Detection of Cryptosporidium, Giardia, fecal indicator bacteria, and total bacteria in commercial jar water in Kathmandu Valley, Nepal

Introduction: Jar water is a convenient and common source of drinking water in the Kathmandu Valley. However, studies including detailed microbial analyses of this source of potable water are lacking. In this study, jar water samples were examined for the occurrence of Cryptosporidium, Giardia, fecal indicator bacteria, and total bacteria. Methods: Thirty different brands of jars were collected in September 2014. Escherichia coli and total coliforms w ere determined using a Colilert reagent. Ten of the 30 brands w ere selected to test for Cryptosporidium, Giardia, and total bacteria. Bacterial DNA extraction from water samples w as performed using the Cica Geneus DNA Extraction Kit, follow ed by quantitative polymerase chain reaction (qPCR) targeting the 16S rRNA gene of bacterial DNA. Protozoan detection was accomplished by concentrating the samples using the electronegative membrane vortex method, followed by immunomagnetic separation and fluorescent staining. Results: E. coli w as detected in 10% of the samples, with a maximum concentration of 2 most probable number (MPN)/100 mL, whereas total coliforms were detected in 97% of the samples, with a maximum and mean concentration of 7.3 × 10 2 and 3.8 × 10 1 MPN/100 mL, respectively. Total coliforms concentrations in 40% of the samples ranged from 10 2 to 10 3 MPN/100 mL. Cryptosporidium and Giardia w ere not detected in any of the tested samples. Concentrations of total bacteria in the samples ranged from 10 4 to 10 6 cells/100 mL. Conclusions: Ninety-seven percent of the jar water brands were unsuitable for drinking without proper treatment based on the guideline values of the National Drinking Water Quality Standards (NDW Q S) of Nepal. There is no guideline value for total bacteria in NDW Q S however, high concentrations can be indicative of poor control on regrowth of bacteria and recontamination or inefficient water treatment methods.Malla B, Ghaju Shrestha R, Bhandari D, Tandukar S, Shrestha S, Yoshinaga H, Inoue D, Sei K, Nishida K, Tanaka Y, Sherchand JB, Haramoto

Gut microbiome transition across a lifestyle gradient in Himalaya

Published: November 15, 2018The composition of the gut microbiome in industrialized populations differs from those living traditional lifestyles. However, it has been difficult to separate the contributions of human genetic and geographic factors from lifestyle. Whether shifts away from the foraging lifestyle that characterize much of humanity's past influence the gut microbiome, and to what degree, remains unclear. Here, we characterize the stool bacterial composition of four Himalayan populations to investigate how the gut community changes in response to shifts in traditional human lifestyles. These groups led seminomadic hunting-gathering lifestyles until transitioning to varying levels of agricultural dependence upon farming. The Tharu began farming 250-300 years ago, the Raute and Raji transitioned 30-40 years ago, and the Chepang retain many aspects of a foraging lifestyle. We assess the contributions of dietary and environmental factors on their gut-associated microbes and find that differences in the lifestyles of Himalayan foragers and farmers are strongly correlated with microbial community variation. Furthermore, the gut microbiomes of all four traditional Himalayan populations are distinct from that of the Americans, indicating that industrialization may further exacerbate differences in the gut community. The Chepang foragers harbor an elevated abundance of taxa associated with foragers around the world. Conversely, the gut microbiomes of the populations that have transitioned to farming are more similar to those of Americans, with agricultural dependence and several associated lifestyle and environmental factors correlating with the extent of microbiome divergence from the foraging population. The gut microbiomes of Raute and Raji reveal an intermediate state between the Chepang and Tharu, indicating that divergence from a stereotypical foraging microbiome can occur within a single generation. Our results also show that environmental factors such as drinking water source and solid cooking fuel are significantly associated with the gut microbiome. Despite the pronounced differences in gut bacterial composition across populations, we found little differences in alpha diversity across lifestyles. These findings in genetically similar populations living in the same geographical region establish the key role of lifestyle in determining human gut microbiome composition and point to the next challenging steps of determining how large-scale gut microbiome reconfiguration impacts human biology.Aashish R. Jha, Emily R. Davenport, Yoshina Gautam, Dinesh Bhandari, Sarmila Tandukar, Katharine M. Ng, Gabriela K. Fragiadakis, Susan Holmes, Guru Prasad Gautam, Jeff Leach, Jeevan Bahadur Sherchand, Carlos D. Bustamante, Justin L. Sonnenbur

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 × 10(-5)M) was the most effective anti-mycobacterial agent; clofazimine (1.25 × 10(-5)M and 3.13 × 10(-6)M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 × 10(-5)M) was filaricidal, but minocycline was more effective and at a lower concentration (5 × 10(-5)M and 1.25 × 10(-5)M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode

Epidemiology and determinants of vitamin D deficiency in Eastern Nepal: a community-based, cross-sectional study

AbstractObjectiveTo estimate the prevalence of vitamin D deficiency in the Eastern the part of Nepal and identify the sociodemographic factors associated with it.MethodsA descriptive cross-sectional study was carried out among 324 participants between the ages of 18 to 65 years from the Sunsari and Morang districts of Nepal. A semi-structured questionnaire helped obtain sociodemographic data followed by anthropometric measurements and blood sampling. 25(OH)D level was measured by Chemiluminescence immunoassay (CLIA) via a fully automated Maglumi 1000 analyzer (SNIBE Co, Ltd, China). Serum 25(OH)D was classified as deficient, insufficient, and sufficient (&lt;20 ng/ml, 20-29 ng/ml, and 30–100 ng/ml respectively). The Chi-square test was used to analyze the sociodemographic variables followed by a post-hoc analysis. Significant variables were subject to multivariate logistic regression.Result181(55.9%) of the study population had vitamin D deficiency. There was significant association between vitamin D status and time of maximum sun exposure (Chi-sq = 11.1, p=0.02), duration of sun exposure (Chi-sq = 15.1, p=0.004), type of meat intake (Fischer’s exact test is 16.4, p=0.01), frequency of fish intake (Fischer’s exact test is 19.3, p=0.001), frequency of dairy intake (Chi-sq=11.2, p=0.02). In multivariate regression, consumption of dairy products ≥3/week had lower OR (95% CI) [0.3(0.1-0.8) p:0.02] and weekly fish consumption had lower OR (95% CI) [0.06(0.008-0.6) p: 0.01] for vitamin D deficiency.ConclusionThe prevalence of vitamin D deficiency was relatively high in eastern Nepal. This highlights the need to create public awareness regarding the importance of bare skin sun exposure and other sources of vitamin D as well as the need to implement food fortification policies by the government.</jats:sec

Epidemiology and Determinants of Vitamin D Deficiency in Eastern Nepal: A Community-Based, Cross-Sectional Study

Objective. To estimate the prevalence of vitamin D deficiency in the eastern part of Nepal and identify the sociodemographic factors associated with it. Methods. A descriptive cross-sectional study was carried out among 324 participants between the ages 18 and 65 years from the Sunsari and Morang districts of Nepal. A semi-structured questionnaire helped obtain sociodemographic data followed by anthropometric measurements and blood sampling. 25(OH)D level was measured by chemiluminescence immunoassay (CLIA) via a fully automated Maglumi 1000 analyzer (SNIBE Co., Ltd., China). Serum 25(OH)D was classified as deficient, insufficient, and sufficient (<20 ng/ml, 20–29 ng/ml, and 30–100 ng/ml, respectively). The chi-square test was used to analyze the sociodemographic variables followed by a post hoc analysis. Significant variables were subjected to multivariate logistic regression. Result. 181(55.9%) of the study population had vitamin D deficiency. There was significant association between vitamin D status and time of maximum sun exposure (chi square test = 11.1, p=0.02), duration of sun exposure (chi-square test = 15.1, p=0.004), type of meat intake (Fisher’s exact test is 16.4, p=0.01), frequency of fish intake (Fisher’s exact test is 19.3, p=0.001), and frequency of dairy intake (chi-square test=11.2, p=0.02). In multivariate regression, consumption of dairy products ≥3/week had lower OR (95% CI) (0.3(0.1–0.8) p:0.02) and weekly fish consumption had lower OR (95% CI) (0.06(0.008–0.6) p:0.01) for vitamin D deficiency. Conclusion. The prevalence of vitamin D deficiency was relatively high in eastern Nepal. This highlights the need to create public awareness regarding the importance of bare skin sun exposure, nutritional sources of vitamin D, and the need to implement food fortification policies by the government