Dinesh Bhandari

Cyclospora Infection among School Children in Kathmandu, Nepal: Prevalence and Associated Risk Factors

Background: The intestinal coccidian protozoa Cyclospora cayetanensis has emerged as an important cause of parasitic diarrhea among children living in developing countries. This study aimed to determine the prevalence of Cyclospora among the school children of Kathmandu with reference to various associated risk factors. Methodology: A total of five hundred and seven stool samples from students between the age of 3–14 years, studying in 13 different schools in Kathmandu were collected during the study period (May–November, 2014) and processed at the Public Health Research Laboratory, Institute of Medicine, Kathmandu, Nepal. A modified acid fast staining technique (Kinyoun’s method) was used to detect oocyst of Cyclospora from the formal-ether concentrated stool samples. Results: Cyclospora was detected in 3.94% (20/507) of the stool samples examined. The prevalence was found to be highest among the students in the 3–5 year age group i.e. 10.15% (13/128), peaking during the rainy season (June–August). The detection rate was found to be significantly higher (p < 0.05) among children presenting with diarrheal symptoms, household keeping livestock and consumers of raw vegetables/fruits, showing a prevalence of 10.57% (11/104), 10.11% (9/89) and 7.25% (14/193) respectively. Conclusion: Consumption of untreated drinking water, fresh produce (raw fruits/vegetables) without proper washing and the presence of livestock at home were found to be predisposing factors for higher susceptibility of infection due to Cyclospora. This finding confirms the existence of a public-health issue with potentially serious consequences whereby children can be infected through exposure to oocysts in contaminated food and water and get ill as a result.

Next-generation sequencing identification of pathogenic bacterial genes and their relationship with fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal

Bacteriological analysis of drinking water leads to detection of only conventional fecal indicator bacteria. This study aimed to explore and characterize bacterial diversity, to understand the extent of pathogenic bacterial contamination, and to examine the relationship between pathogenic bacteria and fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal. Sixteen water samples were collected from shallow dug wells (n=12), a deep tube well (n=1), a spring (n=1), and rivers (n=2) in September 2014 for 16S rRNA gene next-generation sequencing. A total of 525 genera were identified, of which 81 genera were classified as possible pathogenic bacteria. Acinetobacter, Arcobacter, and Clostridium were detected with a relatively higher abundance (>0.1% of total bacterial genes) in 16, 13, and 5 of the 16 samples, respectively, and the highest abundance ratio of Acinetobacter (85.14%) was obtained in the deep tube well sample. Furthermore, the blaOXA23-like genes of Acinetobacter were detected using SYBR Green-based quantitative PCR in 13 (35%) of 37 water samples, including the 16 samples that were analyzed for next-generation sequencing, with concentrations ranging 5.3-7.5logcopies/100mL. There was no sufficient correlation found between fecal indicator bacteria, such as Escherichia coli and total coliforms, and potential pathogenic bacteria, as well as the blaOXA23-like gene of Acinetobacter. These results suggest the limitation of using conventional fecal indicator bacteria in evaluating the pathogenic bacteria contamination of different water sources in the Kathmandu Valley.

Potential for Bacteriophage Endolysins to Supplement or Replace Antibiotics in Food Production and Clinical Care

There is growing concern about the emergence of bacterial strains showing resistance to all classes of antibiotics commonly used in human medicine. Despite the broad range of available antibiotics, bacterial resistance has been identified for every antimicrobial drug developed to date. Alarmingly, there is also an increasing prevalence of multidrug-resistant bacterial strains, rendering some patients effectively untreatable. Therefore, there is an urgent need to develop alternatives to conventional antibiotics for use in the treatment of both humans and food-producing animals. Bacteriophage-encoded lytic enzymes (endolysins), which degrade the cell wall of the bacterial host to release progeny virions, are potential alternatives to antibiotics. Preliminary studies show that endolysins can disrupt the cell wall when applied exogenously, though this has so far proven more effective in Gram-positive bacteria compared with Gram-negative bacteria. Their potential for development is furthered by the prospect of bioengineering, and aided by the modular domain structure of many endolysins, which separates the binding and catalytic activities into distinct subunits. These subunits can be rearranged to create novel, chimeric enzymes with optimized functionality. Furthermore, there is evidence that the development of resistance to these enzymes may be more difficult compared with conventional antibiotics due to their targeting of highly conserved bonds.

Presence of Human Enteric Viruses, Protozoa, and Indicators of Pathogens in the Bagmati River, Nepal

Quantification of waterborne pathogens in water sources is essential for alerting the community about health hazards. This study determined the presence of human enteric viruses and protozoa in the Bagmati River, Nepal, and detected fecal indicator bacteria (total coliforms, Escherichia coli, and Enterococcus spp.), human-fecal markers (human Bacteroidales and JC and BK polyomaviruses), and index viruses (tobacco mosaic virus and pepper mild mottle virus). During a one-year period between October 2015 and September 2016, a total of 18 surface water samples were collected periodically from three sites along the river. Using quantitative polymerase chain reaction, all eight types of human enteric viruses tested—including adenoviruses, noroviruses, and enteroviruses, were detected frequently at the midstream and downstream sites, with concentrations of 4.4–8.3 log copies/L. Enteroviruses and saliviruses were the most frequently detected enteric viruses, which were present in 72% (13/18) of the tested samples. Giardia spp. were detected by fluorescence microscopy in 78% (14/18) of the samples, with a lower detection ratio at the upstream site. Cryptosporidium spp. were detected only at the midstream and downstream sites, with a positive ratio of 39% (7/18). The high concentrations of enteric viruses suggest that the midstream and downstream regions are heavily contaminated with human feces and that there are alarming possibilities of waterborne diseases. The concentrations of enteric viruses were significantly higher in the dry season than the wet season (p < 0.05). There was a significant positive correlation between the concentrations of human enteric viruses and the tested indicators for the presence of pathogens (IPP) (p < 0.05), suggesting that these IPP can be used to estimate the presence of enteric viruses in the Bagmati River water.

A hospital based surveillance of metallo-beta-lactamase producing gram negative bacteria in Nepal by imipenem-EDTA disk method

BackgroundA rising threat of the rapid spread of acquired metallo-beta-lactamases (MBLs) among major Gram-negative pathogens is a matter of public health concern worldwide. Hence, for a low income nation like Nepal, surveillance data on MBL producing clinical isolates via a cost effective technique is necessary to prevent their dissemination as well as formulation and regulation of antimicrobial stewardship policy.MethodsThe prospective study was conducted at Nepal Medical College, Kathmandu from May to October, 2014 to assess the prevalence of MBL production among ceftazidime-resistant Gram-negative rods (GNRs) isolates. The samples were processed according to standard microbiological procedure following the Manual of clinical Microbiology. Isolated GNRs were subjected to susceptibility testing against the selected panel of antibiotics by Kirby- Bauer disc diffusion method and interpretation made in conformity with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Ceftazidime-resistant isolates were subjected to the detection of MBL production by imipenem—EDTA combined disc (CD) method.ResultsAmong the Gram-negative isolates, 5.80% (21/362) were found to be MBL positive with Acinetobacter spp. showing the highest prevalence i.e. 85.71% (18/21), followed by P. aeruginosa i.e. 14.29% (3/21). None of the other cefazidime resistant gram negative bacteria tested were found to be positive for MBL production with all the positive isolates determined to be Multidrug resistant (MDR) strains.ConclusionThis study demonstrated a higher rate of resistance among P. aeruginosa and Acinetobacter spp. to a wide variety of antibiotic categories with an additional burden of MBL production within them, warranting a need for strict surveillance and rapid detection of MBL production among the GNRs.

Gut microbiome transition across a lifestyle gradient in Himalaya

The 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/modernization. Here, we characterize the stool bacterial composition of four Himalayan populations to investigate how the gut community changes in response to shifts in human lifestyles. These groups led seminomadic hunting-gathering lifestyles until transitioning to varying 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 microbiota and find that the gut microbiome composition is significantly associated with lifestyle. The Chepang foragers harbor elevated abundance of taxa associated with foragers around the world. Conversely, the gut microbiomes of 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. For example, our results show that 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 populations. 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 isolating dietary effects from other factors that change during modernization.

Validation of host‐specific Bacteroidales quantitative PCR assays and their application to microbial source tracking of drinking water sources in the Kathmandu Valley, Nepal

To validate host‐specific Bacteroidales assays to identify faecal‐source contamination of drinking water sources in the Kathmandu Valley, Nepal.

Microbiological profile of corneal ulcer cases diagnosed in a tertiary care ophthalmological institute in Nepal

BackgroundCorneal ulcer, a major cause of monocular blindness in developing countries has consistently been listed as the major cause of blindness and visual disability in many of the developing nations in Asia, Africa and the Middle East, ranking second only to cataract. This study was carried out to determine the microbiological profile of corneal ulcer cases diagnosed among patients visiting Tilganga Institute of Ophthalmology (TIO), Nepal.MethodsA total of 101 corneal scrapping samples were tested for routine culture and antibiotic susceptibility at the pathology department of TIO Nepal from April to October 2014. Microorganisms were identified by using standard microbiological procedures following the manual of American Society for Microbiology (ASM) and their antibiotic susceptibility test, performed by Kirby-Bauer disc diffusion method in conformity with the CLSI guideline.ResultsOut of 101 samples analyzed, 44.6% (45/101) showed positive growth with bacterial isolates i.e., 56% (25/45), more prevalent than fungus i.e., 44% (20/45). Among bacteria Streptococcus pneumoniae (31.1%, N = 14) was isolated in highest number whereas Fusarium (13.4%, N = 6) was the most common fungus species. Pseudomonas aeruginosa was the only Gram negative bacteria isolated from corneal ulcer cases. All bacterial isolates were found to be susceptible to the quinolone group of antibiotics (moxifloxacin followed by ofloxacin and ciprofloxacin).ConclusionsThese findings showcase the current trend in the microbiological etiology of corneal ulcer in Nepal, which have important public health implications for the treatment as well as prevention of corneal ulceration in the developing world.