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

AIMS: To validate host-specific Bacteroidales assays to identify faecal-source contamination of drinking water sources in the Kathmandu Valley, Nepal. METHODS AND RESULTS: A total of 54 composite faecal-source samples were collected from human sewage, ruminants, pigs, dogs, chickens and ducks, which were analysed by quantitative polymerase chain reaction using human-specific (BacHum, HF183 SYBR, gyrB and HF183 TaqMan), ruminant-specific (BacCow and BacR), pig-specific (Pig2Bac and PF163) and dog-specific assays (BacCan SYBR). The BacHum, BacR and Pig2Bac assays were judged the best performing human-specific, ruminant-specific and pig-specific assays respectively. The BacCan SYBR assay highly cross-reacted with other species, resulting in poor performance. Furthermore, these validated assays were applied to microbial source tracking (MST) of 74 drinking water samples. Out of these, 20, 12 and 4% samples were judged contaminated by human, ruminant and pig faeces respectively. Detection ratios of human and ruminant faecal markers were relatively higher in built-up and agricultural areas respectively. CONCLUSION: BacHum, BacR and Pig2Bac assays were found suitable for MST and both, human and animal faecal contaminations of drinking water sources were common in the valley. SIGNIFICANCE AND IMPACT OF THE STUDY: MST could be an effective tool for preparing the faecal pollution strategies as these are site specific.

A Rare Case of Port-a-Cath Migration into the Mediastinum.

Port-a-cath is commonly used in patients who require frequent administration of intravenous medications. We describe a rare case of port-a-cath migration into the mediastinum 16 months after its insertion.

Enteric opportunistic parasitic infections among HIV seropositive patients in Kathmandu, Nepal.

BACKGROUND: Enteric opportunistic parasitic infections are the major source of diarrheal disease in developing countries mainly in Human Immunodeficiency virus (HIV) infected patients. OBJECTIVE: The study was to detect enteric parasites causing diarrhea and their association with immune status in HIV-seropositive patients. METHODS: The present study was conducted in Dirgh-Jeevan Health Care Research Center and Tribhuvan University Teaching Hospital, Public Health Research Laboratory, Kathmandu, Nepal between June 2010 and May 2011 involving 146 Human Immunodeficiency virus (HIV) positive patients. Serostatus from these patients were detected by Enzyme Linked Immunosorbent assay. CD4+ T cell counts were done by flow cytometry. Stool was examined for enteric parasites by microscopy with special staining methods. RESULTS: A total of 146 HIV sero-positive patients with and without diarrhea age between 20 to 45 years were included in the study. Of the 146 patients, the protozoan parasitic infection was found in 30.13% (44/146). Out of 146 patients, 78 had diarrhea in which parasitic infection was 39 (50%) and 7.35% (5/68) protozoal parasites positive cases did not have diarrhea. A significant difference (p less than 0.05) was observed in the level of infection of intestinal protozoan between the HIV seropositive with diarrhea and HIV-seropositive without diarrhea. Out of 43 patients whose CD4+ T cells were less than 200/µl, 29 (67.4%) had opportunistic parasitic infection whereas out of 103 patients whose CD4+ T cells were =200/mcl, only 15 (14.56%) had opportunistic parasitic infection (P less than 0.05). CONCLUSION: Enteric opportunistic parasitic infections were detected in 30.1% among HIV-seropositive patients and low CD4+ T count indicated high enteric opportunistic infection. Early detection of enteric parasitic infections will help in the management and to improve the quality of life for HIV-infected individuals.

Hospital-based study in children with rotavirus gastroenteritis and other enteropathogens.

BACKGROUND: Rotavirus is the most common cause of life threatening gastroenteritis in infants and young children in the world. The objective of the study is to find out current trends and incidents of rotavirus, including other enteropathogens related with children diarrhoea and lastly identify the most common rotavirus serotypes that circulate in Nepal. METHODS: A total of 1721 stool samples from less than 5 years of children were collected. Rotavirus in the stool samples were detected by Enzyme Immuno Assay (EIA) and strains were genotyped by Reverse-Transcription Polymerase Chain Reaction (RT-PCR). Bacteria and parasites were detected by following standard microbiological procedures. RESULTS: In between 2009 to 2010, of the total 1721, the prevalence of rotavirus was 24.7%. Of them, 906 (52.6%) were collected in the year 2009 and 815(47.5%) in the year 2010. Rotavirus was frequently detected in inpatients (31.6%) than outpatient (16.8%). Rotavirus detection was higher in female (26.4%) than male (23.7%). The prevalence was seen higher in age group 0-23 months in both years. Among six different bacterial isolates, Escherichia coli was most frequently isolated (6.5%). Similarly, Giardia lamblia (1.3%) was most common among six different parasites detected. A total of rotavirus positive 425 stool samples were detected over 2 years (2009-230, and 2010-195), G12P6 was the predominant strain circulating in both (45% in 2009 and 28% in 2010) years. G9P6 emerged in 2010 (6%). There were significant numbers of mixed infections (14.0% in 2009 and 29.8% in 2010). Thirty five samples were partially typed and 15 were completely untyped over the two year period. CONCLUSIONS: The study helps comprehend the prevalence of rotavirus along with other intestinal pathogens including bacteria and parasites. Major genotypes of rotavirus are also introduced in the study.

Bacterial etiology of acute diarrhea in children under five years of age.

BACKGROUND: Diarrheal diseases are major problem of developing countries. Though precise data on childhood mortality associated with diarrheal diseases in Nepal is not available, it has been estimated that approximately 25% of child death are associated with diarrheal disease, particularly acute diarrhea. The purpose of this study was to assess the incidence of bacterial pathogens causing acute diarrhea in children under 5 years of age. METHODS: A total of 525 children with acute diarrhea in a children's hospital of Kathmandu, Nepal were enrolled between April 2011 to September 2011. Feacal specimens for culture were inoculated to the several media. The organisms were identified by different biochemical tests and serotyping. Their antibiotic sensitivity tests were performed by Kirby-Bauer's disc diffusion method as recommended by CLSI. RESULTS: Out of total 525 enrolled cases bacterial infection was found to be 46 (8.8%). Bacterial infection was found to be of highest, 36 (78.3%) in the age group between 6-24 months. Among the total enrolled cases the prevalence of Shigella species was 24 (4.6%) followed by Escherichia coli 12 (2.3%) and Salmonella species 10 (1.9%). Chloramphenicol and Tetracycline showed efficacy in 9 (90.0%) isolates of Salmonella species, Gentamycin showed efficacy in 22 (91.7%) isolates of Shigella species and Chloramphenicol showed 100% efficacy against Escherichia coli whereas 7 (70.0%) isolates of Salmonella species were resistant to ampicillin in vitro. MDR was highest 7 (70.0%) in Salmonella species. CONCLUSIONS: The bacterial pathogens were found to be a significant cause of acute diarrhea. The most common causative organism for acute diarrhea were Shigella spp. Awareness of improving hygiene and infectious diseases may reduce the burden of infection.

Antimicrobial susceptibility pattern and serotyping of Streptococcus pneumoniae isolated from Kanti Children Hospital in Nepal.

BACKGROUND: Invasive pneumococcal disease is a significant cause of morbidity and mortality worldwide and it is a major cause for childhood deaths in Nepal. OBJECTIVES: The aim of this study was to establish the antimicrobial susceptibility pattern of Streptococcus pneumoniae and perform serotype responsible for pneumococcal disease in Nepal. MATERIALS AND METHODS: All together 3774 children from 2 to 60 months who fulfilled the enrollment criteria for suspect of bacterial pneumonia, sepsis or meningitis were enrolled for etiologic studies of severe illness. During the study period 60 isolates of Streptococcus pneumoniae were isolated and the antimicrobial susceptibility testing and serotyping were performed. RESULTS: The study showed that 24 (52. 17%) isolates were resistant to Cotrimoxazole, 3 (6. 5%) isolates were intermediately resistant to Penicillin but no Penicillin resistant strains were isolated. The 1 (2. 17%) isolate was recorded as Erythromycin and Chloramphenicol resistant and only 1 (2. 17%) isolate was found intermediately resistant to Cefotaxime. Of the 60 isolates, serotyping result was available only for 46 isolates. The most common serotypes were serotype 1 (27. 65%) followed by serotype 5 (19. 14%) and serotype 4 (8. 5%) respectively followed by serotype 39, 23F, 7F, 19B, 12A, 14, 18F, 6B, 32, 16, 19F and 25F. CONCLUSIONS: Alarming level of Cotrimoxazole resistance demands revision of pneumonia treatment policy in Nepal and rising tendency of other drug resistance against Streptococcus pneumoniae showed use of these drugs for the treatment of meningitis, pneumonia and other serious infections needs extended research. The common serotype 1, 5 and 4 need to be incorporated in pneumococcal vaccine to immunise children in Nepal.