The challenges and successes of implementing a sustainable antimicrobial resistance surveillance programme in Nepal.

BACKGROUND: Antimicrobial resistance (AMR) is a major global public health concern and its surveillance is a fundamental tool for monitoring the development of AMR. In 1998, the Nepalese Ministry of Health (MOH) launched an Infectious Disease (ID) programme. The key components of the programme were to establish a surveillance programme for AMR and to develop awareness among physicians regarding AMR and rational drug usage in Nepal. METHODS: An AMR surveillance programme was established and implemented by the Nepalese MOH in partnership with the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) from 1998 to 2003. From 2004 to 2012, the programme was integrated and maintained as a core activity of the National Public Health Laboratory (NPHL) and resulted in an increased number of participating laboratories and pathogens brought under surveillance. The main strategies were to build national capacity on isolation, identification and AMR testing of bacterial pathogens, establish laboratory networking and an External Quality Assessment (EQA) programme, promote standardised recording and reporting of results, and to ensure timely analysis and dissemination of data for advocacy and national policy adaptations. The programme was initiated by nine participating laboratories performing AMR surveillance on Vibrio cholerae, Shigella spp., Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae. RESULTS: The number of participating laboratories was ultimately increased to 13 and the number of pathogens under surveillance was increased to seven (Salmonella spp. was added to the surveillance programme in 2002 and extended spectrum ß-lactamase producing Escherichia coli in 2011). From 1999 to 2012, data were available on 17,103 bacterial isolates. During the AMR programme, we observed changing trends in serovars/species for Salmonella spp., Shigella spp. and V. cholerae and changing AMR trend for all organisms. Notably, N. gonorrhoeae isolates demonstrated increasing resistance to ciprofloxacin. Additionally, the performance of the participating laboratories improved as shown by annual EQA data evaluation. CONCLUSIONS: This Nepalese AMR programme continues and serves as a model for sustainable surveillance of AMR monitoring in resource limited settings.

Phenotypic and genetic characterization of Vibrio cholerae O1 clinical isolates collected through national antimicrobial resistance surveillance network in Nepal.

Cholera occurs in sporadic cases and outbreaks in Nepal each year. Vibrio cholerae O1 (n = 522) isolated during 2007-2010 from diarrheal patients at 10 different hospital laboratories in Nepal were characterized. Biochemical and serologic identifications showed that all the isolates belonged to serogroup O1, El Tor biotype. Except 72 isolates of Inaba serotype isolated in the year 2007, all the remaining isolates were of Ogawa serotype. All isolates were resistant to nalidixic acid and furazolidone. Resistance to tetracycline, ciprofloxacin, erythromycin and co-trimoxazole were 21, 4, 16 and 90 % respectively. Seventy-seven of these isolates were selected for further characterization for ctxB gene and MLVA typing. Two different variants of classical type cholera toxin were observed. Ogawa strains from 2007 and 2010-Western Nepal outbreak harbored CTX-3 type cholera toxin, whereas Inaba serotypes in 2007 and the remaining Ogawa serotypes in 2008-2010 harbored CTX 3b-type toxin. MLVA analysis showed circulation of four different groups of altered V. cholerae O1 El Tor strains. Two different profiles were seen among 2007 Inaba (9, 3, 6, x, x) and Ogawa (10, 7, 6, x, x) isolates. The MLVA profile of 2008 and 2009 Ogawa isolates were similar to those of Inaba strains of 2007. Isolates from 2010 also showed three different MLVA profiles; profile 9, 3, 6, x, x in 3 isolates, 11, 7, 6, x, x among 2010 Western Nepal outbreak strains and profile 8, 3, 6, x, x among isolates from Butwal and Kathmandu.

Emergence and properties of fluoroquinolone resistant Salmonella enterica serovar Typhi strains isolated from Nepal in 2002 and 2003.

A total of 171 Salmonella enterica serovar Typhi strains isolated from Nepal, mostly from patients with typhoid fever in 2002-2003, were tested for antimicrobial susceptibility by disk diffusion assay. Selected S. enterica serovar Typhi isolates were tested for MICs by E-test for ceftriaxone, ciprofloxacin and ofloxacin. Mutations of DNA gyrase gyrA and gyrB and topoisomerase IV parC and parE were identified by sequencing of PCR amplicons. By disk diffusion assay, 75/171 S. enterica serovar Typhi isolates were resistant to nalidixic acid, ampicillin, choramphenicol, streptomycin, tetracycline, sulfisoxazole, and trimethroprim/sulfamethoxazoles. Multiple drug resistance to the 7 antimicrobials was most predominant among S. enterica serovar Typhi isolates in this study. Resistance to nalidixic acid was detected in 76/111 and 56/60 of total isolates collected in 2002 and 2003, respectively. Nalidixic acid-resistant isolates in 2002 and 2003 showed MIC range for ciprofloxacin of 0.125-0.250 mg/l. Nalidixic acid-resistant isolates contained point mutations in gyrA and parC but not gyrB and parE. The gyrA mutation of nalidixic acid-resistant isolates obtained in 2002 and 2003 had amino acid substitution at position 83 of Serine-->Tyrosine and Serine-->Phenylalanine, respectively. Two different mutations of gyrA were detected among nalidixic acid-resistant isolates. Thus it is necessary to monitor mutation in DNA topoisomerase associated with increases in quinolones resistance.

Typhoid fever: a massive, single-point source, multidrug-resistant outbreak in Nepal.

BACKGROUND: In the summer of 2002, a total of 5963 cases of typhoid fever were recorded in Bharatpur, Nepal (population, 92,214) during a 7-week period. A team from the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, and the CIWEC Travel Medicine Clinic (Kathmandu, Nepal) assisted the Nepal National Public Health Laboratory (Kathmandu, Nepal) in the further investigation of this large, explosive febrile disease outbreak. METHODS: Investigators conducted a thorough epidemiologic and laboratory investigation to assess the size and scope of the outbreak. In addition to subculturing of previously collected samples, blood samples were obtained from 100 febrile patients, and culture and susceptibility testing were done by standard laboratory methods. Pulsed field gel electrophoresis (PFGE) and plasmid analysis were done. RESULTS: The majority of the isolates, including 1 from the municipal water supply, were multidrug resistant. The minimum inhibitory concentrations (MICs) of ciprofloxacin ranged from 0.19 microg/mL to 0.125 microg/mL. With use of PFGE, all isolates, including isolates from the water supply, showed an analytical similarity of 96%-100%. Multidrug-resistant isolates had a plasmid encoding for resistance, and those with resistance to nalidixic acid had a single-point mutation. CONCLUSIONS: To the best of our knowledge, this outbreak is the largest single-point source outbreak of multidrug-resistant typhoid fever yet reported, and it was molecularly traced to the city's single municipal water supply. Isolates were uniformly resistant to nalidixic acid, there was a decrease in their susceptibility as measured by MIC of fluoroquinolones, and 90% of isolates obtained were resistant to >1 antibiotic.

Intestinal parasitic infection among the HIV-infected patients in Nepal.

INTRODUCTION: Intestinal parasitic infection has been a significant problem in HIV patients, worldwide. In this study, we aimed to measure the prevalence and identify the factors associated with intestinal parasitic infection in people infected with HIV and attending National Public Health Laboratory in Kathmandu, Nepal, for CD4 T-cell count. METHODOLOGY: An analytical cross-sectional study in 745 HIV-infected people attending for CD4 T-cell count was conducted. RESULTS: The prevalence of intestinal parasitic infection was 22.4% (95% CI 19.5 to 25.5). In univariate analysis, age, sex, longer time since diagnosis of HIV, CD4 T-cell count of <200/µL, diarrhoea, marital status, and being under tuberculosis (TB) treatment were significantly associated with increased odds of intestinal parasite infection. However, in the logistic regression model, only the CD4 T-cell count of <200/µL (adjusted OR=4.2, 95% CI 2.5 to 7.0), diarrhoea (adjusted OR=2.8, 95% CI 1.8 to 4.3) and being under TB treatment (adjusted OR=2.9, 95% CI 1.8 to 4.6) remained as significant predictors. On stratification, CD4 T-cell count of <200/ µL was independently associated with higher odds of protozoal as well as helminthes infection. The parasites Cryptosporidium and Cyclospora were observed only in participants with CD4 T-cell counts <200/µL. CONCLUSIONS: Both protozoal and helminthic intestinal parasitic infections are common in HIV-infected people seeking care in healthcare facilities. The poor immune status as indicated by low CD4 T-cell count and TB may account for such a high risk of parasitic infection.

Improvement in immunological parameters in patients receiving highly active anti-retroviral therapy in Nepal.

Highly active anti-retroviral therapy (HAART) has been freely available in Nepal since 2004. In the present longitudinal study, we followed two distinct cohorts of human immunodeficiency virus-infected participants, those receiving HAART and those under assessment of eligibility for HAART, during the period 2005-2007 in Kathmandu, Nepal. The median change in CD4+ T-cell count among participants receiving HAART after 12 months of the initiation of therapy was +118 T cells/µl (95% confidence interval [CI], +91 to +145 T cells/µl) and that among participants under assessment of eligibility for HAART was -74 T cells/µl (95% CI, -103 to -44 cells/µl). However, the median CD8+ T-cell count after 12 months remained stable in both the cohorts (P > 0.05). The CD4+/CD8+ T-cell ratio increased from 0.16 to 0.26 after 12 months of therapy (P < 0.001). The multivariate regression model revealed that participants >30 years of age, and injection drug users had significantly lower increases in the CD4+ T-cell count in response to therapy. We observed a high proportion of loss to follow-up after 12 months of therapy; however, the associated factors were unknown. In conclusion, we observed a significant improvement in the CD4+ T-cell count in participants receiving HAART; however, the CD4+/CD8+ T-cell ratio remained <0.5 after 12 months of treatment.

Factors associated with high prevalence of pulmonary tuberculosis in HIV-infected people visiting for assessment of eligibility for highly active antiretroviral therapy in Kathmandu, Nepal.

BACKGROUND: Tuberculosis is the leading cause of deaths among HIV patients. In this study, we estimated the prevalence of pulmonary tuberculosis (PTB) and identified the factors/co-morbidities associated with active PTB in HIV-infected people visiting the national public health laboratory to assess their eligibility to receive highly active antiretroviral therapy. METHODS: A cross-sectional study was conducted to measure the prevalence of pulmonary tuberculosis. Data on probable risk factors in patients with and without PTB were compared, calculating the odds ratio as a measure of association. Factors showing significant association in univariate analyses were included in a stepwise backward logistic regression model to adjust for confounding. RESULTS: The prevalence of pulmonary tuberculosis was 32.4 % (95% confidence interval (CI) 30.25-34.56). In the univariate analysis, patients with PTB were more likely to be older, married, and have a longer duration since the diagnosis of HIV, diarrhoea, parasitic infection, lower CD4 T-cell counts, and lower CD4/CD8 ratio. However, the backward stepwise logistic regression revealed that only the CD4 T-cell count < 200/µL (AOR 11.69, 95% CI 6.23-21.94), CD4 T-cell count 200-350/µL (AOR 2.52, 95% CI 1.30-4.89), diarrhoea (AOR 2.77, 95% CI 1.78-4.31), parasitic infection (AOR 3.34, 95% CI 2.02-5.50) and 'sex with partner' as probable modes of transmission (AOR 0.44, 95% CI 0.20-0.93) were independently associated with pulmonary tuberculosis. CONCLUSION: A high prevalence of pulmonary tuberculosis was observed. Participants with tuberculosis were significantly more likely to have lower CD4 counts, diarrhoea, and parasitic infections. HIV treatment programmes should consider these factors for better outcomes.

Antimicrobial susceptibilities of enteric bacterial pathogens isolated in Kathmandu, Nepal, during 2002-2004.

INTRODUCTION: The prevalence and antimicrobial susceptibility patterns of the bacterial enteropathogens Vibrio cholerae, Salmonella species and Shigella species were investigated. METHODOLOGY: A total of 877 stool samples were received for culture at the National Public Health Laboratory (NPHL), Kathmandu, Nepal, during January 2002 to December 2004, from diarrhoea patients attending Shukraraj Tropical Infectious Hospital and referral outpatients. All samples collected were processed for isolation and antibiotic susceptibility testing of Vibrio cholerae, Salmonella spp. and Shigella spp. RESULTS: Of the 877 stool samples, 148 (16.8%) were culture positive for one of the three bacterial enteropathogens investigated. Among them, Vibrio cholerae, Shigella spp. and Salmonella spp. accounted for 98/877 (11.1%), 41/877 (4.6%), 9/877 (1.02%) of the isolates respectively. A year-to-year variation was seen in the type of predominant organism, with Shigella spp. being the most prevalent in 2002 and 2003 and Vibrio spp. in 2004. In all three years, Vibrio cholerae were encountered only during the months of April to June while Salmonella spp. and Shigella spp. were isolated throughout the whole year. All Vibrio cholerae and Salmonella isolates were susceptible to ciprofloxacin.  All Shigella isolates were susceptible to ceftriaxone. Ciprofloxacin resistance was observed among isolates of Shigella dysenteriae type-1 isolated after 2003. CONCLUSION: Vibrio cholerae, Salmonella and Shigella infections are prevalent in Kathmandu, Nepal. A gradual increase in resistance to commonly used antimicrobials was seen among bacterial enteropathogens. Antimicrobial resistance surveillance is necessary to guide empirical treatment.

Cholera incidence among patients with diarrhea visiting National Public Health Laboratory, Nepal.

The major objective of this study was to deliver vital statistics related to cholera to health authorities so as to aid in their attempt to prioritize communicable diseases in Nepal. A laboratory-based surveillance was conducted from mid-June 2008 to mid-January 2009 at the National Public Health Laboratory, Nepal. Diarrheal samples alone were processed for Vibrio cholerae. Isolation and identification of the organisms were carried out as per standard protocol. Antimicrobial susceptibility tests were done according to the guidelines of the Clinical and Laboratory Standards Institute. The incidence of cholera was found to be 27.1%. Only V. cholerae O1 Ogawa biotype El Tor was found during the study. No variation was observed in the percentage of cases between genders (P<0.05). The 15-30 year age group was found to be more susceptible to cholera (P<0.05). The period from mid-June to mid-July had the highest incidence of cholera (P<0.05). Ampicillin, tetracycline, ciprofloxacin, and erythromycin were highly effective, while 100% resistance was observed for furazolidone, nalidixic acid, and cotrimoxazole.

Field evaluation of commercial immunoglobulin M antibody capture ELISA diagnostic tests for the detection of Japanese encephalitis virus infection among encephalitis patients in Nepal.

OBJECTIVES: Japanese encephalitis (JE) is a devastating disease with high rates of death and disability that occurs particularly in resource-limited, rural regions of Asia. Simple, accurate and inexpensive diagnostics tests are vital for quantifying the burden of illness. This field study evaluated two commercial JE immunoglobulin M antibody capture (MAC) ELISA kits using samples from routine JE surveillance. METHODS: Positive (n=132) and negative (n=218) sera were randomly selected from patient samples collected as part of JE surveillance in Nepal in 2005. Samples were tested in a national public health laboratory with commercial kits produced by XCyton and Inverness (Panbio). Results were compared with those of the research lab-based reference standard, the Armed Forces Research Institute of Medical Sciences JE MAC ELISA. RESULTS: Positive and negative predictive values and 95% confidence intervals were 90% (82-95%) and 85% (79-89%) for Panbio1, 94% (88-98%) and 89% (87-93%) for Panbio2, and 84% (77-90%) and 96% (92-98%) for XCyton kits, respectively. Sensitivities of Panbio1, Panbio2, and XCyton kits were 71% (63-79%), 80% (72-87%), and 93% (88-97%); specificities were 95% (91-98%), 97% (94-99%), and 89% (85-93%), respectively. Overall percent agreement was 86% for Panbio1 and 91% for both Panbio2 and XCyton. CONCLUSIONS: Both commercial kits had good predictive values when single serum samples from encephalitis cases were tested in a national laboratory. Either kit can be used in similar JE-endemic settings where co-transmission of dengue virus, a flavivirus which has strong cross-reactivity with JE, is limited. These results can inform decisions by countries and the World Health Organization laboratory networks on national-level use of these kits for JE surveillance.

Laboratory-based Japanese encephalitis surveillance in Nepal and the implications for a national immunization strategy.

We report on two years of Japanese encephalitis (JE) surveillance in Nepal and the implications for a national immunization strategy. From May 2004 to April 2006, 4,652 patients with encephalitis were evaluated. A serum or cerebrospinal fluid specimen was collected from 3198 (69%) patients of which 1,035 (32%) were positive by Japanese encephalitis IgM ELISA. Most cases (N = 951, 92%) were from the 24 Terai districts (i.e., southern plains, 12.3 million persons) with the majority (N = 616, 65%) from four western Terai districts (population = 1.8 million). The case fatality ratio was 14.7% and 6.3% and the proportion of cases under 15 years old was 52% and 62% in the four western and 20 non-western Terai districts, respectively. Japanese encephalitis immunization targeting residents one year of age and older in the western districts and one through 14 years old in the non-western Terai districts may have reduced Japanese encephalitis cases by 84% and deaths by 92%, nationally.

Factors associated with high prevalence of pulmonary tuberculosis in HIV-infected people visiting for assessment of eligibility for highly active antiretroviral therapy in Kathmandu, Nepal.

Background: Tuberculosis is the leading cause of deaths among HIV patients. In this study, we estimated the prevalence of pulmonary tuberculosis (PTB) and identified the factors/co-morbidities associated with active PTB in HIV-infected people visiting the national public health laboratory to assess their eligibility to receive highly active antiretroviral therapy. Methods: A cross-sectional study was conducted to measure the prevalence of pulmonary tuberculosis. Data on probable risk factors in patients with and without PTB were compared, calculating the odds ratio as a measure of association. Factors showing significant association in univariate analyses were included in a stepwise backward logistic regression model to adjust for confounding. Results: The prevalence of pulmonary tuberculosis was 32.4 % (95% confidence interval (CI) 30.25–34.56). In the univariate analysis, patients with PTB were more likely to be older, married, and have a longer duration since the diagnosis of HIV, diarrhoea, parasitic infection, lower CD4 T-cell counts, and lower CD4/CD8 ratio. However, the backward stepwise logistic regression revealed that only the CD4 T-cell count < 200/μL (AOR 11.69, 95% CI 6.23–21.94), CD4 T-cell count 200–350/μL (AOR 2.52, 95% CI 1.30–4.89), diarrhoea (AOR 2.77, 95% CI 1.78–4.31), parasitic infection (AOR 3.34, 95% CI 2.02–5.50) and ‘sex with partner’ as probable modes of transmission (AOR 0.44, 95% CI 0.20–0.93) were independently associated with pulmonary tuberculosis. Conclusion: A high prevalence of pulmonary tuberculosis was observed. Participants with tuberculosis were significantly more likely to have lower CD4 counts, diarrhoea, and parasitic infections. HIV treatment programmes should consider these factors for better outcomes.

Genetic relatedness of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated in south Asia.

OBJECTIVES: The aim of the present study was to determine the clonal relationships of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated from south Asia, and S. dysenteriae 1 strains associated with epidemics in 1978, 1984 and 1994. METHODS: The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing. RESULTS: Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae 1, except for the presence of a new plasmid of approximately 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. The current ciprofloxacin-resistant strains belong to five subtypes (A3-A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India. CONCLUSIONS: PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.