Multiplex PCR for bacterial, viral and protozoal pathogens in persistent diarrhoea or persistent abdominal pain in Côte d'Ivoire, Mali and Nepal.

In contrast to acute diarrhoea, the aetiology of persistent digestive disorders (≥ 14 days) is poorly understood in low-resource settings and conventional diagnostic approaches lack accuracy. In this multi-country study, we compared multiplex real-time PCR for enteric bacterial, parasitic and viral pathogens in stool samples from symptomatic patients and matched asymptomatic controls in Côte d'Ivoire, Mali and Nepal. Among 1826 stool samples, the prevalence of most pathogens was highest in Mali, being up to threefold higher than in Côte d'Ivoire and up to tenfold higher than in Nepal. In all settings, the most prevalent bacteria were EAEC (13.0-39.9%) and Campylobacter spp. (3.9-35.3%). Giardia intestinalis was the predominant intestinal protozoon (2.9-20.5%), and adenovirus 40/41 was the most frequently observed viral pathogen (6.3-25.1%). Significantly different prevalences between symptomatic and asymptomatic individuals were observed for Campylobacter, EIEC and ETEC in the two African sites, and for norovirus in Nepal. Multiple species pathogen infection was common in Côte d'Ivoire and Mali, but rarely found in Nepal. We observed that molecular testing detected multiple enteric pathogens and showed low discriminatory accuracy to distinguish between symptomatic and asymptomatic individuals. Yet, multiplex PCR allowed for direct comparison between different countries and revealed considerable setting-specificity.

Urinary Calculi: A Microbiological and Biochemical Analysis at a Tertiary Care Hospital in Eastern Nepal.

BACKGROUND: The occurrence of urinary tract infection in presence of urolithiasis is frequently noted; however, microbial agents of urolithiasis and their antimicrobial susceptibility patterns remain underinvestigated. This study aimed to identify the microorganisms isolated from urine and stone matrices to determine their antimicrobial susceptibility, to find the association between the pathogens of urine and stone matrices, and to perform the biochemical analysis of stones. METHODS: A total of 88 cases of urolithiasis admitted for elective stone removal at Department of surgery, B.P. Koirala Institute of Health Sciences (BPKIHS), were enrolled. Preoperative urine culture and postoperative stone culture were performed. Isolation, identification, and AST were done by the standard microbiological technique. Further qualitative biochemical analysis of stones was also attempted. RESULT: Among 88 stone formers recruited, culture of urine, whole stone, and nidus yielded the growth of bacteria 44, 32, and 30, respectively. Bacteria isolated from urine culture correlated with those from stone matrices with a sensitivity of 90%, specificity of 79.69%, PPV of 63.64%, and NPV of 95.45%. Escherichia coli (46.7%) was the most common bacteria followed by Klebsiella pneumoniae (16.7%) and Proteus mirabilis (13.3%) from urine and stone cultures. Almost all the uropathogens isolated were susceptible to commonly used antibiotics. Calcium oxalate (84.1%) was common biochemical constituent found in stone formers followed by calcium oxalate + phosphate (8%). CONCLUSIONS: The association of microorganism isolated from urine and nidus culture was significant that can predict the source of infective stone; however, in some cases, microorganisms and the antimicrobial susceptibility pattern from urine and nidus were different. This study emphasizes the use of appropriate antimicrobial agents to prevent the regrowth of residual stones and minimize the risk of infectious complications after surgical removal of stones.

Application of PCR and Microscopy to Detect Helicobacter pylori in Gastric Biopsy Specimen among Acid Peptic Disorders at Tertiary Care Centre in Eastern Nepal.

BACKGROUND: Helicobacter pylori infection is most prevalent in developing countries. It is an etiological agent of peptic ulcer, gastric adenocarcinoma, and mucosal-associated lymphoid tissue (MALT) lymphoma. Despite the development of different assays to confirm H. pylori infection, the diagnosis of infection is challenged by precision of the applied assay. Hence, the aim of this study was to understand the diagnostic accuracy of PCR and microscopy to detect the H. pylori in the gastric antrum biopsy specimen from gastric disorder patients. METHODS: A total of 52 patients with gastric disorders underwent upper gastrointestinal endoscopy with biopsy. The H. pylori infection in gastric biopsies was identified after examination by microscopy and 23S rRNA specific PCR. The agreement between two test results were analysed by McNemar's test and Kappa coefficient. RESULT: H. pylori infection was confirmed in 9 (17.30%) patients by both assays, 6.25% in antral gastritis, 22.22% in gastric ulcer, 100% in gastric ulcer with duodenitis, 50% in gastric ulcer with duodenal ulcer, and 33.33% in severe erosive duodenitis with antral gastritis. Out of nine H. pylori infection confirmed patients, 3 patients were confirmed by microscopy and 8 patients by PCR. In case of two patients, both microscopy and PCR assay confirmed the H. pylori infection. The agreement between two test results was 86.54% and disagreed by 13.46% (p value > 0.05). CONCLUSION: We found that PCR assay to detect H. pylori is more sensitive than microscopy. However, we advocate for the combination of both assays to increase the strength of diagnostic accuracy due to the absence of the gold standard assay for H. pylori infection.

PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar.

OBJECTIVE: To compare a PCR assay and direct agglutination test (DAT) for the detection of potential markers of Leishmania infection in 231 healthy subjects living in a kala-azar endemic focus of Nepal. METHODS: The sample was composed of 184 (80%) persons without any known history of KA and not living in the same house as known kala-azar cases (HNK), 24 (10%) Healthy Household Contacts (HHC) and 23 (10%) past kala-azar cases which had been successfully treated (HPK). RESULTS: PCR and DAT positivity scores were, respectively: HNK, 17.6% and 5.6%; HHC, 12.5% and 20.8%; HPK, 26.1% and 95.7%. The ratio PCR-positives/DAT-positives was significantly higher in HNK (ratio = 3.1) than in HHC (ratio = 0.6, P = 0.036) and in HPK (ratio = 0.2, P = 0.012). The ratio PCR-positives/DAT-positives did not significantly differ between HHC (ratio = 0.6) and HPK (ratio = 0.2, P = 0.473). The positive agreement index between PCR and DAT in HNK was 5%; in HHC, 0%; in HPK, 43%. CONCLUSIONS: Our study highlights the specific character of PCR and DAT for the exploration of Leishmania asymptomatic infections. PCR is probably more informative for very recent infections among HNK, while DAT provides more information among HHC and HPK, a feature likely related to the power of serology to track less recent infections.

Epidemiological dynamics of antimonial resistance in Leishmania donovani: genotyping reveals a polyclonal population structure among naturally-resistant clinical isolates from Nepal.

Pentavalent antimonials (SbV) are the first line drug against leishmaniasis worldwide, but drug resistance is increasingly reported, particularly in the Indian sub-continent, where it represents a major threat for the control of anthroponotic visceral leishmaniasis (VL). In order to understand the epidemiological dynamics of antimonial resistance in anthroponotic VL, we analysed here the population structure of 24 Leishmania donovani stocks isolated from anthroponotic VL-patients from Eastern Nepal: 13 SbV-naturally resistant and 11 SbV-sensitive, as demonstrated by in vitro drug susceptibility assays. The parasites were genotyped by PCR-RFLP analysis of kDNA minicircles and by microsatellite analysis and the encountered polymorphism revealed a polyclonal structure among resistant isolates. Furthermore, analysis of paired samples obtained from the same patients before treatment and after failure revealed primary as well as acquired resistance. The hypothesis of independent events of drug resistance emergence is proposed and confronted to alternative explanations. Our results show the dynamics of drug resistance epidemiology and highlight the importance of surveillance networks.

Chromoblastomycosis: report of two cases from Nepal.

Chromoblastomycosis is a chronic fungal infection of the skin and subcutaneous tissue caused by dematiaceous fungi. The first case, a 67-year-old male farmer, presented with itchy hyperkeratotic, scaly plaques with scarring and black dots on the lateral aspects of his left arm and dorsum of his left hand of 28 years duration. The case was clinically diagnosed as chromoblastomycosis. The second case, a 75-year-old farmer, presented with erythematous, crusted, scaly plaques on the dorsum of the left foot of 30 years duration. Initially, a clinical diagnosis of lupus vulgaris was made, but treatment with anti tuberculosis therapy showed no improvement. On the basis of histopathological examinations of skin biopsies and isolation of fungus on culture, both cases were diagnosed as chromoblastomycosis. To the best of our knowledge, these two cases are the first case reports of chromoblastomycosis from Nepal and are presented for their academic interest.