Safety and immunogenicity of the Euvichol-S oral cholera vaccine for prevention of Vibrio cholerae O1 infection in Nepal: an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial.

BACKGROUND: In October, 2017, WHO launched a strategy to eliminate cholera by 2030. A primary challenge in meeting this goal is the limited global supply capacity of oral cholera vaccine and the worsening of cholera outbreaks since 2021. To help address the current shortage of oral cholera vaccine, a WHO prequalified oral cholera vaccine, Euvichol-Plus was reformulated by reducing the number of components and inactivation methods. We aimed to evaluate the immunogenicity and safety of Euvichol-S (EuBiologics, Seoul, South Korea) compared with an active control vaccine, Shanchol (Sanofi Healthcare India, Telangana, India) in participants of various ages in Nepal. METHODS: We did an observer-blind, active-controlled, randomised, non-inferiority, phase 3 trial at four hospitals in Nepal. Eligible participants were healthy individuals aged 1-40 years without a history of cholera vaccination. Individuals with a history of hypersensitivity reactions to other preventive vaccines, severe chronic disease, previous cholera vaccination, receipt of blood or blood-derived products in the past 3 months or other vaccine within 4 weeks before enrolment, and pregnant or lactating women were excluded. Participants were randomly assigned (1:1:1:1) by block randomisation (block sizes of two, four, six, or eight) to one of four groups (groups A-D); groups C and D were stratified by age (1-5, 6-17, and 18-40 years). Participants in groups A-C were assigned to receive two 1·5 mL doses of Euvichol-S (three different lots) and participants in group D were assigned to receive the active control vaccine, Shanchol. All participants and site staff (with the exception of those who prepared and administered the study vaccines) were masked to group assignment. The primary immunogenicity endpoint was non-inferiority of immunogenicity of Euvichol-S (group C) versus Shanchol (group D) at 2 weeks after the second vaccine dose, measured by the seroconversion rate, defined as the proportion of participants who had achieved seroconversion (defined as ≥four-fold increase in V cholerae O1 Inaba and Ogawa titres compared with baseline). The primary immunogenicity endpoint was assessed in the per-protocol analysis set, which included all participants who received all their planned vaccine administrations, had no important protocol deviations, and who provided blood samples for all immunogenicity assessments. The primary safety endpoint was the number of solicited adverse events, unsolicited adverse events, and serious adverse events after each vaccine dose in all ages and each age stratum, assessed in all participants who received at least one dose of the Euvichol-S or Shanchol. Non-inferiority of Euvichol-S compared with Shanchol was shown if the lower limit of the 95% CI for the difference between the seroconversion rates in Euvichol-S group C versus Shanchol group D was above the predefined non-inferiority margin of -10%. The trial was registered at ClinicalTrials.gov, NCT04760236. FINDINGS: Between Oct 6, 2021, and Jan 19, 2022, 2529 healthy participants (1261 [49·9%] males; 1268 [50·1%] females), were randomly assigned to group A (n=330; Euvichol-S lot number ES-2002), group B (n=331; Euvichol-S ES-2003), group C (n=934; Euvichol-S ES-2004]), or group D (n=934; Shanchol). Non-inferiority of Euvichol-S versus Shanchol in seroconversion rate for both serotypes at 2 weeks after the second dose was confirmed in all ages (difference in seroconversion rate for V cholerae O1 Inaba -0·00 [95% CI -1·86 to 1·86]; for V cholerae O1 Ogawa -1·62 [-4·80 to 1·56]). Treatment-emergent adverse events were reported in 244 (9·7%) of 2529 participants in the safety analysis set, with a total of 403 events; 247 events were reported among 151 (9·5%) of 1595 Euvichol-S recipients and 156 events among 93 (10·0%) of 934 Shanchol recipients. Pyrexia was the most common adverse event in both groups (57 events among 56 [3·5%] of 1595 Euvichol-S recipients and 37 events among 35 [3·7%] of 934 Shanchol recipients). No serious adverse events were deemed to be vaccine-related. INTERPRETATION: A two-dose regimen of Euvichol-S vaccine was non-inferior to the active control vaccine, Shanchol, in terms of seroconversion rates 2 weeks after the second dose. The simplified formulation and production requirements of the Euvichol-S vaccine have the potential to increase the supply of oral cholera vaccine and reduce the gap between the current oral cholera vaccine supply and demand. FUNDING: The Bill & Melinda Gates Foundation. TRANSLATION: For the Nepali translation of the abstract see Supplementary Materials section.

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.

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.

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.

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.

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.

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.

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.

Maternal and severe anaemia in delivering women is associated with risk of preterm and low birth weight: A cross sectional study from Jharkhand, India.

BACKGROUND AND OBJECTIVES: Haemoglobin content is the well accepted indicator for anaemia assessment. The high prevalence of anaemia, maternal health care issues and adverse delivery outcome in Jharkhand, we investigated whether delivering women with anaemia would present a modifiable risk of preterm (PTB) and low birth weight (LBW). METHODS: A facility-based cross-sectional study involving pregnant women, with screening for pregnancy endpoints and haemoglobin assay, were conducted. Anaemia was classified according to World Health Organization's definition of anaemia in pregnancy. Confounding variables were adjusted in a logistic model. The adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were used for analyzing the association among maternal anaemia, PTB and LBW. RESULTS: We observed a high prevalence of anaemia (78.45%) in delivering women, whereas high prevalence of preterm birth (34.75%) and LBW (32.81%) in delivering women overall. In the adjusted analysis, overall anaemia in pregnancy was strongly associated with preterm birth (OR, 3.42; 95% CI, 1.98-5.88; P ≤ .0001) as compared to LBW (OR, 1.12; 95% CI, 0.65-1.61; P = .0003). The risk of PTB and LBW were dependent on the stratification of the anaemia group, as the strongest association was observed in severe (OR, 4.86) followed by mild (OR, 3.66) and moderate (OR, 3.18) anaemia in PTB; whereas risk of LBW was found in severe (OR, 2.5) followed by moderate (OR, 1.11) and mild (OR, 0.57) anaemia. The risk of PTB and LBW across six pregnancy haemoglobin groups were compared, haemoglobin of 10-10.9 g/dl (OR, 1.25) and ≤ 8 g/dl (OR, 1.03) have shown association with PTB and LBW, respectively. However, high haemoglobin concentration was not associated with either PTB or LBW. CONCLUSIONS: Anaemia in delivering women was associated with an elevated risk of PTB and LBW and the risk increased with the severity of anaemia in pregnant women.

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.

Genetic diversity and allelic variation in MSP3α gene of paired clinical Plasmodium vivax isolates from Delhi, India.

BACKGROUND: Plasmodium vivax malaria accounts for 80% of the malaria cases in Delhi, India. The gene merozoite surface protein 3 alpha (MSP3α) is highly polymorphic and has been used as marker in many P. vivax population studies. METHODS: MSP3α has been used to assess the genetic diversity of P. vivax samples from Delhi (India) having more than one malaria episode (s) i.e. clinically identified relapse cases using PCR-RFLP and sequencing. RESULTS: Three major genotypes 2.0 kb (A), 1.4 kb (B) and 1.2 kb (C) were amplified from 72 isolates with frequencies of 72.2%, 19.44% and 9.72% respectively. One sample out of 72 showed mixed infection having both A and B type genotypes. 82.05% patients showed same genotype while only 17.94% patients showed different genotypes after subsequent malaria episodes. 18 different genotypes with Alu I and 35 with Hha I were identified among 72 samples analyzed by restriction fragment length polymorphism (RFLP). 18 Pvmsp3α nucleotide sequences were analyzed and it did not reveal any distinct intragenic differences within sequences of the same type, however, allelic diversity among the three types (Π = 0.029703) was observed. Phylogenetic analysis showed allelic family types A, B and C were not clustered but distributed in different branches. The results indicate that the P. vivax parasite population is highly diverse in Delhi, India. A large number of amino acid substitutions were found at the locus of the isolates when compared with the Belem Strain (Π = 0.030528). The substantial sequence diversity is largely restricted to certain domains of encoded protein. Analysis of synonymous and nonsynonymous substitutions suggested that different selection forces were operating on different regions of the protein molecule. CONCLUSION: We propose that genotyping of the PvMSP-3α gene as one of the molecular tools for differentiating relapse from new infection in epidemiological settings. The analyses of sequence polymorphism in PvMSP-3α gene enable it as potential candidate for inclusion in a P.vivax vaccine research.

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.

Determination of CD4+ T- Lymphocytes in Healthy Children of Kathmandu.

BACKGROUND: The cluster differentiation (CD) of T-cell is the good marker for the immunological competence study. Nepal does not have a reference value for CD4+ T cell count and percentage for children, which severely limits the prospect of pediatric prognosis. METHODS: This cross-sectional study was conducted in Kathmandu valley where total 207 children of age 0-14 year age group were recruited in this study. We analyzed 50 cord blood and 157 peripheral blood samples in order to calculate the absolute count of CD4+ T lymphocyte using Fluorescence-activated cell sorting methodology. RESULTS: The reference range for absolute CD4+ T cell count was found to be 634-4040 cells/µL(mean1470; median: 1335 and 95% CI [1322-1617]) for male children and 491-2922 cells/µL (mean: 1443 median: 1326 and95% CI [1298-1588]) for the female children.We also observed elevated CD4 to the CD3 ratio in younger children (0.67 from cord blood Vs 0.53 from 10-14yr) compared to older ones. CONCLUSIONS: The observed CD4+ T cell counts among healthy children of Kathmandu highlights the gender differences skewed for male as well the need of defining specific reference values for other lymphocyte subsets as well in a country like Nepal which has a population with diverse genetic and socio-cultural parameters.

Estimation of Transmission of Porphyromonas Gingivalis from Mother to Child through Saliva.

INTRODUCTION: Chronic periodontitis is an infectious disease. Porphyromonas gingivalis is the major pathogen associated with it and can be found in all ecosystems in the oral cavity. The presence of this organism is highly correlated with preterm and low birth weight babies. So, this study aimed to assess vertical transmission of P.gingivalis from pregnant women to their new born. METHODS: Forty six pregnant women with chronic periodontitis were recruited for this cross-sectional study. Whole unstimulated saliva was collected from them before delivery and from their new-borns within forty eight hours of birth. Quantification of P.gingivalis in the saliva samples was carried out by quantitative real time polymerase chain reaction. The obtained data were analysed by SPSS 16 program. RESULTS: The results showed a significant correlation (P=0.002) between the number of P.gingivalis present in the mother's saliva with that of the new-borns' saliva. DNA copies of more than 5000/µl of P.gingivalis was found in 20 (43.5%) maternal saliva and 21 (45.7%) in new-borns' saliva. Both Plaque index and Extent and Severity index showed no correlation (P>0.05) with DNA copies of P.gingivalis in new-borns' saliva. CONCLUSIONS: The DNA copies of P.gingivalis found in new-borns' saliva are in par with mother saliva, as the saliva sample obtained from new-borns' were within forty eight hours of birth, no other environmental factor can have a direct role in its transmission. Thus, it can be concluded that P.gingivalis is vertically transmitted from mother to child.

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.

Profile of the 2016 dengue outbreak in Nepal.

OBJECTIVE: The objective of this study was to obtain clinical, virological and demographic data detailing the 2016 dengue outbreak in Nepal. RESULTS: Dengue disease was first reported in Nepal in 2004 and several major outbreaks have occurred since then, with a significant impact on public health. An outbreak of dengue fever occurred in Nepal during June to November 2016, with a peak number of cases reported in September. 1473 patients with laboratory confirmed DENV infections visited or were admitted to hospitals during this period. The most common clinical symptoms included fever, headache, joint pain and thrombocytopenia. Serotyping of 75 serum samples from patients having fever for less than 4 days was carried out with a dengue virus (DENV) serotype-specific RT-PCR strategy. Our results indicate that the dengue outbreak in Nepal during 2016 was caused predominantly, if not exclusively, by DENV-1, representing a shift in the prevailing serotype from DENV-2, the dominant serotype characterizing the 2013 dengue epidemic in Nepal. Hopefully, this report will assist Nepalese public health agencies in developing improved dengue-related programs including mosquito-vector control, DENV surveillance, and diagnosis and treatment of dengue fever patients, in order to reduce the impact of future dengue epidemics.

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.

Emergence of dengue in Nepal.

Dengue virus is a major health problem in Nepal. The endogenous dengue appeared in 2006 in the country with reported outbreaks in 2010, 2013 and 2016. Eleven years vertical data show there were sporadic cases in all the years and mostly adults between 25 and 40 years of age were infected with dengue virus. Compared with primary infections, secondary infections were observed in relatively larger numbers during the period of 2008-2016. Most of the cases had symptoms of dengue fever; while 7 and 19 cases demonstrated dengue hemorrhagic fever/dengue shock syndrome in 2010 and 2013 respectively. The proportion of dengue hemorrhagic fever amongst all cases of dengue fever was 2.5:4.7% in 2010 and 2013. We found there is shift of serotype from dengue virus serotype-1 (DENV-1) in 2010, DENV-2 in 2013 and DENV-1 in 2016. We feel there is urgent need for better community, hospital and laboratory based surveillance system capable of monitoring the circulating dengue virus (DENV) serotypes in different districts of Nepal. With improvement in surveillance system and efficient management of cases, the case fatality rate due to severe dengue can be reduced.