Krishna Das Manandhar

Phylogenetic study reveals co-circulation of Asian II and Cosmopolitan genotypes of Dengue virus serotype 2 in Nepal during 2013.

The re-emergence of dengue virus in Nepal and the recent widespread disease epidemics of unprecedented magnitude have raised a great public health concern. There are very few reports on Dengue virus (DENV) strains circulating in the country, especially at the molecular phylogenetics level. In this study, clinical samples from an outbreak in Nepal in 2013, which were positive for DENV serotype 2, were characterized by targeted genome sequencing. Envelope protein (E) coding region from fifteen samples were sequenced and compared with DENV-2 sequences of strains from different geographic regions obtained from the GenBank. Compared to the prototype New Guinea C strain, the samples had a total of eleven non-synonymous substitutions in the envelope protein coding region leading to amino acid change at positions 47, 52, 71, 126, 129, 149, 164, 390, 402, 454 and 462. However, none of these sites were found to be positively selected. A major observation was the presence of two distinct genotypes (Cosmopolitan Genotype IVa and Asian II) in the outbreak as seen by the phylogenetic analysis. It gives the first evidence of the introduction of Cosmopolitan Genotype IVa in Nepal. These strains replace the Genotype IVb strains prevalent earlier since 2004. Both genotypes had closer genetic relation to strains from other countries indicating possibility of exotic introduction. The Genotype IVa strain seems to be more adapted in C6/36 mosquito cells as indicated by its marginally increased replication rate than the Asian II strain in in vitro infection kinetics assays. The genotype replacement and co-circulation of two distinct genotypes may have significant consequences in dengue epidemiology and disease dynamics in Nepal in years to come.

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.

Dengue virus infection in a French traveller to the hilly region of Nepal in 2015: a case report

BackgroundDengue viral infections are known to pose a significant risk during travel to tropical regions, but it is surprising to find dengue transmission in the hilly region of Nepal, which is over 1800mtr above sea level.Case presentationA 43-year-old Caucasian female traveler from France presented with fever and abdominal pain following a diarrheal illness while visiting the central hilly region of Nepal. Over the course of 9 days, she developed fever, body aches, and joint pain, with hemorrhagic manifestation. She was hospitalized in India and treated with supportive care, with daily monitoring of her platelets. An assessment by enzyme-linked immunosorbent assay showed that she was positive for dengue non-structural protein 1. Upon her return to France, dengue virus was confirmed by reverse transcriptase-polymerase chain reaction.ConclusionThe district where this dengue case was reported is in the hilly region of Nepal, neighboring the capital city Kathmandu. To the best of our knowledge, there has previously been no dengue cases reported from the district. This study is important because it aims to establish a potential region of dengue virus circulation not only in the tropics, but also in the subtropics as well, which in Nepal may exceed elevations of 1800mtr. This recent case report has raised alarm among concerned health personnel, researchers, and organizations that this infectious disease is now on the way to becoming established in a temperate climate.

Characterization of home-made silver sulphide based iodide selective electrode

Polycrystalline silver sulphide/silver iodide ion selective electrodes (ISEs) with four different compositions, 9:1, 2:1, 1:1, 1:9 Ag(2)S-AgI mole ratios, have been fabricated in the laboratory and characterized by X-ray diffractometry (XRD), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). X-ray diffraction studies show the presence of Ag(3)SI, Ag(2)S and AgI crystalline phases in the electrode material. The electrode surfaces have been found to become smoother and lustrous with increasing percentage of silver sulphide in silver iodide. ISE 1:1, ISE 2:1 and ISE 9:1 all responded in Nernstian manner with slopes of about 60 mV/decade change in iodide ion concentration in the linear range of 1 x 10(-1) to 1 x 10(-6)M while ISE 1:9 showed sub-Nernstian behavior with slope of about 45 mV up to the concentration 1 x 10(-5)M. Two capacitive loops, one corresponding to the charge transfer process at metal electrode and the back contact and a second loop corresponding to the charge transfer process at membrane-electrolyte interface have been observed at high and low frequency ranges, respectively. Mott-Schottky analysis shows that the materials are n-type semiconductors with donor defect concentrations in the range of 5.1 x 10(14) to 2.4 x 10(19)/cm(3).

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.

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

BackgroundInfection 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.MethodsWe 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.ResultsWe 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.ConclusionsHIV-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.

Human papillomavirus genotype distribution in cervical cancer biopsies from Nepalese women

BackgroundCervical cancer (CC) is the leading cause of morbidity and mortality from cancer in Nepalese women. Nearly all cases of CC are caused by infection with certain genotypes of human papillomavirus (HPV). Data on HPV genotype distribution in Nepalese CC patients is sparse. We aimed to determine the distribution of HPV genotypes in biopsies of CC tissue from Nepalese women.MethodsThis study examined 248 archived paraffin-embedded tissue specimens from CC cases from patients of B.P. Koirala Memorial Cancer Hospital, Bharatpur, Chitwan, Nepal. DNA was extracted from the biopsies and HPV detection performed by PCR. HPV genotyping was then carried out by a reverse line hybridization technique capable of identifying 36 distinct HPV genotypes.ResultsMost of the samples were from tumors that had been designated by hospital pathologists as squamous cell carcinoma (77.6%). 165 of the 248 samples contained DNA of sufficient quality for rigorous PCR testing. All the analyzable specimens were positive for HPV. The most common HPV genotypes, in decreasing order of frequency were 16, 18, 45, 33, 52, 56 and 31; most were found as single infections (94.5%). Together, HPV types 16, 18, and 45 were found in 92% of the tumor samples.ConclusionThis study strengthens the knowledge-base of HPV genotype distribution in CC cases in Nepal. Hopefully, this information will be useful to the medical community and public health policy-makers in generating improved HPV-surveillance, −prevention and -treatment strategies in Nepal.

Biomarkers in Immune Reconstitution Inflammatory Syndrome (IRIS) among People Living with Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS)

Background: An Immune Reconstitution Inflammatory Syndrome (IRIS) event is a presentation or a paradoxical worsening of a pre-existing infection following initiation of anti-retroviral therapy in the presence of a decreasing viral load and features consistent with an inflammatory process. This study was conducted to find out the significance of different clinical parameters like hemoglobin, albumin, viral load, erythrocyte, body mass index in the people living with HIV/AIDS with IRIS in Nepalese population. Methods: The study was descriptive with control group. The study included patients who experienced IRIS after initiation of highly active antiretroviral therapy (HAART) with control group as patients who were HIV positive without HAART treatment. This study was carried out on 44 HIV infected individual who initiated HAART and then suffered from IRIS and compared with 56 control HIV infected person without IRIS visiting National Public Health Laboratory (NPHL) for routine HIV viral load testing and CD4 count between April and August, 2014. Results: The patients were categorized into highly active antiretroviral therapy (HAART) naive (n=56) and on HAART with immune reconstitution inflammatory syndrome (IRIS) (n=44). Among 56 individuals naive HAART, viral load 1000 copies/ml among which 02 were female (34.83 ± 2.030 years) and 10 were male (41.34 ± 1.462 years). The comparison of CD4 count between the naive and patients enrolled for HAART; the risk of having CD4 count <200 cell/mm3 is significantly greater in male than that of female. The BMI ratio of HAART to naive patients (19.88 ± 0.7290) was lower than that of HAART enrolled patients (21.78 ± 0.3546). The hemoglobin value showed significant (P value<0.0001) difference among PLHIV having CD4 level less than 200 (9.9 ± 2.156), between 200-500 (11.63 ± 1.946) and more than 500 CD4 level (12.71 ± 1.850). Significant (P<0.0001) viral load suppression showed among HAART initiated female patients with IRIS when compared with naive female patients without IRIS. BMI, hemoglobin level, total leukocyte count, albumin level, HDL level, ESR value, CRP level and absolute eosinophil level less than 351 cells/mm3 showed significant (P<0.05) difference among HAART naive and on HAART female patients with IRIS. Significant (P<0.0001) viral load suppression showed among HAART initiated male patients with IRIS when compared with naive female patients without IRIS. BMI (18-26), hemoglobin level below 8 g/dl, TLC, serum albumin level below 5 g/dl, HDL level below 61 mg/dl, ESR level, CRP value and absolute eosinophil count showed significant (P<0.05) difference between HAART naive and on HAART male patients with IRIS. Hemoglobin level, HDL, TLC, ESR, CD8, AEC, viral load, BMI and serum albumin level showed significant (P<0.0001) difference among HAART initiated patients with IRIS when compared to different level of CD4 T cell count. Conclusion: Prevalence of anemia was high in HAART naive patients while leucopenia prevalence was higher in patients on HAART and their prevalence increased as the CD4 count decreased. HIV Patients should be investigated for hematological and immunological changes following with appropriate therapeutic interventions. The study findings reemphasize the importance of nutritional and immunological parameters to assess the stage of the disease, initiate antiretroviral therapy and monitor the response in disease progression.

Iodide Ion Sensor for Indirect Determination of As (III) in Water

A simple, low cost and portable iodide sensor for the indirect determination of As(III) has been developed from the polycrystalline material obtained by co-precipitation of silver sulphide and silver iodide from aqueous solution. The Ag2S-AgI sensor was found to behave well up to the iodide concentration of 1×10-6 M with a Nernstian slope 57.5 mV per decade change in concentration. The selectivity coefficient of sensor containing iodide ion in the presence of Br- and Cl- ions was found to be 1.77 × 10-2 and 7.94 × 10-3, respectively indicating that these ions do not interfere in the response of iodide sensor to iodide ion. The electrode was successfully used for the direct determination of iodide and indirect determination of As(III) by titrating with cerric ammonium sulphate at pH 2. The results obtained from this method are comparable to the existing methods for the estimation of As(III).