In vitro Temocillin efficacy against extended spectrum ß-lactamase producing multidrug resistant gram negative bacterial isolates from Nepal.

Temocillin is relatively more stable against most ß-lactamases and requires re-evaluation to include it in common clinical practice as a therapeutic alternative. At the National Reference Laboratory of Nepal, we evaluated multidrug resistance (MDR) and extended spectrum ß-lactamase (ESBL) phenotypes among 292 gram-negative clinical bacterial isolates of 18 different genera during 2009/2010 by Kirby-Bauer disc diffusion method following CLSI guidelines. ESBL screen positive isolates were tested for Temocillin efficacy by disc diffusion method following British Society of Antimicrobial Chemotherapy (BSAC) guidelines and other antibiotics following Clinical and Laboratory Standards Institute (CLSI) guidelines. Of the 292 isolates, 75.0% isolates were MDR, among which 61.6% were primarily screened positive for ESBL production but only 38.8% were confirmed as ESBL producers. We report relatively lower Temocillin resistance of 28.9% and 15.6% among MDR and ESBL positive populations, respectively. Among ESBL positive isolates, no Proteus mirabilis, 19.7% Escherichia coli and 33.3% Klebsiella oxytoca showed resistance to Temocillin, although such resistance was higher among Acinetobacter spp. (66.7%) and K. pneumoniae 50.0%. Among ESBL negative isolates, none of the K. oxytoca and few (13.3%) Acinetobacter spp. were resistant to Temocillin, while all Citrobacter freundii, Pseudomonas aeruginosa (85.7%) and K. pneumoniae (66.7%) showed Temocillin resistance. Only 14.8% and 3.0% of total MDR isolates were resistant to Imipenem and Meropenem, respectively. However, Imipenem resistance was remarkably high (86.7%) among ESBL negative Acinetobacter spp. than Meropenem (13.3%). Temocillin showed comparable efficacy against MDR and ESBL producing bacterial isolates and could be a next therapeutic option.

Application of WHONET in the Antimicrobial Resistance Surveillance of Uropathogens: A First User Experience from Nepal.

INTRODUCTION: WHONET is a freely downloadable, Windows-based database software which is used for the management and analysis of microbiology data, with a special focus on the analysis of antimicrobial susceptibility test results. Urinary Tract Infections (UTI) are a common medical problem and they are responsible for notable morbidity among young and sexually active women. OBJECTIVES: The major objective of this study was the utilization and application of the WHONET program for the Antimicrobial Resistance (AMR) surveillance of uropathogens. METHODS: A total of 3209 urine samples were collected from patients who visited Manipal Teaching Hospital with a clinical suspicion of UTI, during December 2010 to July 2011. The isolation and characterization of the isolates were done by conventional methods. Antimicrobial Susceptibility Testing (AST) was performed by Kirby Bauer's disc diffusion method. The data entry and analysis were done by using the WHONET 5.6 software. RESULTS: Out of the 3209 specimens, 497 bacterial isolates were obtained and they were subjected to AST. Escherichia coli (66.2%) was the commonest bacterial isolate, followed by Enterococcus species (9.3%), Staphylococcus aureus (5.0%), and Klebsiella pneumoniae (4.2%). Among the gram-negative enteric bacilli, a high prevalence of resistance was observed against ampicillin and ciprofloxacin. The gram negative nonfermenters exhibited a high degree of resistance to ceftazidime. Staphylococcus species. showed a moderately high resistance to co-trimoxazole. One isolate was Vancomycin Resistant Enterococci (VRE). CONCLUSION: This study, a first of its kind which was done in Nepal, was carried out by using the WHONET software to monitor, analyze and share the antimicrobial susceptibility data at various levels. This study was also aimed at building a surveillance network in Nepal, with the National Public Health Laboratory, Nepal, acting as a nodal centre. This would help in the formulation of antibiotic policies and in identifying hospital and community outbreaks at the nodal centre, as well as in sharing information with the clinicians at the local level.

Antibiotic resistance pattern and plasmid profiling of thermotolerant Escherichia coli isolates in drinking water.

BACKGROUND: Antibiotic resistant Escherichia coli is potential source of transmission of resistance to other water borne pathogens where plasmid borne resistance is most significant. METHODS: Drinking water samples were collected from different water sources: that is to say- tap, well and spring from different places of Kathmandu where E. coli and thermotolerant E. coli were isolated using membrane filtration technique. Antibiotic susceptibility was determined using a modified Kirby Bauer disc diffusion method and thermotolerant E. coli isolates from tap water were subjected for plasmid profiling. RESULTS: Type of water sources were not associated with the presence of coliform (P=0.155) and thermotolerant coliform (P=0.235) but the significant association was observed in thermotolerant coliform and thermotolerant E. coli for all sources tap (P=0.029), well (P=0.028), spring (P=0.05) but total coliform and E. coli association was found for well (P=0.01). All E. coli and thermotolerant E. coli isolates were susceptible to Ofloxacin, Chloramphenicol and Cotrimixazole. Resistance to Cefexime, Amikacin, Nalidixic acid, Amoxicillin, Tetracycline were 17 (54.8%), 9 (29%), 11 (35.5%), 25 (80.6%), 29 (93.5%) and 19 (57.6%), 12 (36.4%), 13 (39.4%), 31 (94%), 33 (100%) was observed in E. coli and thermotolerant E. coli respectively where 25 (75.8%) thermotolerant E. coli and 22 (70.9%) E. coli were observed with multiple drug resistance patterns. Single band of plasmid were observed in three MDRs and one non-MDR isolates and size varied from 2kb to >10kb. All Nalidixic acid resistant thermotolerant E. coli were found to harbor a plasmid. CONCLUSIONS: Presence of plasmid in Nalidixic acid resistant thermotolerant E. coli heightens public health issue and the need of monitoring Quinolone resistance bacteria in environment.

Current fluoroquinolone susceptibility criteria for Salmonella needs re-evaluation.

BACKGROUND: Disc diffusion technique is the routine susceptibility testing procedure for isolates of enteric fever, the most common clinical diagnosis among febrile patients in Nepal. OBJECTIVE: To evaluated the current fluoroquinolones (FQs) susceptibility criteria and nalidixic acid screening test in Salmonella enterica serovar Typhi and Paratyphi A. METHODS: S. Typhi and Paratyphi A strains isolated from 443 suspected enteric fever patients visiting National Public Health Laboratory (NPHL) during April through October 2008 were analyzed. All isolates were confirmed by standard microbiological procedures including serotyping. Antibiotic susceptibility testing was performed by using Kirby Bauer disc diffusion method and Clinical and Laboratory Standards Institute (CLSI) approved interpretive criteria. Agar dilution method was used to determine Minimum Inhibitory Concentration (MIC) of ciprofloxacin, ofloxacin and nalidixic acid. RESULT: Out of 41 Salmonella isolates, 80.49% were nalidixic acid resistant, with S. Paratyphi A showing higher resistance rate (88.23%) compared to S. Typhi (75%). The difference in both MIC and zone diameter in nalidixic acid susceptible and nalidixic acid resistant isolates was found to be significant (P < 0.001) and decreased susceptibility to FQs was strongly correlated (sensitivity and specificity of 100%) with resistance to nalidixic acid. Regression analysis of MIC against zone diameter based on the current CLSI recommended guidelines suggests that accommodation of current susceptible and resistant MIC requires increase in the zone diameter of ciprofloxacin and ofloxacin. CONCLUSION: Before using these drugs for management of enteric fever, appropriate identification of Salmonella isolates with reduced susceptibility to FQs is essential to limit the possible treatment failure and development of highly resistant strains. The current FQs susceptibility break point criteria for Salmonella need re-evaluation.

Studies on extended spectrum beta lactamase (ESBL) producing salmonella isolates from clinical samples of Nepal.

Present study was carried out among the patients of age 2-60 years from November 2009 to November 2011 to assess the production of Extended Spectrum Beta Lactamase (ESBL) by salmonella enterica serotype (Typhi and paratyphi A) at Kanti Children's Hospital (KCH), Nepal Medical College (NMC), Kathmandu Medical College (KMC), National Public Health Laboratory (NPHL) and Sankata Laboratory (SKL) of Nepal. Blood cultures were obtained from 4,820 patients with febrile illnesses. 400 strains of salmonella enterica were isolated. Antibiotic susceptibility testing was carried out using disk diffusion method by Kirby-Bauer technique and ESBL screening were done by Double Disk Synergy Test (DDST), following the Clinical and Laboratory Standard Institute (CLSI) recommendations for Escherichia coli. Male had higher infection (56%) than female (44%). Highest number of culture positive cases were (37%) in 10-19 years of age groups. Highest episodes of enteric fever cases occurred during April - June (45%) followed by July-September (35%). Among the total isolates 286 (72%) were Multidrug resistance (MDR). All the Multidrugs resistance salmonella, resistant to third generation Cephlospornis were ESBL producers.

The burden of dengue infection in some vulnerable regions of Nepal.

Dengue is an emerging mosquito borne disease of public health importance in Nepal. A descriptive cross sectional study was carried out to estimate sero-prevalence and distribution pattern of dengue in certain vulnerable regions of Nepal from June to September 2009. A total of 460 venous blood samples were collected from individuals experiencing a febrile illness clinically consistent with dengue infection visiting nearby hospitals of Kanchanpur, Kailali, Banke, Dang and Chitwan districts. The sero-prevalence of dengue virus specific IgM was determined by enzyme linked immunosorbent assay (ELISA) kit. The anti-dengue IgM positivity was found to be 12.17%. The higher frequency of positive cases (16.4%) were from age group 20-40 years followed by < 20 years age group with 9.7% and 5.3% from > 40 years age group. The association between dengue infection and age is found to be statistically significant (p < 0.05). The male:female ratio was determined as 1.3:1 in IgM positive population. Among sampling areas, Kanchanpur showed highest prevalence of dengue infection (15.5%) followed by Chitwan (11.7%), Kailai (11.1%), Banke (10.7%) and Dang (8.3%). Similarly, 94.6% of the positive cases were indigeneous and had no history of travel to other countries. Dengue is firmly established in terai region with increasing trends of infection and expansion into newer areas raising a public health threat. Regular epidemiological studies are suggested which could further reveal the contributing factors associated with dengue virus infection and help in formulating strategies in reducing the transmission rate and control of the infection.

Extended spectrum â-lactamase producing multidrug resistant clinical bacterial isolates at National Public Health Laboratory, Nepal.

Extended Spectrum â-lactamase (ESBL) producing multidrug resistant bacteria complicate therapeutic management and limit treatment options. Therefore, detection of ESBL-producing multidrug resistant (MDR) pathogens has a paramount importance. Between April 2009 and January 2010, a prospective study was carried out in National Public Health Laboratory with an objective to determine the status of ESBL producing MDR bacterial isolates from different clinical samples. Identification of the isolates was done by standard microbiological techniques and antibiotic susceptibility testing was done by Kirby Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL screening among MDR isolates was done using Ceftriaxone, Aztreonam, Cefotaxime, Ceftazidime and Cefpodoxime followed by confirmation using MASTDISCS ID ES2L Detection Discs (CPD10). Data analysis was done by SPSS 16 software. Of the 314 bacterial isolates from 1601 different clinical specimens, 199 (63.4%) were MDR. Cefotaxime was found the reliable screening agent for ESBL detection with sensitivity and positive predictive value of 98.6% and 76.4% respectively. Sixtey nine (62.7%) isolates of the 110 tested MDR isolates were ESBL positive with at least one of the Combined Disk (CD) Assays. Escherichia coli (80%) was the major ESBL producer followed by Klebsiella pneumoniae (5.8%). A statistically significant relationship was found between increasing spectrum of drug resistance and ESBL production (p<0.05). Thus it is concluded that a higher rate of ESBL production prevail among MDR clinical bacterial isolates underscoring the need for routine ESBL detection in clinical laboratories.

Study of rifampicin and isoniazid resistance mutation genes of M. tuberculosis isolates in Nepal.

We studied the rifampicin (RIF) and isoniazid (INH) resistant genes in Mycobacterium tuberculosis isolated from sputum samples of re-treated TB cases using phenotypic drug susceptibility test (DST) and rapid molecular method (Genotype; MTBDR plus, Hains Life Science, Nehren, Germany). Of the total 207 isolates included in this study, 90 (42.0%) were RIF resistant, 107 (50.7%) were INH resistant whereas 86 (41.5%) were multidrug resistant (MDR) by DST. According to genotypic analysis, 37.0% (77/207) were RIF resistant, 50.2% (105/207) INH resistant and 70 (33.8%) were MDR. Among the total 90 RIF resistant isolates, 85.5% (77/90) showed mutation in 81 bp of rpoB gene and the mutation locus were distributed as follows; MUT1 Asp516Val (16.7%; 15/90), MUT2A His526Tyr (6.6%; 6/90), MUT2B His526Asp (5.5%; 5/90) and MUT3 Ser531Lue (56.7%; 51/90). Among the total 107 INH resistant isolates, the mutation located in katG gene was 98.1% (105/107) and in inhA MUT1 gene was (16.8%; 18/107). The mutation locus were distributed as follows; MUT1 Ser315Thr (77.6%; 83/107), MUT2 Ser315Ile (3.7%; 4/107) in katG and in inhA MUT1Cys15Thr (16.8%; 18/107). The highest frequency of mutations in rpoB gene was found at the region of codon Ser531Leu (56.7%) while the frequency of mutation in katG and inhA gene were found at the region of codon Ser315Thr (77.6%) and Cyst15Thr (17.1%), respectively. The rapid molecular test kit used in this study was found to be sensitive as well as specific for detection of RIF and INH resistant gene and also helpful in early detection of MDR-TB.

Salmonella enterica serovar Paratyphi A: an emerging cause of febrile illness in Nepal.

With an aim to evaluate the isolation rate and antibiotic susceptibility pattern in Salmonella enterica serovar Typhi and S. Paratyphi A, 656 blood samples collected from clinically diagnosed enteric fever patients at National Public Health Laboratory, Nepal during January through December 2008 were processed. Isolates were identified by standard microbiological procedures including serotyping. Antibiotic susceptibility testing was performed by disc diffusion method and minimum inhibitory concentration (MIC) to ciprofloxacin, ofloxacin and nalidixic acid was determined by agar dilution method following CLSI guidelines. Altogether 59 isolates of S. Typhi (49.15%) and S. Paratyphi A (50.85%) were recovered. A total of 80% isolates were resistant to nalidixic acid with S. Paratyphi A (93%) showing significantly higher resistance (P < 0.05) compared to S. Typhi (66%). The nalidixic acid resistant S. Paratyphi A strains required significantly higher MICs (P < 0.001) to quinolone (MIC expressed as mean +/- SD for nalidixic acid 477.87 +/- 87.02 microg/mL, ofloxacin 1.8 +/- 0.63 microg/mL, ciprofloxacin 0.62 +/- 0.3 microg/mL) compared with that of S. Typhi (nalidixic acid 173.18 +/- 72.03 microg/mL, ofloxacin 0.43 +/- 0.11 microg/mL, ciprofloxacin 0.25 microg/mL). Increased MIC of fluoroquinolone (FQ) is of particular concern in emerging strains of S. Paratyphi A as exposure to these drugs fuels up further development of full FQ resistant populations. Use of FQs as the first-line drugs for empirical therapy and management of enteric fever in areas where these strains are prevalent is questionable and requires an urgent review.

Resistotypes of Vibrio cholerae 01 Ogawa Biotype El Tor in Kathmandu, Nepal.

Cholera continued to be a major diarrheal illness in Nepal and antibiotic resistance has appeared as a serious problem in cholera management. The study aimed at analyzing the distribution pattern of the resistotypes (R-types) of Vibrio cholerae in the Kathmandu valley, Nepal. During June 2008 to January 2009, 210 diarrheal specimens received at National Public Health Laboratory from suspected cholera patients were subjected to standard bacteriological investigation including biotyping and serotyping. Antimicrobial susceptibility pattern of V. cholerae isolates was determined by Kirby Bauer disc diffusion method following CLSI guidelines. A total of 57 (27%) V. cholerae isolated were recovered, all of which belonged to 01 Ogawa Biotype EL Tor. Based on antibiogram, V. cholerae isolates in our study revealed three distinct R-types: R-type I, R-type II and R-type III. All three R types showed resistance to furazolidone, nalidixic acid and cotrimoxazole while sensitive to ciprofloxacin and tetracycline. Additional resistance to ampicillin and erythromycin was observed respectively in R-type II and III. Different R-types showed unique month wise variations (P < 0.05). Differentiation of V. cholerae strains into R-types is an important tool. In addition to direct patient management, it may have implication in identifying the source and spread of infection, and understanding the distribution pattern in a particular geographical region.

Acute dengue infection in the western terai region of Nepal.

INTRODUCTION: Dengue fever is an emerging mosquito borne disease in Nepal claiming substantial morbidity and mortality. The objective of the study was to find out frequency of acute dengue infection in patients from the hospitals of the western Nepal. METHODS: The study was conducted between August 2007 and July 2008 in patients visiting hospitals of the western terai of Nepal with chief complains of fever. The sero-diagnosis of acute dengue infection was determined by enzyme linked immunosorbent assay among 239 patients visiting Lumbini Zonal Hospital, Butwal; Bheri Zonal Hospital, Nepalgunj; Bardiya District Hospital, Bardiya and Mahakali Zonal Hospital, Mahendranagar. RESULTS: The anti-dengue IgM positivity was 29.3%. There was slight male preponderance with a male to female ratio of 1.2:1. Out of the total positive cases, the highest positive cases (75.7%) were from the age group 15 - 50 years followed by < 15 years old (15.7%). Out of four hospitals, the highest positive cases (54.3%) were in Lumbini Zonal Hospital, Butwal. The age and gender were independent predictors to dengue virus infection. The highest numbers of dengue positive cases were in October (52.6%). The association between dengue disease and the month was statistically significant. CONCLUSIONS: The dengue positivity was estimated in acute patients from hospitals of western Nepal by enzyme immunoassay. Therefore, the serological marker can be used to diagnose acute patients of dengue during outbreaks.

Bacteriology and antimicrobial susceptibility of adult chronic dacryocystitis.

INTRODUCTION: Chronic dacryocystitis results in chronic infection and inflammation of the lacrimal sac. OBJECTIVE: To identify the aetiology of chronic dacryocystitis and to determine their antimicrobial susceptibility pattern. MATERIALS AND METHODS: A cross - sectional study was undertaken including 120 lacrimal swab materials collected from patients aged above 15 years suffering from chronic dacryocystitis. STATISTICS: Data analysis was done by using software "Win pepi'' ver 7.9. RESULTS: The bacteria of eight different species were isolated from 76.66 % (92/120) culture positive samples. 85.86 % showed a single and 14.13 % showed a mixed growth pattern. Coagulase negative staphylococci were the most common bacteria (P= 0.018) accounting for 33.96 % followed by Staphylococcus aureus (25.46 %), Streptococcus pneumoniae ( 19.81 % ), Streptococci viridans (5.66 %), Escherichia coli (5.66 %), Haemophilus spp ( 4.71 % ), Streptococcus pyogenes (3.77 %) and Bacillus spp (0.94 % ). Staphylococcus aureus were the most predominant bacteria in mixed growth. Rate of infection was higher in males 81.39 % than in females 74.02 %. Infection was higher in the age group of above 31 years. In the antimicrobial susceptibility test, except staphylococcus aureus, all the Gram positive isolates were 100 % sensitive to chloramphenicol and were least sensitive to tobramycin, but Gram negative isolates were equally sensitive to Chloramphenicol and Nalidixic acid. CONCLUSION: Coagulase negative staphylococci are the most frequently isolated bacteria. Staphylococcus aureus is predominantly found in mixed growth. Chloramphenicol is the most effective drug of choice for chronic dacryocystitis.

Serotyping, PCR, phage-typing and antibiotic sensitivity testing of Salmonella serovars isolated from urban drinking water supply systems of Nepal.

AIMS: To study the occurrence and diversity of Salmonella serovars in urban water supply systems of Nepal. METHODS AND RESULTS: Occurrence of Salmonella was detected in 42 out of 300 water samples by enrichment culture technique in selenite F broth followed by plating on Salmonella Shigella agar. A total of 54 isolates identified to genus level by standard tests were subsequently confirmed by serotyping, phage typing and PCR detection of virulence genes (inv A and spv C). The predominant serotype was Salmonella Typhimurium, followed by Salm. Typhi, Salm. Paratyphi A and Salmonella Enteritidis. Most of the Salm. Typhi isolates were E1 phage type followed by UVS4, A and UVS1. All isolates of Salm. Paratyphi A and Salm. Enteritidis were an untypable (UT) phage type. The majority of isolates were multi-drug resistant as revealed by Kirby-Bauer disc diffusion technique. Ceftriaxone resistant isolates of Salm. Enteritidis indicated the presence of one of the ESBL genes, blaSHV, whereas the genes blaTEM and blaCTX were absent. CONCLUSIONS: The microbiological quality of the urban water supply is poor and indicates possibility of fatal outbreaks of enteric fever and related infections in Nepal. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study will be useful in water borne disease control and prevention strategy formulation in Nepal and in the global context.