D) indicate recent transmission and late diagnosis, respectively, which necessitate active and early case detection. This operational research was performed to identify approaches best suited for early case detection, determine community-based leprosy epidemiology, and identify hidden leprosy cases early and respond with prompt treatment. METHODS: Active case detection was undertaken in two Nepali provinces with the greatest burden of leprosy, Madhesh Province (40% national cases) and Lumbini Province (18%) and at-risk prison populations in Madhesh, Lumbini and Bagmati provinces. Case detection was performed by (1) house-to-house visits among vulnerable populations (n = 26,469); (2) contact examination and tracing (n = 7608); in Madhesh and Lumbini Provinces and, (3) screening prison populations (n = 4428) in Madhesh, Lumbini and Bagmati Provinces of Nepal. Per case direct medical and non-medical costs for each approach were calculated. RESULTS: New case detection rates were highest for contact tracing (250), followed by house-to-house visits (102) and prison screening (45) per 100,000 population screened. However, the cost per case identified was cheapest for house-to-house visits [Nepalese rupee (NPR) 76,500/case], followed by contact tracing (NPR 90,286/case) and prison screening (NPR 298,300/case). House-to-house and contact tracing case paucibacillary/multibacillary (PB:MB) ratios were 59:41 and 68:32; female/male ratios 63:37 and 57:43; pediatric cases 11% in both approaches; and grade-2 disabilities (G2D) 11% and 5%, respectively. Developing leprosy was not significantly different among household and neighbor contacts [odds ratios (OR) = 1.4, 95% confidence interval (CI): 0.24-5.85] and for contacts of MB versus PB cases (OR = 0.7, 95% CI 0.26-2.0). Attack rates were not significantly different among household contacts of MB cases (0.32%, 95% CI 0.07-0.94%) and PB cases (0.13%, 95% CI 0.03-0.73) (χ2 = 0.07, df = 1, P = 0.9) and neighbor contacts of MB cases (0.23%, 0.1-0.46) and PB cases (0.48%, 0.19-0.98) (χ2 = 0.8, df = 1, P = 0.7). BCG vaccination with scar presence had a significant protective effect against leprosy (OR = 0.42, 0.22-0.81). CONCLUSIONS: The most effective case identification approach here is contact tracing, followed by house-to-house visits in vulnerable populations and screening in prisons, although house-to-house visits are cheaper. The findings suggest that hidden cases, recent transmission, and late diagnosis in the community exist and highlight the importance of early case detection.

A Novel, Comprehensive A129 Mouse Model for Investigating Dengue Vaccines and Evaluating Pathogenesis.

In search of a mouse model for use in evaluating dengue vaccines, we assessed A129 mice that lacked IFN-α/ß receptors, rendering them susceptible to dengue virus (DENV) infection. To our knowledge, no reports have evaluated dengue vaccine efficiency using A129 mice. A129 mice were given a single intraperitoneal (IP) or subcutaneous (SC) injection of the vaccine, Dengvaxia. After 14 days of immunization via the IP or SC injection of Dengvaxia, the A129 mice exhibited notably elevated levels of anti-DENV immunoglobulin G and neutralizing antibodies (NAb) targeting all four DENV serotypes, with DENV-4 displaying the highest NAb levels. After challenge with DENV-2, Dengvaxia and mock-immunized mice survived, while only the mock group exhibited signs of morbidity. Viral genome levels in the serum and tissues (excluding the brain) were considerably lower in the immunized mice compared to those in the mock group. The SC administration of Dengvaxia resulted in lower viremia levels than IP administration did. Therefore, given that A129 mice manifest dengue-related morbidity, including viremia in the serum and other tissues, these mice represent a valuable model for investigating novel dengue vaccines and antiviral drugs and for exploring dengue pathogenesis.

Seroprevalence of SARS-CoV-2 infection in the general population of Nepal during the first and second generalized waves of the COVID-19 pandemic-2020-2021.

Few seroprevalence studies have been conducted on coronavirus disease (COVID-19) in Nepal. Here, we aimed to estimate seroprevalence and assess risk factors for infection in the general population of Nepal by conducting two rounds of sampling. The first round was in October 2020, at the peak of the first generalized wave of COVID-19, and the second round in July-August 2021, following the peak of the wave caused by the delta variant of SARS-CoV-2. We used cross-sectional probability-to-size (PPS)-based multistage cluster sampling to estimate the seroprevalence in the general population of Nepal at the national and provincial levels. We tested for anti-SARS-CoV-2 total antibody using the WANTAI SARS-CoV-2 Ab ELISA kit. In Round 1, the overall national seroprevalence was 14.4%, with provincial estimates ranging from 5.3% in Sudurpaschim to 27.3% in Madhesh Province. In Round 2, the estimated national seroprevalence was 70.7%, with the highest in the Madhesh Province (84.8%) and the lowest in the Gandaki Province (62.9%). Seroprevalence was comparable between males and females (Round 1, 15.8% vs. 12.2% and Round 2, 72.3% vs. 68.7%). The seroprevalence in the ecozones-Terai, hills, and mountains-was 76.3%, 65.3%, and 60.5% in Round 2 and 17.7%, 11.7%, and 4.6% in Round 1, respectively. In Nepal, COVID-19 vaccination was introduced in January 2021. At the peak of the first generalized wave of COVID-19, most of the population of Nepal remained unexposed to SARS-CoV-2. Towards the end of the second generalized wave in April 2021, two thirds of the population was exposed.

Possible high risk of transmission of the Nipah virus in South and South East Asia: a review.

Nipah virus (NiV) is a zoonotic, single-stranded RNA virus from the family Paramyxoviridae, genus Henipavirus. NiV is a biosafety-level-4 pathogen that is mostly spread by Pteropus species, which serve as its natural reservoir host. NiV is one of the major public health challenges in South and South East Asia. However, few molecular studies have been conducted to characterise NiV in a specific region. The main objective of this review is to understand the epidemiology, pathogenesis, molecular surveillance, transmission dynamics, genetic diversity, reservoir host, clinical characteristics, and phylogenetics of NiV. South and South East Asian nations have experienced NiV outbreaks. Phylogenetic analysis confirmed that two primary clades of NiV are in circulation. In humans, NiV causes severe respiratory illness and/or deadly encephalitis. NiV is mainly diagnosed by ELISA along with PCR. Therefore, we recommend that the governments of the region support the One Health approach to reducing the risk of zoonotic disease transmission in their respective countries.

Co-Circulation of Dengue Virus Serotypes 1, 2, and 3 during the 2022 Dengue Outbreak in Nepal: A Cross-Sectional Study.

The largest dengue outbreak in the history of Nepal occurred in 2022, with a significant number of casualties. It affected all 77 districts, with the nation's capital, Kathmandu (altitude 1300 m), being the hardest hit. However, the molecular epidemiology of this outbreak, including the dengue virus (DENV) serotype(s) responsible for this epidemic, remain unknown. Here, we report the epidemic trends, clinico-laboratory features, and virus serotypes and their viral load profiles that are associated with this outbreak in Nepal. Dengue-suspected febrile patients were investigated by routine laboratory, serological, and molecular tools, including a real-time quantitative polymerase chain reaction (qRT-PCR). Of the 538 dengue-suspected patients enrolled, 401 (74.5%) were diagnosed with dengue. Among these dengue cases, 129 (32.2%) patients who required hospital admission had significant associations with myalgia, rash, diarrhea, retro-orbital pain, bleeding, and abdominal pain. DENV-1, -2, and -3 were identified during the 2022 epidemic, with a predominance of DENV-1 (57.1%) and DENV-3 (32.1%), exhibiting a new serotype addition. We found that multiple serotypes circulated in 2022, with a higher frequency of hospitalizations, more severe dengue, and more deaths than in the past. Therefore, precise mapping of dengue and other related infections through integrated disease surveillance, evaluation of the dynamics of population-level immunity and virus evolution should be the urgent plans of action for evidence-based policy-making for dengue control and prevention in the country.

Forty years (1980-2019) of visceral leishmaniasis in Nepal: trends and elimination challenges.

BACKGROUND: Visceral leishmaniasis (VL) is an important re-emerging neglected tropical disease associated with poverty. Despite the elimination initiative started in 2005, VL cases have been expanding into geographic areas in Nepal. The present study aims at exploring the trends of VL from 1980 to 2019. METHODS: This retrospective analysis covers 40 y of VL cases reported by the Epidemiology Diseases Control Division, Nepal. Subgroup analyses for annual incidence were performed by age, sex, seasons, districts and provinces, and VL cases were visualized on in-country maps. RESULTS: A total of 34 564 cases and 584 deaths of VL were reported during 1980-2019. VL persistently increased until 2006 and was reported from all seven provinces of the country. The highest number of confirmed cases (n=2229) was reported in 2003 and the lowest (n=60) in 1983. VL cases expanded from 12 to 23 endemic districts. The key components of the VL elimination program are early diagnosis; enhanced surveillance; integrated vector management; social mobilization; research and treatment. CONCLUSIONS: Expansion of VL towards the hilly and mountain regions of Nepal has posed challenges to the elimination program. Urgent VL control measures are required to achieve the elimination goals.

Has COVID-19 suppressed dengue transmission in Nepal?

Following the report of the first COVID-19 case in Nepal on 23 January 2020, three major waves were documented between 2020 and 2021. By the end of July 2022, 986 596 cases of confirmed COVID-19 and 11 967 deaths had been reported and 70.5% of the population had received at least two doses of a COVID-19 vaccine. Prior to the pandemic, a large dengue virus (DENV) epidemic affected 68 out of 77 districts, with 17 932 cases and six deaths recorded in 2019. In contrast, the country's Epidemiology and Disease Control Division reported 530 and 540 dengue cases in the pandemic period (2020 and 2021), respectively. Furthermore, Kathmandu reported just 63 dengue cases during 2020 and 2021, significantly lower than the 1463 cases reported in 2019. Serological assay showed 3.2% positivity rates for anti-dengue immunoglobulin M antibodies during the pandemic period, contrasting with 26.9-40% prior to it. Real-time polymerase chain reaction for DENV showed a 0.5% positive rate during the COVID-19 pandemic which is far lower than the 57.0% recorded in 2019. Continuing analyses of dengue incidence and further strengthening of surveillance and collaboration at the regional and international levels are required to fully understand whether the reduction in dengue incidence/transmission were caused by movement restrictions during the COVID-19 pandemic.

How an Outbreak of COVID-19 Circulated Widely in Nepal: A Chronological Analysis of the National Response to an Unprecedented Pandemic.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The first COVID-19 case was reported in Wuhan, China, in December 2019. In March 2020, the World Health Organization (WHO) declared COVID-19 a global pandemic. The first COVID-19 case in Nepal was reported in January 2020 in a Nepalese man who had returned from Wuhan to Nepal. This study aims to evaluate the government of Nepal's (GoN) response to the COVID-19 pandemic and explore ways to prevent COVID-19 and other pandemic diseases in the future. As of May 2022, a total of 979,140 cases and 11,951 deaths associated with COVID-19 have been reported in Nepal. To prevent the spread of the virus, the GoN initiated various preventive and control measures, including lockdown strategies. The effects of COVID-19 are expected to persist for many years; the best strategies a resource-limited country such as Nepal can implement to control pandemic diseases such as COVID-19 in the pre-vaccine stage are to increase testing, tracing, and isolation capacity.

High prevalence of rheumatoid arthritis and its risk factors among Tibetan highlanders living in Tsarang, Mustang district of Nepal.

BACKGROUND: In Tsarang (at 3560 m), which is located in Mustang, 62.7% of the residents answered that they had a subjective medical history of arthritis, and 41.1% of the residents answered that their families had a subjective medical history of arthritis on a survey conducted in 2017. The expression of hypoxia-inducible factor (HIF) and its effects are deeply involved in hypoxic adaptation in Tibetan highlanders. At the same time, HIF is also related to the onset of rheumatoid arthritis. Therefore, the adaptive mechanism acquired by Tibetan highlanders may promote the development of rheumatoid arthritis. The prevalence of rheumatoid arthritis is estimated to be approximately 0.5-1.0% worldwide. The objective of this study was to estimate the prevalence of rheumatoid arthritis in Tsarang residents using existing diagnostic criteria and to explore its risk factors. METHODS: An epidemiological survey was conducted in Tsarang in 2019. Data obtained from anthropometry and questionnaires were statistically analyzed. Biochemical measurements using blood samples were also performed, and the results were used to assess arthritis status. Residents' joint status was scored, and arthritis was assessed based on the clinical disease activity index and ACR/EULAR 2010 criteria. RESULTS: Twenty-seven males and 50 females participated in this survey. In Tsarang, ACR/EULAR 2010 classified 4.3% of males and 7.1% of females as having rheumatoid arthritis, indicating a very high estimated prevalence. We also performed a multivariate analysis to explore its risk factors, and two factors, older age (standardized parameter estimate = 4.84E-01, 95% CI = [9.19E-02, 8.76E-01], p = 0.0170) and a history of living in urban areas (standardized parameter estimate = - 5.49E-01, 95% CI = [- 9.21E-01, 1.77E-01], p = 0.0050), significantly contributed to the higher ACR/EULAR 2010 score in females. In addition, three factors, having no spouse (standardized parameter estimate = 3.17E-01, 95% CI = [5.74E-02, 5.77E-01], p = 0.0179), having a smoking habit (standardized parameter estimate = 2.88E-01, 95% CI = [1.71E-02, 5.59E-01], p = 0.0377), and a history of living in urban areas (standardized parameter estimate = - 3.69E-01, 95% CI = [- 6.83E-01, - 5.60E-02], p = 0.0219), resulted in significantly higher clinical disease activity index scores in females. Furthermore, smoking habits were found to significantly increase blood hyaluronic acid in both males (standardized parameter estimate = 6.03E-01, 95% CI = [3.06E-01, 9.01E-01], p = 0.0020) and females (standardized parameter estimate = 4.87E-01, 95% CI = [5.63E-02, 9.18E-01], p = 0.0291). CONCLUSIONS: In this study, we evaluated the symptoms of arthritis and estimated the prevalence of rheumatoid arthritis using classification criteria for Tibetan highlanders who have adapted to the hypoxic environment and fostered their own culture. The high prevalence of rheumatoid arthritis among Tsarang residents suggests that the hypoxic adaptation mechanism involving HIF in Tibetan highlanders may promote the onset or exacerbation of rheumatoid arthritis. The high prevalence of rheumatoid arthritis among Tibetan highlanders may be related not only to the environmental factors analyzed in this study but also to hypoxic adaptation genes. Further investigation is needed to clarify the genetic factors involved.

Insights into transmission dynamics of Mycobacterium tuberculosis complex in Nepal.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis complex (MTBC) in humans and animals. Numbers of multi drug resistance TB (MDR-TB), extrapulmonary TB (EPTB) and zoonotic TB cases are increasingly being reported every year in Nepal posing a major public health problem. Therefore, the Government of Nepal should act immediately to strengthen the screening facilities across the country to be able to identify and treat the TB infected patients as well as detect zoonotic TB in animal species. Endorsement of One Health Act by the Government of Nepal is an opportunity to initiate the joint programs for TB surveillance among human and animal species using one health approach to reduce the TB burden in Nepal.

Emergence of cutaneous leishmaniasis in Nepal.

BACKGROUND: Cutaneous leishmaniasis (CL) is endemic in 70 countries worldwide. Nepal is considered non-endemic for CL and hence the control program is targeted to visceral leishmaniasis (VL) only. Here, we report the emergence of CL cases in different parts of Nepal. METHODS: We analyzed the CL and VL cases reported to Epidemiology and Diseases Control Division (EDCD), Ministry of Health and Population, Nepal through District Health Information System 2 (DHIS-2) and Early Warning and Reporting System (EWRS) during the past 4 years (2016-2019). Any laboratory-confirmed case was included in the study. Demographic and clinical details of each patient were transcribed into Excel sheets, verified with the case report forms and analyzed. RESULTS: VL has been reported in Nepal since 1980, but CL was reported very recently. From 2016 to 2019, 42 CL cases were reported from 26 different hospitals to EDCD which had been diagnosed on the basis of clinical presentation, and laboratory findings (demonstration of amastigotes in Giemsa-stained smears and rK39 test results). Majority of the patients (31.0%, 13/42) visited to the hospital within 1-6 months of onset of lesions. Facial region (38.1%, 16/42) was the common place where lesions were found ompared to other exposed parts of the body. CL was successfully treated with miltefosine for 28 days. The majority of CL patients did not have history of travel outside the endemic areas and there was no report of sandfly from these areas. CONCLUSION: These evidences highlight that the Government of Nepal need to pay more efforts on CL and include it in differential diagnosis by clinicians, and plan for an active surveillance when the country is targeting leishmaniasis elimination by the year 2025 with the decreasing number of VL cases.

An Outbreak of Dengue Virus Serotype 2 Cosmopolitan Genotype in Nepal, 2017.

Dengue virus (DENV) is one of the most prevalent neglected tropical diseases, with half of the world's population at risk of infection. In Nepal, DENV was first reported in 2004, and its prevalence is increasing every year. The present study aimed to obtain and characterize the full-length genome sequence of DENV from the 2017 outbreak. Hospital-based surveillance was conducted in two provinces of Nepal during the outbreak. Acute-phase serum samples were collected from 141 clinically suspected dengue patients after the rainy season. By serological and molecular techniques, 37 (26.9%) and 49 (34.8%), respectively, were confirmed as dengue patients. The cosmopolitan genotype of DENV-2 was isolated from 27 laboratory-confirmed dengue patients. Genomic analysis showed many amino acid substitutions distributed mainly among the E, NS3, and NS5 genes. Phylogenetic analyses of the whole genome sequence revealed two clades (Asian and Indian) among DENV-2 isolates from Nepal. The DENV isolates from hilly and Terai areas were similar to Asian and Indian strains, respectively. Further genomic study on different DENV serotypes is warranted to understand DENV epidemics in Nepal, where there are limited scientific resources and infrastructure.