Burden of human metapneumovirus infections among children with acute respiratory tract infections attending a Tertiary Care Hospital, Kathmandu.

BACKGROUND: Acute respiratory infections (ARIs) are one of the most common causes of mortality and morbidity worldwide. Every year millions of children suffer from viral respiratory tract infections (RTIs) ranging from mild to severe illnesses. Human Metapneumovirus (HMPV) is among the most frequent viruses responsible for RTIs. However, HMPV infections and their severity among children have not been explored yet in Nepal. PURPOSE: Therefore, the study was focused on HMPV infections and other potential viral etiologies or co-infections using multiplex PCR among children attending Kanti Children's Hospital and assessed the clinical characteristics of the infections as well as found the co-infections. A hospital-based cross-sectional study was designed and a convenience sampling method was used to enroll children of less than 15 years with flu-like symptoms from both outpatients and inpatients departments over three months of the study period. RESULTS: HMPV infection (13.3%) was the most predominant infection among the different viral infections in children with ARIs in Kanti Children's Hospital. The HMPV was more prevalent in the age group less than three years (21.8%). Cough and fever were the most common clinical features present in all children infected with HMPV followed by rhinorrhea, sore throat, and wheezing. HMPV-positive children were diagnosed with pneumonia (42.9%), bronchiolitis (28.5%), upper respiratory tract infections (14.3%), and asthma (14.3%). The prevalence of HMPV was high in late winter (14.3%) followed by early spring (13.5%). CONCLUSIONS: This study provides the baseline information on HMPV and associated co-infection with other respiratory viruses for the differential diagnosis based on molecular methods and also the comparison of clinical presentations among the different respiratory syndromes.

Etiology of Coinfections in Children with Influenza during 2015/16 Winter Season in Nepal.

Acute respiratory infections (ARIs) are one of the major public health problems in developing countries like Nepal. Besides the influenza, several other pathogens are responsible for acute respiratory infection in children. Etiology of infections is poorly characterized at the course of clinical management, and hence empirical antimicrobial agents are used. The objective of this study was to characterize the influenza and other respiratory pathogens by real-time PCR assay. A total of 175 throat swab specimens of influenza-positive cases collected at National Influenza Center, Nepal, during the 2015/16 winter season were selected for detecting other respiratory copathogens. Total nucleic acid was extracted using Pure Link viral RNA/DNA mini kit (Invitrogen), and multiplex RT-PCR assays were performed. Influenza A and B viruses were found in 120 (68.6%) and 55 (31.4%) specimens, respectively, among which coinfections were found in 106 (60.6%) specimens. Among the influenza A-positive cases, 25 (20.8%) were A/H1N1 pdm09 and 95 (79.2%) were A/H3 subtypes. Viruses coinfected frequently with influenza virus in children were rhinovirus (26; 14.8%), respiratory syncytial virus A/B (19; 10.8%), adenovirus (14; 8.0%), coronavirus (CoV)-HKU1 (14; 8.0%), CoV-OC43 (5; 2.9%), CoV-229E (2; 1.1%), metapneumovirus A/B (5; 2.9%), bocavirus (6; 3.4%), enterovirus (5; 2.9%), parainfluenza virus-1 (3; 1.7%), and parainfluenza virus-3 (2; 1.1%). Coinfection of Mycoplasma pneumoniae with influenza virus was found in children (5; 2.8%). Most of the viral infection occurred in young children below 5 years of age. In addition to influenza virus, nine different respiratory pathogens were detected, of which coinfections of rhinovirus and respiratory syncytial virus A/B were predominantly found in children. This study gives us better information on the respiratory pathogen profile and coinfection combinations which are important for diagnosis and treatment of ARIs.

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.

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.

Characterization of influenza A(H1N1)pdm09 viruses isolated from Nepalese and Indian outbreak patients in early 2015.

We characterized influenza A(H1N1)pdm09 isolates from large-scale outbreaks that occurred in Nepal and India in early 2015. Although no specific viral features, which may have caused the outbreaks, were identified, an S84N substitution in hemagglutinin was frequently observed. Chronological phylogenetic analysis revealed that these Nepalese and Indian viruses possessing the S84N substitution constitute potential ancestors of the novel genetic subclade 6B.1 virus that spread globally in the following (2015/16) influenza season. Thus, active surveillance of circulating influenza viruses in the Southern Asia region, including Nepal and India, would be beneficial for detecting novel variant viruses prior to their worldwide spread.

Detection of Anti-Leptospira IgM Antibody in Serum Samples of Suspected Patients Visiting National Public Health Laboratory, Teku, Kathmandu.

Leptospirosis is a globally distributed zoonosis with varied clinical outcomes and multiorgan involvement in humans. In this study conducted from July 2011 to December 2011, 178 serum samples from patients suspected of leptospirosis were tested by Panbio IgM ELISA at National Public Health Laboratory, Kathmandu, out of which 51 (28.65%) were positive for anti-Leptospira IgM antibody. Leptospirosis was more common in people in their 2nd and 3rd decades of their life which together comprised 56.86% of the total positive cases. Most of those tested positive were farmers followed by students and housewives. Both animal contact and water contact seemed to play significant roles in disease transmission. Symptoms were vague with the most common being fever, headache, myalgia, abdominal pain, vomiting, jaundice, and diarrhoea. Life style heavily dominated by agronomical and farming activities in Nepal is conducive to leptospirosis transmission. Leptospirosis seems to be a significant public health problem in Nepal but is underestimated. In resource poor countries like Nepal where laboratories performing MAT or maintaining cultures are rarely available, serological test like ELISA could well depict the scenario of the disease prevalence.

Population genetics of Vibrio cholerae from Nepal in 2010: evidence on the origin of the Haitian outbreak.

Cholera continues to be an important cause of human infections, and outbreaks are often observed after natural disasters, such as the one following the 2010 earthquake in Haiti. Once the cholera outbreak was confirmed, rumors spread that the disease was brought to Haiti by a battalion of Nepalese soldiers serving as United Nations peacekeepers. This possible connection has never been confirmed. We used whole-genome sequence typing (WGST), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing to characterize 24 recent Vibrio cholerae isolates from Nepal and evaluate the suggested epidemiological link with the Haitian outbreak. The isolates were obtained from 30 July to 1 November 2010 from five different districts in Nepal. We compared the 24 genomes to 10 previously sequenced V. cholerae isolates, including 3 from the Haitian outbreak (began July 2010). Antimicrobial susceptibility and PFGE patterns were consistent with an epidemiological link between the isolates from Nepal and Haiti. WGST showed that all 24 V. cholerae isolates from Nepal belonged to a single monophyletic group that also contained isolates from Bangladesh and Haiti. The Nepalese isolates were divided into four closely related clusters. One cluster contained three Nepalese isolates and three Haitian isolates that were almost identical, with only 1- or 2-bp differences. Results in this study are consistent with Nepal as the origin of the Haitian outbreak. This highlights how rapidly infectious diseases might be transmitted globally through international travel and how public health officials need advanced molecular tools along with standard epidemiological analyses to quickly determine the sources of outbreaks.

Outbreak of pandemic influenza A/H1N1 2009 in Nepal.

BACKGROUND: The 2009 flu pandemic is a global outbreak of a new strain of H1N1 influenza virus. Pandemic influenza A (H1N1) 2009 has posed a serious public health challenge world-wide. Nepal has started Laboratory diagnosis of Pandemic influenza A/H1N1 from mid June 2009 though active screening of febrile travellers with respiratory symptoms was started from April 27, 2009. RESULTS: Out of 609 collected samples, 302 (49.6%) were Universal Influenza A positive. Among the influenza A positive samples, 172(28.3%) were positive for Pandemic influenza A/H1N1 and 130 (21.3%) were Seasonal influenza A. Most of the pandemic cases (53%) were found among young people with ≤ 20 years. Case Fatality Ratio for Pandemic influenza A/H1N1 in Nepal was 1.74%. Upon Molecular characterization, all the isolated pandemic influenza A/H1N1 2009 virus found in Nepal were antigenically and genetically related to the novel influenza A/CALIFORNIA/07/2009-LIKE (H1N1)v type. CONCLUSION: The Pandemic 2009 influenza virus found in Nepal were antigenically and genetically related to the novel A/CALIFORNIA/07/2009-LIKE (H1N1)v type.

Detection of Salmonella invA gene in shrimp enrichment culture by polymerase chain reaction.

Contamination of seafood with salmonellae is a major public health concern. Detection of Salmonella by standard culture methods is time consuming. In this study, an enrichment culture step prior to polymerase chain reaction (PCR) was applied to detect 284 bp fragment of Salmonella invA in comparison with the conventional culture method in 100 shrimp samples collected from four different shrimp farms and fresh food markets around Bangkok. Samples were pre-enriched in non-selective lactose broth (LB) and selective tetrathionate broth (TTB). PCR detection limit was 10 pg and 10(4) cfu/ml of viable salmonellae with 100% specificity. PCR assay detected 19 different Salmonella serovars belonging to 8 serogroups (B, C1, C2-C3, D1, E1, E4 and K) commonly found in clinical and environmental samples in Thailand. The detection rate of PCR following TTB enrichment (24%) was higher than conventional culture method (19%). PCR following TTB, but not in LB enrichment allowed salmonella detection with 84% sensitivity, 90% specificity and 89% accuracy. Shrimp samples collected from fresh food markets had higher levels of contaminated salmonellae than those from shrimp farms. The results indicated that incorporation of an enrichment step prior to PCR has the potential to be applied for detection of naturally contaminated salmonellae in food, environment and clinical samples.