Application of Hepatitis E Virus-Related Markers on Samples from a Developing Country.

BACKGROUND: Nepal is an endemic area for hepatitis E virus (HEV) epidemics. The research on viral hepatitis in Nepal is limited. METHODS: Serum samples from 170 patients presenting with symptoms of hepatitis were collected from April to May 2014 in Biratnagar, Nepal, and then transported to Xiamen, China, for further evaluation. All samples were tested for HEV RNA, HEV antigen, anti-HEV IgM, anti-HEV IgG and anti-HBc IgM, anti-HCV IgG, and anti-HAV IgM. RESULTS: Sixteen patients were identified as acute hepatitis E with the presence of ≥ 2 HEV acute phase markers (antigen, RNA, and anti-HEV IgM). HEV infection was the major cause of potential active viral hepatitis (59.2%, 16 of 27), followed by HBV (25.9%, 7 of 27, anti-HBc IgM positive), HAV (18.5%, 5 of 27, anti-HAV IgM positive), and HCV (3.7%, 1 of 27, anti-HCV antibodies). All 16 confirmed HE cases were positive for HEV antigen, while 5 cases were HEV RNA positive, as well as 15 anti-HEV IgM positive. The low positive rate of RNA might be related to the collection and/or the transportation of these samples. CONCLUSIONS: This study showed that HEV is a major cause of acute hepatitis in developing countries and regions. Application of immunoassay diagnostic kits, especially the HEV antigen tests, showed great potential for HE detection in these countries and regions.

Dengue periodic outbreaks and epidemiological trends in Nepal.

Dengue is a global health problem and expansion of its endemics towards new territories in the hilly regions in Nepal is a serious concern. It appeared as a new disease in Nepal in 2004 from Japanese traveler with sporadic cases every year and massive outbreaks in 2010, 2013 and 2016. The serotype was responsible for outbreak in particular year was dengue virus serotype-1 (DENV-1) in 2010, 2016; and DENV-2 in 2013. Nepal lacks basic health related infrastructure in rural areas and does not have a stringent health care policy. With severances of epidemic like dengue, a new surveillance or an upgrading of existing one are direly needed to better challenge the possible outbreaks. This review paper aims to explain the dengue trend in last one decade in Nepal and warrants concerted and timely public health interventions to minimize the deleterious effects of the disease.

The Dengue virus in Nepal: gaps in diagnosis and surveillance.

BACKGROUND: The introduction of the dengue virus (DENV) in Nepal is recent, first reports date back to 2004 from a Japanese traveller and limited information is available about DENV infection in the Nepali population. Within a decade after the first DENV detection, it is now endemic in multiple districts of Nepal with approximately 11.2 million people residing in the Terai belt being at risk of DENV infection. Sporadic cases of DENV infection have been reported every year for the past decade during the monsoon season, mainly in the Terai region. METHODS: Medline/Embase/Cochrane databases were reviewed for reports on the burden of dengue infection, diagnostic methods, and national surveillance. RESULTS: Four outbreaks were reported since 2004 including the diagnosis of all serotypes in 2006 and predominance of a single serotype in 2010 (DENV-1), 2013 (DENV-2), and 2016 (DENV-1). The clinical diagnoses showed a predominance of dengue fever while 4/917 (0.4%), 8/642 (1.2%) and 8/1615 (0.4%) dengue haemorrhagic fever/dengue shock syndrome cases were identified during the outbreaks in 2010, 2013 and 2016, respectively. The number of cases reported in males was significantly higher (67.4%) than in females. Disease occurrence was primarily found in the Terai region until 2010 and was increasingly detected in the Hilly region in 2016. CONCLUSION: In Nepal currently weak diagnostic facilities, very limited research on mosquitoes vectors, and poor surveillance of dengue leading to inappropriate detection and control of DENV. We surmise that improved basic research and epidemiological training courses for local scientists and laboratory personal at national and international level will help better understand the evolution and distribution of DENV transmission and its eventual control.

Prevalence and risk of hepatitis E virus infection in the HIV population of Nepal.

BACKGROUND: Infection with the hepatitis E virus (HEV) can cause acute hepatitis in endemic areas in immune-competent hosts, as well as chronic infection in immune-compromised subjects in non-endemic areas. Most studies assessing HEV infection in HIV-infected populations have been performed in developed countries that are usually affected by HEV genotype 3. The objective of this study is to measure the prevalence and risk of acquiring HEV among HIV-infected individuals in Nepal. METHODS: We prospectively evaluated 459 Human Immunodeficiency Virus (HIV)-positive individuals from Nepal, an endemic country for HEV, for seroprevalence of HEV and assessed risk factors associated with HEV infection. All individuals were on antiretroviral therapy and healthy blood donors were used as controls. RESULTS: We found a high prevalence of HEV IgG (39.4%) and HEV IgM (15.3%) in HIV-positive subjects when compared to healthy HIV-negative controls: 9.5% and 4.4%, respectively (OR: 6.17, 95% CI 4.42-8.61, p < 0.001 and OR: 3.7, 95% CI 2.35-5.92, p < 0.001, respectively). Individuals residing in the Kathmandu area showed a significantly higher HEV IgG seroprevalance compared to individuals residing outside of Kathmandu (76.8% vs 11.1%, OR: 30.33, 95% CI 18.02-51.04, p = 0.001). Mean CD4 counts, HIV viral load and presence of hepatitis B surface antigen correlated with higher HEV IgM rate, while presence of hepatitis C antibody correlated with higher rate of HEV IgG in serum. Overall, individuals with HEV IgM positivity had higher levels of alanine aminotransferase (ALT) than IgM negative subjects, suggesting active acute infection. However, no specific symptoms for hepatitis were identified. CONCLUSIONS: HIV-positive subjects living in Kathmandu are at higher risk of acquiring HEV infection as compared to the general population and to HIV-positive subjects living outside Kathmandu.

Comparative Study of GeneXpert MTB/RIF Assay and Multiplex PCR Assay for Direct Detection of Mycobacterium tuberculosis in Suspected Pulmonary Tuberculosis Patients.

Pulmonary tuberculosis (PTB) is one of the major infectious diseases in developing countries. The objective of this study was to compare rapid diagnostics technique, GeneXpert MTB/RIF (GeneXpert) and Multiplex PCR assay (MPCR) targeting IS6110 segment and mpb64 gene for direct detection of Mycobacterium tuberculosis (MTB) in suspected PTB patients. A cross sectional study was carried among 105 sputum samples from suspected PTB patients to evaluate GeneXpert and Multiplex PCR who visited National Tuberculosis Center, Nepal. The patient's sputum samples were used directly for the GeneXpert whereas DNA extraction by CTAB method was followed to process the sample for MPCR. The sensitivity and specificity of GeneXpert and MPCR in smear positive cases was 78.6, 33.3, and 100.0%, 66.7%, respectively (P = 0.125). However, in smear negative cases sensitivity and specificity of both methods exhibited 90.9, 95.2, and 100.0%, 100.0% respectively (P = 0.625). Finally, the sensitivity and specificity of GeneXpert and MPCR were 82.9, 95.3 and 100.0%, 98.5% respectively, (P = 0.549) in pulmonary cases. Comparatively, we observed higher sensitivity and specificity for MPCR than GeneXpert for both smear positive and negative samples. Thus, we recommend MPCR alongside GeneXpert for the better diagnostic accuracy of PTB in a resource-limited country where tuberculosis is endemic.

Negative feedback circuit for toll like receptor-8 activation in human embryonic Kidney 293 using outer membrane vesicle delivered bi-specific siRNA.

BACKGROUND: TLR8 assists in antiviral approach by producing Type 1 INF via MyD88 dependent IRF7 pathway. However, over expression of INFα/ß molecule poses threat by developing tolerance in chronic infection cases and enhancing inflammatory response. Here we report a bi-specific siRNA based complex which differentially activates and silences the TLR8 and MYD88 respectively in a negatively regulated fashion. RESULTS: Outer membrane vesicle from Escherichia coli used for siRNA delivery was observed more efficient when attached with invasive protein Ail along with OmpA (P<0.001) in HEK293-TLR8 cell line. siRNA complexed with p19 protein was efficient in activating TLR8, confirmed by the increment of INFß molecules (P<0.001) in HEK293-TLR8 compared to its counterpart. Fusion of lipid bilayer of endosomal compartment was significant at pH 4.5 when fusogenic peptides (diINF-7) were incubated in membrane vesicle, thus facilitating the escape of siRNA complex to the host cytoplasm in order to silence MyD88 transcript (P<0.001). CONCLUSIONS: We investigated the activation of TLR8 by bi-specific si-RNA for the production of INFß. In the same setting we showed that bi-specific si-RNA was able to silence MyD88 transcript in a delayed manner. For the cases of auto immune disease and inflammation where over activation of endosomal TLRs poses serious threat, bi specific siRNA could be used as negative feedback controlled system.

Evaluation of sensitivity and specificity of ELISA against Widal test for typhoid diagnosis in endemic population of Kathmandu.

BACKGROUND: Widal test, which has poor predictive outcomes in predominant typhoid population, is not standard enough to predict accurate diagnosis. This study aims to compare the diagnostic accuracy of Widal test to ELISA using blood culture as gold standard. METHODS: The blood samples were collected in Capital Hospital, Kathmandu, Nepal from febrile patients having ≥48 h fever in 3 years study period for blood culture, Widal test and IgG-IgM ELISA. RESULTS: Amongst 1371 febrile cases, 237 were Salmonella typhi positive to blood culture and 71.4 % typhoid fever patient were of 46-60 years old with male to female ratio of 2:1. Blood culture confirmed patients had ≥1:40 anti-TH and anti-TO titre in 45.56 % (n = 108) and 43.88 % (n = 104) patients respectively. The sensitivity and specificity of IgG (0.96 and 0.95) and IgM (0.95 and 0.94) at 95 % confidence level were significant compared to Widal anti-TH (0.72 and 0.58) and TO (0.80 and 0.51) test (p value, 0.038) at titre level ≥1:200. Further the PPV of Widal TH and TO (0.38 and 0.23) was low compared to IgG and IgM ELISA (0.78 and 0.77) (p value, 0.045). CONCLUSION: Widal test is not sensitive enough for an endemic setting like Nepal and thus should be either replaced with more accurate test like ELISA or follow an alternative diagnostic methodology.

Re-emergence of dengue virus serotype 2 strains in the 2013 outbreak in Nepal.

BACKGROUND & OBJECTIVES: Epidemiological interventions and mosquito control are the available measures for dengue control. The former approach uses serotype and genetic information on the circulating virus strains. Dengue has been frequently reported from Nepal, but this information is mostly lacking. The present study was done to generate a comprehensive clinical and virological picture of a dengue outbreak in Nepal during 2013. METHODS: A hospital-based study involving patients from five districts of Nepal was carried out. Demographic information, clinical details and dengue serological status were obtained. Viral RNA was characterized at the molecular level by reverse-transcription polymerase chain reaction (RT-PCR), nucleotide sequencing and phylogenetic analysis. RESULTS: From among the 2340 laboratory-confirmed dengue cases during the study period, 198 patients consented for the study. Clinically they had fever (100%), headache (59.1%), rashes (18.2%), retro-orbital pain (30.3%), vomiting (15.1%), joint pain (28.8%) and thrombocytopenia (74.3%). Fifteen (7.5%) of them had mucosal bleeding manifestations, and the rest were uncomplicated dengue fever. The patients were mostly adults with a mean age of 45.75 ± 38.61 yr. Of the 52 acute serum samples tested, 15 were positive in RT-PCR. The causative virus was identified as DENV serotype 2 belonging to the Cosmopolitan genotype. INTERPRETATIONS & CONCLUSIONS: We report here the involvement of DENV serotype 2 in an outbreak in Nepal in 2013. Earlier outbreaks in the region in 2010 were attributed to serotype 1 virus. As serotype shifts are frequently associated with secondary infections and severe disease, there is a need for enhancing surveillance especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in the region.

Safety and immunogenicity of conjugate vaccine for typhoid (Vi-DT): Finding from an observer-blind, active-controlled, randomized, non-inferiority, phase III clinical trial among healthy volunteers.

Typhoid fever is a significant public health concern with most of the sufferers between 15 and 25 y of age in Nepal. We undertook this study to demonstrate Vi polysaccharide conjugated with diphtheria toxoid (Vi-DT) conjugate vaccine which is non-inferior to Typbar typhoid conjugate vaccine, a Vi polysaccharide vaccine conjugated with tetanus toxoid (Vi-TT) with a focus on the adult population from Dhulikhel Hospital which was one of the total four sites in Nepal. In this study, we assigned the eligible participants in 1:1:1:1 ratio by block randomization, and stratified into three age groups (6 months to less than 2 y, 2 y to less than 18 y, and 18 y to 45 y), allotted to Group A, B, C, and D. Group A, B, and C received 25 µg (0.5 mL) of Vi-DT study vaccine and participants in Group D received 25 µg (0.5 mL) Vi-TT vaccine. We descriptively analyzed safety in all the participants receiving one dose of the investigational vaccine. The anti-Vi-IgG seroconversion rate in Vi-DT recipients was 99.71% (97.5% CI 98.04-99.96; 344 of 345 participants) and 99.13% (94.27-99.87; 114 of 115) in Vi-TT recipients which indicates that Vi-DT vaccine is non-inferior to Vi-TT vaccine. In safety aspect, 16.81% of total subject had at least one solicited adverse reaction and 22.61% of the Vi-TT participants experienced at least one solicited adverse reaction with most of them being local adverse reactions. None of the enrolled participants reported serious adverse events. Our study shows that a single dose of the Vi-DT vaccine is immunogenic, safe to administer and non-inferior to the Vi-TT vaccine four weeks after vaccination.

A randomized, observer-blind, controlled phase III clinical trial assessing safety and immunological non-inferiority of Vi-diphtheria toxoid versus Vi-tetanus toxoid typhoid conjugate vaccine in healthy volunteers in eastern Nepal.

Typhoid remains one of the major serious health concerns for children in developing countries. With extremely drug-resistant cases emerging, preventative measures like sanitation and vaccination, including typhoid conjugate vaccines (TCV) remain the mainstay in its prevention and control. Different types of TCVs are being developed to meet the global demand. This report outlines the results from a study done to assess the immunogenicity and safety of Vi-Diphtheria toxoid (Vi-DT) TCV in Nepal. The study was a randomized, active-controlled, immunological non-inferiority and safety study. Eligible participants from Sunsari and Morang districts of eastern Nepal were randomized into 4 study groups (A-D) within 3 age strata (6 months to <2 years, 2 to <18 years, and 18 to 45 years). Groups A to C received a single dose (25 µg) of Vi-DT test vaccine from any of the 3 lots, while group D received the comparator, Typbar-TCV®, Vi-tetanus toxoid (Vi-TT) vaccine (25 µg) in 1:1:1:1 ratio and evaluated at 4 weeks postvaccination with 6 months follow-up. Amongst 400 randomized participants, anti-Vi-IgG seroconversion rates for all age strata in Vi-DT pooled groups (A+B+C) were 100.00% (97.5% CI 98.34-100.00) vs 98.99% (97.5% CI 93.99-99.85) in Vi-TT group (D) at 4 weeks. Comparable safety events were reported between the groups. Three serious adverse events (1 in Vi-DT; 2 in Vi-TT group) were reported during the 6 months follow-up, none being related to the investigational product. Thus, Vi-DT vaccine is safe, immunogenic, and immunologically non-inferior to Vi-TT when analyzed at 4 weeks postvaccination.

Factors affecting willingness to participate in vaccine clinical trials in an underdeveloped country: perspective from Nepal.

Due to the inherent complex nature of clinical trials, individual's willingness to participate and hence, enrollment in a clinical trial maybe challenging. When it comes to vaccine clinical trial in children, informed consent needs to be secured from the parents or legally acceptable representatives (LARs). Some of the factors which contribute to hesitancy in taking part in clinical trials are based on the level of education, living standards, part of the world they live, associated burden of disease, fear of different procedures in clinical trial, side effects, limited understanding, limited time, and mistrust with Investigational product. This study included 201 parents/LARs, who approached Kanti Children Hospital site in Kathmandu with the interest to get their children enrolled in a vaccine clinical trial with objectives of describing the reasons for agreeing or disagreeing to participate in the vaccine clinical trial, factors affecting decision making, and finding the major concerns of parents/LARs. The acceptance for the study vaccine was 136 (67.7%) whereas denial was 65 (32.3%). This study showed that age, education level, family structure, advice from family and friends, and medical guidance play important roles in willingness of parents to get their child enrolled in the trial. If a proper counseling is done, fear of blood sampling is not a big factor which is contrary to the belief among clinical researchers. Safety of vaccine, frequency of injections, and cost of vaccine were the main concerns of the parents, which need to be addressed extensively while planning for any clinical trial in children.

Immune non-interference and safety study of Vi-DT typhoid conjugate vaccine with a measles, mumps and rubella containing vaccine in 9-15 months old Nepalese infants.

BACKGROUND: Typhoid fever is a common disease in developing countries especially in the Indian subcontinent and Africa. The available typhoid conjugate vaccines (TCV) have been found to be highly immunogenic in infants and children less than 2 years of age. Many countries are planning to adopt TCV in their routine EPI programs around 9 months of age when measles containing vaccines are given. Therefore, Vi-DT TCV was tested in 9-15 months aged healthy infants in Nepal to demonstrate non-interference with a measles containing vaccine. METHODS: This was a randomized, open label, phase III study to assess the immune non-interference, safety, and reactogenicity of Vi-DT typhoid conjugate vaccine when given concomitantly with measles, mumps and rubella (MMR) vaccine. A total of 360 participants aged 9-15 months were enrolled and randomized equally into Vi-DT + MMR (180 participants) or MMR alone (180 participants) group and were evaluated for immunogenicity and safety 28 days post vaccination. RESULTS: Using the immunogenicity set, difference between proportions (95% CI) of the Vi-DT + MMR group vs MMR alone group were -2.73% (-8.85, 3.38), -3.19% (-11.25, 4.88) and 2.91% (-3.36, 9.18) for sero-positivity rate of anti-measles, anti-mumps and anti- rubella, respectively. Only the lower bound of the range in difference of the proportions for sero-positivity rate of anti-mumps did not satisfy the non-inferiority criteria as it was above the -10% limit, which may not be of clinical significance. These results were confirmed in the per protocol set. There were no safety concerns reported from the study and both Vi-DT + MMR and MMR alone groups were comparable in terms of solicited and unsolicited adverse events . CONCLUSIONS: Results indicated that there is non-interference of MMR vaccine with Vi-DT and Vi-DT conjugate vaccine could be considered as an addition to the EPI schedule among children at risk of contracting typhoid.

Safety and immunogenicity of the Vi-DT typhoid conjugate vaccine in healthy volunteers in Nepal: an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial.

BACKGROUND: Typhoid fever is an endemic disease in many low-income and middle-income countries. The 2018 WHO position paper recommends that countries should consider typhoid vaccination in high-risk groups and for outbreak control. To address the typhoid vaccine supply and demand gap, a typhoid Vi polysaccharide-diphtheria toxoid (Vi-DT) conjugate vaccine development effort was undertaken to achieve WHO prequalification and contribute to the global supply of typhoid conjugate vaccine. The main aim of this study was to show immune non-inferiority of the Vi-DT vaccine compared with the WHO prequalified Vi polysaccharide-tetanus toxoid (Vi-TT) conjugate vaccine (Typbar TCV; Bharat Biotech India, Hyderabad, India) in participants of various ages from an endemic country. METHODS: We did an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial at four hospitals in Kathmandu, Dhulikhel, Dharan, and Nepalgunj in Nepal. Eligible participants were healthy individuals aged 6 months to 45 years for whom informed consent was obtained, were willing to follow the study procedures and were available for the duration of the study. Patients with an acute or chronic illness that could interfere with interpretation of the study endpoints, or who were involved in any other clinical trial were excluded. Participants were randomly assigned (1:1:1:1) by block randomisation (block size of four and eight), stratified by age (6 months to <2 years, 2 years to <18 years, and 18 years to 45 years), into one of four groups (A-D). Participants in groups A-C received a single dose (25 µg; 0·5 mL) of Vi-DT test vaccine via intramuscular injection from one of three good manufacturing practice lots (group A received lot 1, group B received lot 2, and group C received lot 3), and those in group D received a single dose (25 µg; 0·5 mL) of the Vi-TT vaccine via intramuscular injection. All participants, site staff (except for those who administered the study vaccines), and those assessing the outcomes were masked to group assignment. The co-primary endpoints were: (1) non-inferiority of immunogenicity of the Vi-DT vaccine (pooled groups A-C) versus the Vi-TT vaccine (group D), measured by the anti-Vi IgG seroconversion rate at 4 weeks after vaccination; and (2) the lot-to-lot consistency of the Vi-DT vaccine, measured by immune equivalence of the anti-Vi IgG geometric mean titre (GMT) at 4 weeks after receipt of the three Vi-DT vaccine lots (lot 1 vs lot 2, lot 1 vs lot 3, and lot 2 vs lot 3). Non-inferiority of the Vi-DT vaccine compared with the Vi-TT vaccine was shown if the lower limit of the 97·5% CI for the difference between the seroconversion rates in Vi-DT vaccine groups A-C combined versus Vi-TT vaccine group D was above the predefined non-inferiority margin of -10%. Lot-to-lot immune equivalence was shown if the upper and lower bounds of the two-sided 99·17% CI around the GMT ratio for each pairwise lot-to-lot comparison was between 0·67 and 1·50, which is the predefined equivalence margin recommended by WHO. The co-primary immunogenicity endpoints were assessed in all randomised participants who had received their assigned vaccine and had completed at least one post-baseline immunogenicity assessment. Safety was descriptively summarised by group and age strata, and was assessed in all participants who had received one dose of the investigational vaccine. The trial is registered with ClinicalTrials.gov, NCT03933098. FINDINGS: Between Nov 20, 2019, and March 10, 2020, 1854 individuals were screened, of whom 1800 were enrolled and randomly assigned to groups A-D (450 participants in each group). 1786 (99·2%; 443 in group A, 450 in group B, 447 in group C, and 446 in group D) were included in the immunogenicity assessments at 4 weeks post vaccination, and all 1800 participants were included in the safety analysis. In the immunogenicity analysis, the anti-Vi-IgG seroconversion rate in all age strata was 99·33% (97·5% CI 98·61 to 99·68; 1331 of 1340 participants) in Vi-DT vaccine groups A-C and 98·88% (97·10 to 99·57; 441 of 446) in Vi-TT vaccine group D. The difference in seroconversion rates between Vi-DT vaccine groups A-C combined versus Vi-TT group D was 0·47% (97·5% CI -0·68 to 1·61), indicating non-inferiority of the Vi-DT vaccine. Anti-Vi-IgG GMT ratios at 4 weeks post-vaccination were 1·02 (99·17% CI 0·85 to 1·22) for lot 1 versus lot 2, 1·02 (0·85 to 1·23) for lot 1 versus lot 3, and 1·01 (0·84 to 1·21) for lot 2 versus lot 3, indicating lot-to-lot equivalence according to the predefined, WHO-recommended equivalence margin. The proportion of participants reporting adverse events was similar between Vi-DT vaccine groups A-C and Vi-TT vaccine group D; 260 (19·3%) of 1350 participants in Vi-DT vaccine groups A-C and 115 (25·6%) of 450 in Vi-TT vaccine group D reported solicited adverse events within 7 days after vaccination, and 208 (15·4%) in Vi-DT vaccine groups A-C and 76 (16·9%) in Vi-TT vaccine group D reported unsolicited adverse events within 4 weeks after vaccination. Seven serious adverse events (four [0·3%] participants in Vi-DT vaccine groups A-C and three [0·7%] in Vi-TT vaccine group D), including one death in the Vi-TT vaccine group, were reported during the 24-week follow-up period, none of which were considered related to the investigational product. INTERPRETATION: When administered as a single dose, the Vi-DT test vaccine was safe, immunogenic, and non-inferior to the Vi-TT vaccine at 4 weeks post vaccination. Equivalent immunogenicity of the three lots of Vi-DT vaccine was also shown, supporting the manufacturing process of this vaccine. Once prequalified by WHO, this vaccine could be an option for purchase by UN agencies. FUNDING: The Bill & Melinda Gates Foundation. TRANSLATION: For the Nepali translation of the abstract see Supplementary Materials section.

Whole Genome Sequencing of Dengue Virus Serotype 2 from Two Clinical Isolates and Serological Profile of Dengue in the 2015-2016 Nepal Outbreak.

Dengue virus (DENV) is the cause of one of the most prevalent neglected tropical diseases, and up to half of the world's population is at risk for infection. Recent results from clinical trials have shown that DENV vaccination can induce the development of severe dengue disease and/or prolong hospitalization after natural infection in certain naive populations. Thus, it is crucial that vaccine development takes into account the history of DENV exposure in the targeted population. In Nepal, DENV infection was first documented in 2004, and despite the increasing prevalence of DENV infection, the population remains relatively naive. However, it is not known which of the four DENV serotypes circulate in Nepal or whether there is evidence of repeated exposure to DENV in the Nepali population. To address this, we studied 112 patients who presented with symptomology suspicious for DENV infection at clinics throughout Nepal during late 2015 and early 2016. Of the 112 patients examined, 39 showed serological and/or genetic evidence of primary or secondary DENV infection: 30 were positive for DENV exposure by IgM/IgG ELISA, two by real-time reverse-transcription PCR (RT-PCR), and seven by both methods. Dengue virus 1-3, but not DENV4, serotypes were detected by RT-PCR. Whole genome sequencing of two DENV2 strains isolated from patients with primary and secondary infections suggests that DENV was introduced into Nepal through India, with which it shares a porous border. Further study is needed to better define the DENV epidemic in Nepal, a country with limited scientific resources and infrastructure.

Challenges and opportunities in setting up a phase III vaccine clinical trial in resource limited settings: Experience from Nepal.

Clinical trials are complicated, time-consuming and costly. From the initial screening, informed consent and recruitment of the participants' to study completion, the sponsor must undertake a wide array of complex and closely monitored operations, complying with international standards for human subject research and local requirements. Conducting these studies in an underdeveloped country, with limited resources, infrastructure, and experience with regulated clinical trials adds to this complexity. The initial site selection, set up and preparatory activities for the clinical trial are crucial to minimizing the risks to both participants and to successful completion during the subsequent study execution.In this paper, we describe the experience and lessons learned of building clinical trial site capacity in terms of infrastructure and human resource development for a Phase III vaccine clinical trial. We believe that sharing the experience of setting up a clinical trial in a resource-limited country will enable other entities contemplating clinical research in these countries, to prepare and plan ahead, to minimize the impact of barriers, and to contribute to bringing more studies to the countries where people live with the burden of vaccine-preventable, poverty-associated diseases.

A case of high altitude cutaneous leishmaniasis in a non-endemic region in Nepal.

A case of cutaneous leishmaniasis was discovered in a 32-year old man with a persistent erythematous plaque. The patient resides in a high altitude (~2000 m above sea level) area that is not endemic for cutaneous leishmaniasis in the Dunai village of Dolpa, Nepal. The patient's lesion was initially misdiagnosed as lupus vulgaris. After response failure to initial treatment, additional testing by histological microscopy revealed the presence of Leishmania amastigotes in tissue from the lesion, and the diagnosis of cutaneous leishmaniasis was confirmed by nested PCR DNA assay of tissue from the lesion, and by a positive rK39 test in blood. Sequencing of the kinetoplast region confirmed the presence of Leishmania donovani complex. The patient responded well to treatments for cutaneous leishmaniasis and the skin lesions regressed after 6 months. This is the first known case of cutaneous leishmaniasis in a patient in Nepal who resides at high altitude in a non-endemic region. Increasing temperatures in this region of Nepal may be expanding the range of vectors that transmit cutaneous leishmaniasis.

Short article: Association between liver fibrosis and hepatitis E seroprevalence among HIV-positive individuals in Nepal.

OBJECTIVE: Hepatitis E virus (HEV) seropositivity may confer an increased risk of liver fibrosis in immunosuppressed individuals. We studied this effect in HIV-infected individuals in Nepal, a country hyperendemic for HEV. PARTICIPANTS AND METHODS: We prospectively evaluated 200 HIV-positive individuals. Serum samples were tested for components of fibrosis scores and cytokeratin-18. RESULTS: Of 200 patients, 43% were HEV-immunoglobulin G+. The mean fibrosis-4 score was 8.02 in the HEV-positive and 1.17 in the HEV-negative group (P<0.001). The mean nonalcoholic fatty liver disease score was 2.12 in the HEV-positive and -2.53 in the HEV-negative group (P=0.02). The mean aminotransferase-platelet ratio index score was 0.37 in the HEV-positive and 0.38 in the HEV-negative group (P=0.9). The mean cytokeratin-18 levels were 119.9 in the HEV-positive group and 158.6 in the HEV-negative group (P=0.08). CONCLUSION: We found higher fibrosis-4 and nonalcoholic fatty liver disease scores in HEV-HIV-positive individuals, suggesting an increased liver fibrosis profile in this group. Further studies using liver stiffness measurements should be carried out.

Hepatitis viruses in Kathmandu, Nepal: hospital-based study.

OBJECTIVE: The objective of this study was to see the aetiology and outcome of sporadic acute viral hepatitis (AVH) in Kathmandu, Nepal. RESULTS: Among 210 patients, 94 (45%) were male and 116 (55%) were female. Mean age was 30 years. 52 (24.7%) out of 210 were positive for either of the hepatitis virus infection. Major causative agent for AVH among hepatitis positive patients were hepatitis E virus (HEV) in 36 (69.2%), followed by hepatitis A virus (HAV) 8 (15.3%), hepatitis B virus (HBV) 7 (13.4%) and hepatitis C virus (HCV) 1 (1.9%). The 158 (75.3%) patient were negative for all hepatitis viral markers. Co-infections with more than one virus were found in 4 (7.6%) patients. All liver-specific enzymes including bilirubin increased in hepatitis-infected patients. We found large number circulation of HEV in Kathmandu, Nepal, indicating that this region is endemic for hepatitis virus infection.

Microfilaria persistent foci during post MDA and the risk assessment of resurgence in India.

BACKGROUND: Pondicherry, a union territory in India, is an endemic district for bancroftian lymphatic filariasis transmitted by Culex quinquefasciatus where eight rounds of mass drug administration (MDA) were completed in 2011 (annually once from 2004 to 2011).The objectives of this study were to conduct a focal survey to assess microfilaria and antigen (Ag) prevalence among young adults and to assess vector infection and infectivity through a focal entomological survey. METHODS: Mosquitoes were collected using gravid traps in Sedurapet village of Pondicherry and dissected to enumerate W. bancrofti larvae stage first larval stage (L1), second larval stage (L2), and third larval stage (L3). Microfilarias (Mf) were detected using blood smears collected from inhabitants. RESULTS: A total of 360 individuals from 67 houses were enrolled in this study of which 290 (80.6%) were surveyed for the presence of Mf. Two Mf carriers were detected yielding an overall prevalence of 0.69% and two out of 85 (2.35%) were Mf antigen positive. Of the 2875 mosquitoes collected by gravid trap, Culex quinquefasciatus (93.9%) was the predominant species, followed by Anopheles subpictus (2.3%) and Culex vishnui (3.8%). The density of Cx. quinquefasciatus was 28.1 per trap-night. A total of 2429 Cx. quinquefasciatus were dissected and microscopically examined for abdominal conditions (gravid 85%, semi-gravid 9.4%, unfed 3.8%, and fully fed 1.9%) and filarial infection. One mosquito (infection rate equal to 0.04%) was found to harbor a second stage filarial larva, and none of the mosquitoes had infective stage larva. CONCLUSION: Our results show no reappearance of infection of lymphatic filariasis in Sedurapet village of Pondicherry after MDA, and thus, no further intervention is required in that area for possible resurgence of lymphatic filariasis. However, monitoring should be continued as part of post MDA activities until the endpoint of complete elimination is achieved. We demonstrated that xenomonitoring can be used to monitor the post MDA situation for possible risk of transmission to initiate control measures.