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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

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.

Competing Interests: We declare no competing interests.

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.

Background: Dengue cases have been continuously reported in Nepal, including some large outbreaks, since its first introduction in 2004. The disease is now expanding towards newer locations above 1400 m high, especially the country's capital city, Kathmandu. In 2019, >14,000 dengue cases including six deaths were reported. This study was aimed at the detection and molecular characterization of dengue virus (DENV) in dengue patients., Methods: A total of 451 patients were enrolled in this study. Demographic, clinical and laboratory information was collected from dengue patients. Dengue infection was confirmed by antibody/antigen detection assays followed by RT-PCR analysis., Results: The DENV patients showed fever, body ache, headache, myalgia, retro-orbital pain and arthralgia. The platelets were decreased, serum liver enzymes were increased and leucopenia was seen. Out of 195 patients, 111 (57.0%) were positive for DENV RNA by consensus PCR. We found DENV-2, 70 (63.1%) as the predominant serotype responsible for the 2019 outbreak, while DENV-3 was detected in two patients., Conclusion: Our findings suggest that DENV-2 was the major serotype causing the 2019 massive outbreak in Nepal. This information will help in disease control programs to understand the molecular epidemiology and its changing trend., (© The Author(s) 2020. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Background: Dengue is one of the newest emerging diseases in Nepal with increasing burden and geographic spread over the years. The main objective of this study was to explore the epidemiological patterns of dengue since its first outbreak (2006) to 2019 in Nepal., Methods: This study is a retrospective analysis that covers the last 14 years (2006-2019) of reported dengue cases from Epidemiology Diseases Control Division (EDCD), Ministry of Health and Population, Government of Nepal. Reported cases were plotted over time and maps of reported case incidence were generated (from 2016 through 2019). An ecological analysis of environmental predictors of case incidence was conducted using negative binomial regression., Results: While endemic dengue has been reported in Nepal since 2006, the case load has increased over time and in 2019 a total of 17 992 dengue cases were reported from 68 districts (from all seven provinces). Compared to the case incidence in 2016, incidence was approximately five times higher in 2018 [incidence rate ratio (IRR): 4.8; 95% confidence interval (CI) 1.5-15.3] and over 140 times higher in 2019 (IRR: 141.6; 95% CI 45.8-438.4). A one standard deviation increase in elevation was associated with a 90% decrease in reported case incidence (IRR: 0.10; 95% CI 0.01-0.20). However, the association between elevation and reported cases varied across the years. In 2018 there was a cluster of cases reported from high elevation Kaski District of Gandaki Province. Our results suggest that dengue infections are increasing in magnitude and expanding out of the lowland areas to higher elevations over time., Conclusions: There is a high risk of dengue outbreak in the lowland Terai region, with increasing spread towards the mid-mountains and beyond as seen over the last 14 years. Urgent measures are required to increase the availability of diagnostics and resources to mitigate future dengue epidemics.

Coronavirus disease 2019 (COVID-19) pandemic has caused significant impact on the health care system. As a consequence, diagnosis and treatment of vector borne diseases including dengue has been equally affected. Nepal is no exception to this, where COVID-19 cases is exponentially increased and all resources are concentrated on its prevention, control and management. Dengue, one of the major vector-borne diseases in Nepal, is apparently overlooked despite approaching the peak season of the disease. The aim of this paper is to describe the double burden of COVID-19 and dengue in Nepal, particularly highlighting the co-circulation and possible coinfections. This has posed higher risk of increased severity, more severe cases and deaths in Nepal. Moreover, potential misdiagnosis of these viral diseases may lead to delayed or, inappropriate treatment and poor allocation of resources.

Background: Chikungunya virus (CHIKV) infection is an emerging viral disease in Nepal, with the reported by our laboratories in 2013. Because of the similarity in signs and symptoms to dengue virus (DENV) and misdiagnosis, the under-reporting of CHIKV infection in DENV-endemic areas of Nepal is very common. The aim of the present study is to identify CHIKV infection among patients with febrile illness during the 2014-2015 outbreaks., Methods: A total of 219 serum samples were tested for CHIKV and DENV by using enzyme-linked immunosorbent assays (ELISAs) to detect immunoglobulin M (IgM) and IgG antibodies, with confirmation by a 50% focal reduction neutralization test (FRNT50)., Results: ELISA results demonstrated that for CHIKV and DENV, 20.5% and 14.2% of samples, respectively, were positive for IgG and/or IgM and 9.6% and 5.9% were positive for IgM alone. Further, FRNT50 results confirmed that 7.3% of samples possessed neutralizing anti-CHIKV antibodies., Conclusion: We conclude that CHIKV infection is settling in Nepal as an endemic disease. It is highly recommended to strengthen the surveillance system for CHIKV and DENV to prevent possible outbreaks., (© The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Dengue fever, an endemic arboviral disease, represents one of the major public health concerns in Nepal. It is transmitted by bites of infected Aedes aegypti and Aedes albopictus, the former being primary vector. The bacterial community plays a significant role in biology of mosquitoes; however, the bacterial communities of primary vector A. aegypti remain unstudied in Nepal. The study was designed to determine the rate of dengue seropositivity and to explore the bacterial diversity of A. aegypti from dengue endemic districts, Kanchanpur and Parsa of Nepal., Methods: A cross-sectional study was conducted between June 2013 and November 2013 at two hospitals of Kanchanpur and Parsa. A total of 221 serum samples were collected from patients (inpatients and outpatients) suspected of suffering from dengue fever and attending Mahakali Zonal Hospital, Mahendranagar, Kanchanpur, and Narayani Zonal Hospital, Birgunj, Parsa. Detection of anti-dengue IgM was performed by using human dengue IgM capture ELISA. The larvae and pupae of mosquitoes (A. aegypti) were collected, reared, and emerged. Then, the bacteria were isolated and identified from the gut of identified mosquitoes by using standard methods., Results: Out of total 221 serum samples collected from patients suspected of suffering from dengue fever, 34 (15.38%) were positive for anti-dengue IgM. Gram-negative bacteria were isolated in largest proportion (63%) followed by gram-positive cocci (23.27%) and gram-positive rods (13.73%). The most common cultivable bacteria isolated were Staphylococcus spp., Pseudomonas spp., and Acinetobacter spp. The average bacterial load in the vectors was 3.91 × 10 4  cfu/ml., Conclusions: High rate of anti-dengue IgM seropositivity was reported in our study. The environmental bacteria were predominantly isolated and identified in A. aegypti. The paratransgenic approach to control vector might be possible by spreading the genetically modified bacteria in larval habitat or shelter of adult mosquitoes.

A descriptive cross-sectional study was carried out among 264 suspected dengue patients in two districts (Dang and Chitwan) of Nepal from June 2013 to November 2013. The anti-dengue IgM positivity was found to be (51/264)19.31% by capture ELISA, of which 21 (41.2%) were male and 30 (58.8%) were female. Symptoms of seropositive cases were fever, anorexia, nausea, headache, retro-orbital pain, skin rashes, and myalgia. Hematological features like thrombocytopenia and leucopenia were found to be significantly associated with the dengue fever (DF). Discarded tires were found as the commonest breeding habitats for the dengue vectors. Higher sero-positivity was recorded from the area having higher Breteau index (BI). The pH, chloride ion concentration and the salinity of the water from breeding habitats were found to be ranging from 6.9±0.82 to 8, 103.33±17.52 mg/L to 140.65 mg/L, and 0.19±0.032 ppt to 0.25 ppt respectively. This study may be helpful for the health authorities and public health workers for early diagnosis of DF and for the improved preventive measures to be adopted in the epidemic and possible epidemic areas.

Background: Dengue fever (DF) is an emerging public health problem in Nepal, and yet neither the magnitude of the DF burden or its epidemiological trends are well understood., Methods: We conducted a sero-epidemiological, seasonal trend and demographic analysis of the trends in DF in the Terai region of Nepal, from 2007 to 2013. In that period, 2002 serum samples were collected from febrile patients suspected of dengue virus infection. Samples were screened by IgM antibody ELISA analysis for the presence of anti-dengue IgM antibodies., Results: The overall prevalence of DF cases was found to range from 26.1 to 55.4%. Infection was found to be more common among adults and males. Seasonal trends revealed that cases peaked in October and November., Conclusions: The study offers perspective on the dengue fever burden before, during and after a major epidemic and can now be used as a basis for formulating strategies by policy makers that will enhance and develop relevant control and preventive measures against dengue fever. The findings of this study reinforce the perceived need for urgent dengue virus surveillance to enhance dengue control strategies that need to be developed for future preparedness. In the light of the recent earthquakes, future major outbreaks of vector borne disease are likely to recur., (© The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dengue is an emerging mosquito borne disease of public health importance in Nepal. A descriptive cross sectional study was carried out to estimate sero-prevalence and distribution pattern of dengue in certain vulnerable regions of Nepal from June to September 2009. A total of 460 venous blood samples were collected from individuals experiencing a febrile illness clinically consistent with dengue infection visiting nearby hospitals of Kanchanpur, Kailali, Banke, Dang and Chitwan districts. The sero-prevalence of dengue virus specific IgM was determined by enzyme linked immunosorbent assay (ELISA) kit. The anti-dengue IgM positivity was found to be 12.17%. The higher frequency of positive cases (16.4%) were from age group 20-40 years followed by < 20 years age group with 9.7% and 5.3% from > 40 years age group. The association between dengue infection and age is found to be statistically significant (p < 0.05). The male:female ratio was determined as 1.3:1 in IgM positive population. Among sampling areas, Kanchanpur showed highest prevalence of dengue infection (15.5%) followed by Chitwan (11.7%), Kailai (11.1%), Banke (10.7%) and Dang (8.3%). Similarly, 94.6% of the positive cases were indigeneous and had no history of travel to other countries. Dengue is firmly established in terai region with increasing trends of infection and expansion into newer areas raising a public health threat. Regular epidemiological studies are suggested which could further reveal the contributing factors associated with dengue virus infection and help in formulating strategies in reducing the transmission rate and control of the infection.

Background: Dengue Fever (DF) is an emerging mosquito-borne disease. It is a nagging public health problem in the low lands of Terai, expanding to new areas of Nepal in recent years., Methods: A cross-sectional study was conducted to determine anti-Dengue IgM positive rate in Mahendranagar, Dhangadi and Dang between August 2008 and November 2009. Serum samples were collected from 283 patients visiting hospitals with history of fever, headache and suspected DF. The samples were examined by ELISA., Results: The anti-Dengue IgM positivity was found to be 9.8 %. The positive rate was highest in Mahendranagar (13.3 %) followed by Dhangadi (9.8 %) (P<0.05). The Dengue positive cases were higher in female (10.9 %) than males (9.0 %). The positivity was higher in Ethnic group Brahman/Chherti (13.1%) as compared to Janajati (5.6 %). The highest positive cases (10.7 %) were from age group above 50 years. The highest numbers of Dengue positive cases were observed in occupation group of agriculture (18.2 %) (P>0.05)., Conclusions: Dengue has substantial expansion in Western and Far Western Terai region of Nepal which was limited to the middle Terai region in the past and mostly infects older people.

Background: Dengue is an emerging vector borne disease in Nepal and rapid diagnostic test is important for early diagnosis of the disease., Objectives: The aim of the study was to evaluate the diagnostic accuracy of commonly used rapid immunochromatographic test kit in Nepal during 2010 dengue epidemic and to assess disease burden of dengue., Methods: A total of 131 acute and nonacute serum samples were collected during recent epidemic of dengue in 2010 from clinically suspected Nepalese patients of different hospitals. Rapid immunochromatographic test kit was used for early diagnosis and enzyme immunosorbent was chosen as a reference assay., Results: The sensitivity and specificity of rapid test was 70% and 76.54% respectively whereas the prevalence of the disease was 38.17%. The odds ratio for males was 1.8 however; the association with the disease was statistically not significant., Conclusion: The diagnostic accuracy of rapid immunochromatographic test for dengue diagnosis was low (k=0.46). So, it should be substituted by highly sensitive test device for prompt diagnosis and health personnel should consider appropriate timing of sample collection for better performance of rapid test.

Introduction: Dengue fever is an emerging mosquito borne disease in Nepal claiming substantial morbidity and mortality. The objective of the study was to find out frequency of acute dengue infection in patients from the hospitals of the western Nepal., Methods: The study was conducted between August 2007 and July 2008 in patients visiting hospitals of the western terai of Nepal with chief complains of fever. The sero-diagnosis of acute dengue infection was determined by enzyme linked immunosorbent assay among 239 patients visiting Lumbini Zonal Hospital, Butwal; Bheri Zonal Hospital, Nepalgunj; Bardiya District Hospital, Bardiya and Mahakali Zonal Hospital, Mahendranagar., Results: The anti-dengue IgM positivity was 29.3%. There was slight male preponderance with a male to female ratio of 1.2:1. Out of the total positive cases, the highest positive cases (75.7%) were from the age group 15 - 50 years followed by < 15 years old (15.7%). Out of four hospitals, the highest positive cases (54.3%) were in Lumbini Zonal Hospital, Butwal. The age and gender were independent predictors to dengue virus infection. The highest numbers of dengue positive cases were in October (52.6%). The association between dengue disease and the month was statistically significant., Conclusions: The dengue positivity was estimated in acute patients from hospitals of western Nepal by enzyme immunoassay. Therefore, the serological marker can be used to diagnose acute patients of dengue during outbreaks.

A cross-sectional study was conducted to determine dengue virus IgM-positive rate in Terai region, Nepal from August to December 2007. Serum samples were collected from 183 symptomatic cases. The samples were examined for dengue virus specific IgM using particle agglutination test. Of 183 serum samples, 55 (30.0%) had positive for dengue IgM antibody. The positive rate was highest (50.0%) in Biratnagar, and lowest (19.6%) in Chitwan male to female ratio was 2:1 in IgM-positive populations. IgM-positive rate was 29.0% at ages 21-30, 25.4% at ages 11-20 and 23.6% at ages 0-10, but 10.9% at ages 31-40, and ages over 40. There was not significant association between occupation of the patients and positive rate among farmer, labour, service, business and student.

Dengue virus transmitted by Aedes mosquito is one of the important cause health problems in world. Dengue fever and more severe and often fatal forms namely dengue hemorrhagic fever and dengue shock syndrome are emerging health problems in many part of the globe. No cases of Dengue virus infection have been reported from Nepal till date and for the first time, we report a case of dengue fever from Nepal.

Twenty-two strains of dengue 2 virus, isolated in China, Latin America, New Guinea and Thailand were subjected to phylogenetic analysis. The UPGMA analysis was carried out on each gene region of dengue virus and demonstrated that outcome from most of the gene regions showed similar results except those from NS4B and YUTR with very short nucleotide length. Among ten regions examined, the results from E gene documented the geographical differences of the virus strains most clearly and all the American strains (Mara 4, IQT1797 and S1) were distantly related to the Asian isolates. As for the 16 Thai strains isolated in 1993, they were clustered into three groups and a strain from a DSS patient formed a distinct branch compared to the other two groups. This finding from phylogenetic analysis is consistent with earlier conclusion and support the severity related subtyping of dengue 2 virus based on amino acid changes.

Comparative nucleotide sequencing was carried out on dengue type 2 virus (DEN-2) strains isolated from patients in Northeast Thailand during the epidemic season in 1993. The patients exhibited different clinical manifestations ranging from dengue fever (DF) to dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS). The results classified 19 DEN-2 strains into 3 subtypes according to nonsynonymous amino acid replacements. The strain isolated from a DSS patient eliciting secondary serological response belonged to subtype I, whereas 13 strains isolated from DHF patients with secondary response and 2 strains from DF patients with primary response belonged to subtype II. On the other hand, 3 strains isolated from DF cases evoking either primary or secondary response belonged to subtype III. These results suggest that subtype III virus infection could result in clinically milder manifestation irrespective of the serological response compared with subtype I or II viruses. The RNA secondary structure predicted for the 3' noncoding region showed 4 different structures (A, B, C, and D). The result also indicates that different subtypes of DEN-2 serotypes are circulating in a single epidemic in Thailand.