Basu Dev Pandey

Molecular Epidemiology of Rotavirus Diarrhea among Children and Adults in Nepal: Detection of G12 Strains with P[6] or P[8] and a G11P[25] Strain

ABSTRACT In anticipation of a rotavirus vaccine in Nepal, this study was undertaken to determine the distribution of the G and P serotypes and electropherotypes of rotaviruses in order to examine if there is any emerging serotype or unusual strain circulating in children and adults in Nepal. Of 1,315 diarrheal stool specimens, rotavirus was detected by an enzyme-linked immunosorbent assay in 116 (17%) of 666 patients less than 5 years of age, in 18 (7%) of 260 patients 5 to 14 years of age, and in 19 (5%) of 358 patients 15 years of age and older. Approximately 75% of rotavirus diarrhea occurred in children less than 5 years of age. Approximately 70% of rotaviruses found in each of the three age groups belonged to serotype G1P[8]. Interestingly, there were 29 (20%) G12 rotaviruses carrying either P[8] or P[6] and one (0.7%) G11 rotavirus carrying an unusual P[25] genotype. RNA polyacrylamide gel electrophoresis discriminated 19 strains (electropherotypes), among which there were three codominant strains carrying G1P[8] and long RNA patterns. Five electropherotypes were discriminated among G12 rotaviruses, all of which had long RNA patterns. The fact that 20% of rotaviruses were G12 strains carrying either P[8] or P[6] and had multiple electropherotypes suggest that G12 strains are not more rare strains but that they pose an emerging challenge to current and future vaccines. The presence of multiple strains as defined by electropherotypes suggests the richness of the rotavirus gene pool in Nepal, where unusual strains may continue to emerge.

Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients

A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.

Molecular characterization of Blastocystis isolates from children and rhesus monkeys in Kathmandu, Nepal.

To investigate the possible transmission of Blastocystis organisms between local rhesus monkeys and children in Kathmandu, Nepal, we compared the subtype (ST) and sequence of Blastocystis isolates from children with gastrointestinal symptoms and local rhesus monkeys. Twenty and 10 Blastocystis isolates were established from 82 and 10 fecal samples obtained from children and monkeys, respectively. Subtype analysis with seven sequence-tagged site (STS) primers indicated that the prevalence of Blastocystis sp. ST1, ST2 and ST3 was 20%, 20% and 60% in the child isolates, respectively. In contrast to human isolates, ST3 was not found in monkey isolates and the prevalence of ST1 and ST2 was 50% and 70%, respectively, including three mixed STs1 and 2 and one isolate not amplified by any STS primers, respectively. Since Blastocystis sp. ST2 has been reported as the most dominant genotype in the survey of Blastocystis infection among the various monkey species, sequence comparison of the 150bp variable region of the small subunit rRNA (SSU rRNA) gene was conducted among ST2 isolates of humans and monkeys. Sequence alignment of 24 clones developed from ST2 isolates of 4 humans and 4 monkeys showed three distinct subgroups, defined as ST2A, ST2B and ST2C. These three subgroups were shared between the child and monkey isolates. These results suggest that the local rhesus monkeys are a possible source of Blastocystis sp. ST2 infection of humans in Kathmandu.

Detection of G12 human rotaviruses in Nepal

Of 731 stool specimens collected from children with diarrhea in Kathmandu, Nepal, from August 2004 through July 2005, 170 (23.3%) tested positive for rotavirus. Reverse transcription–PCR, including a revised G12-specific primer set, identified 56 (32.9%) as G2P[4] and 39 (23.0%) as G12 with P[6], P[8], or P[4].

Severity-related molecular differences among nineteen strains of dengue type 2 viruses.

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

The burden of parasitic zoonoses in Nepal: a systematic review

Background Parasitic zoonoses (PZs) pose a significant but often neglected threat to public health, especially in developing countries. In order to obtain a better understanding of their health impact, summary measures of population health may be calculated, such as the Disability-Adjusted Life Year (DALY). However, the data required to calculate such measures are often not readily available for these diseases, which may lead to a vicious circle of under-recognition and under-funding. Methodology We examined the burden of PZs in Nepal through a systematic review of online and offline data sources. PZs were classified qualitatively according to endemicity, and where possible a quantitative burden assessment was conducted in terms of the annual number of incident cases, deaths and DALYs. Principal Findings Between 2000 and 2012, the highest annual burden was imposed by neurocysticercosis and congenital toxoplasmosis (14,268 DALYs [95% Credibility Interval (CrI): 5450–27,694] and 9255 DALYs [95% CrI: 6135–13,292], respectively), followed by cystic echinococcosis (251 DALYs [95% CrI: 105–458]). Nepal is probably endemic for trichinellosis, toxocarosis, diphyllobothriosis, foodborne trematodosis, taeniosis, and zoonotic intestinal helminthic and protozoal infections, but insufficient data were available to quantify their health impact. Sporadic cases of alveolar echinococcosis, angiostrongylosis, capillariosis, dirofilariosis, gnathostomosis, sparganosis and cutaneous leishmaniosis may occur. Conclusions/Significance In settings with limited surveillance capacity, it is possible to quantify the health impact of PZs and other neglected diseases, thereby interrupting the vicious circle of neglect. In Nepal, we found that several PZs are endemic and are imposing a significant burden to public health, higher than that of malaria, and comparable to that of HIV/AIDS. However, several critical data gaps remain. Enhanced surveillance for the endemic PZs identified in this study would enable additional burden estimates, and a more complete picture of the impact of these diseases.

Molecular Epidemiology of Rotavirus Diarrhea among Children Aged <5 Years in Nepal: Predominance of Emergent G12 Strains during 2 Years

A 2-year surveillance was performed in Kathmandu, Nepal, by collection of stool specimens from 1139 children aged <5 years who were hospitalized for acute diarrhea from November 2005 through October 2007. Of the 1139 samples, 379 (33%) had rotavirus strains identified by enzyme-linked immunosorbent assay; the most prevalent G type was G12, accounting for 50% of typed strains in 2005-2006 and 29% in 2006-2007, followed by G1 (26%) in 2005-2006 and by G9 (28%) and G2 (20%) in 2006-2007. The most prevalent P type was P[8], accounting for 47% of strains in 2005-2006 and 35% in 2006-2007, followed by P[6] (37% in 2005-2006 and 33% in 2006-2007) and P[4] (10% in 2005-2006 and 24% in 2006-2007). Of combined genotypes, G12P[6] was the most prevalent, accounting for 34% of strains in 2005-2006 and 24% in 2006-2007, followed by G1P[8] (23%) in 2005-2006 and G2P[4] (20%) in 2006-2007. An unusually high detection of G12 strains underscores the importance of continued surveillance of rotavirus strains.

Perceived Family Support, Depression, and Suicidal Ideation among People Living with HIV/AIDS: A Cross- Sectional Study in the Kathmandu Valley, Nepal

Background Depression and suicidal thinking occur frequently alongside HIV/AIDS, triggering profound detrimental impacts on quality of life, treatment adherence, disease progression, and mortality. Yet the psychosocial factors contributing to these psychiatric comorbidities remain underexplored, particularly in the developing country context. This study thus examined different dimensions of perceived family support in relation to depression and suicidal ideation among people living with HIV/AIDS (PLWHA) in Nepal. Methods A cross-sectional survey of 322 adult PLWHA residing in the Kathmandu Valley, Nepal was conducted. Data were analyzed using multiple logistic regressions for correlates of Beck Depression Inventory (BDI)-Ia-defined depressive symptoms and suicidal ideation in the past 2 weeks. Perceived family support, measured using the 10-item Nepali Family Support and Difficulty Scale, was entered into separate models, in turn, as a composite score, for each sub-scale (emotional, instrumental, and negative support), and for each individual item. Results Overall, 25.5% of participants registered BDI-Ia-defined depression, with significantly lower rates among those with perceived family support scores in the highest (AOR = 0.19; 95% CI = 0.07, 0.55) and middle (AOR = 0.38; 95% CI = 0.17, 0.86) tertiles relative to those with lowest-tertile scores. Meanwhile, 14.0% reported suicidal thinking, with significantly lower rates among those in the highest perceived family support tertile relative to the lowest (AOR = 0.25; 95% CI = 0.07, 0.91). Broken down by support sub-scale, only negative support (i.e. family difficulty) was significant in its correlations with both outcomes – a trend similarly reflected in the item-wise analyses. Conclusions Our findings highlight an important role for family support in determining experiences of depression and suicidality among PLWHA. Incorporating family counseling and support services – with special focus on ameliorating negative interaction and bolstering emotional support – into HIV care and treatment services may help to improve mental health along with overall wellness and treatment outcomes for HIV-positive populations in Nepal and similar settings.

Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolated in Nepal

ABSTRACT Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.

Physicians are a key to encouraging cessation of smoking among people living with HIV/AIDS: a cross-sectional study in the Kathmandu Valley, Nepal

BackgroundHIV care providers may be optimally positioned to promote smoking behaviour change in their patients, among whom smoking is both highly prevalent and uniquely harmful. Yet research on this front is scant, particularly in the developing country context. Hence, this study describes smoking behaviour among people living with HIV/AIDS (PLWHA) in the Kathmandu Valley of Nepal, and assesses the association between experience of physician-delivered smoking status assessment and readiness to quit among HIV-positive smokers.MethodsWe conducted a cross-sectional survey of PLWHA residing in the Kathmandu Valley, Nepal. Data from 321 adult PLWHA were analyzed using multiple logistic regression for correlates of current smoking and, among current smokers, of motivational readiness to quit based on the transtheoretical model (TTM) of behaviour change.ResultsOverall, 47% of participants were current smokers, with significantly higher rates among men (72%), ever- injecting drug users (IDUs), recent (30-day) alcohol consumers, those without any formal education, and those with higher HIV symptom burdens. Of 151 current smokers, 34% were thinking seriously of quitting within the next 6 months (contemplation or preparation stage of behaviour change). Adjusting for potential confounders, experience of physician-delivered smoking status assessment during any visit to a hospital or clinic in the past 12 months was associated with greater readiness to quit smoking (AOR = 3.34; 95% CI = 1.05,10.61).ConclusionsRoughly one-third of HIV-positive smokers residing in the Kathmandu Valley, Nepal, are at the contemplation or preparation stage of smoking behaviour change, with rates significantly higher among those whose physicians have asked about their smoking status during any clinical interaction over the past year. Systematic screening for smoking by physicians during routine HIV care may help to reduce the heavy burden of smoking and smoking-related morbidity and mortality within HIV-positive populations in Nepal and similar settings.