Emergence of Aminoglycoside Resistance Due to armA methylase in Multi-drug Resistant Acinetobacter Baumannii Isolates in a University Hospital in Nepal.

BACKGROUND: The emergence of multidrug-resistant Acinetobacter baumannii associated with hospital-acquired infections has been increasingly reported worldwide. 16S rRNA methylase producing Gram-negative bacteria are highly resistant to all clinically important aminoglycosides. We analyzed A. baumannii clinical isolates resistant to aminoglycosides from hospitalized patients. The objective of this study was to investigate the emergence of armA in A.baumannii species associated with nosocomial infection in a university hospital in Nepal. METHODS: This was a cross-sectional study conducted at the department of Clinical Microbiology, Tribhuvan University Teaching Hospital (TUTH), from December 2013 to December 2014. A total of 246 Acinetobacter species were isolated from different patients were screened for MDR A. baumannii. Identification at the species level was confirmed by 16S rRNA sequencing. Drug susceptibility testing was performed by Kirby- Bauer disc diffusion method and minimum inhibitory concentrations (MICs) were determined using the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Screening for 16S rRNA methylase-production was done for the isolates resistant to gentamicin and amikacin. Detection of 16S rRNA methylase gene was done by PCR. RESULTS: All 122 multidrug-resistant A. baumanniiisolates were resistant to majority of the antibiotics used except polymyxin and tigecycline. Ninty-six MDR A. baumannii isolates had MICs of > 512 mg/L to amikacin and arbekacin indicating their high resistance to aminoglycosides.Of the 96 pan-aminoglycoside resistant isolates, 75 isolates had 16SrRNAmethylasewith all isolates harboring armA gene. CONCLUSIONS: This is the first report describing multidrug-resistant A. baumannii strains harboring armA from hospitalized patients in Nepal. A methylase gene (armA), conferring high level of resistance to aminoglycosides, was detected in majority of our isolates.

Phenotypic Characterization of Multidrug-resistant Escherichia Coli with Special Reference to Extended-spectrum-beta-lactamases and Metallo-beta-lactamases in a Tertiary Care Center.

INTRODUCTION: The increasing reports on extended-spectrum-beta-lactamase and metallo-beta-lactamase producing Escherichia coli have addressed a potential threat to global health since it is found to be highly resistance to most of the currently available antibiotics including carbapenems. The present study was aimed to determine the antibiogram of extended-spectrum-beta-lactamase and metallo-beta-lactamase producing MDR E. coli isolates from various clinical samples. METHODS: This was a cross-sectional study conducted over a period of seven months from December 2013 to July 2014 at bacteriology laboratory of Tribhuvan University Teaching Hospital. A total of 250 clinical specimens (urine, pus, sputum, blood, body fluid, bile, tissue and central venous pressure line tip) were processed from inpatients, with multidrug-resistant Escherichia coli infections. Standard microbiological techniques were used for isolation and identification of the isolates. The presence of extended-spectrum-beta-lactamase was detected by phenotypic confirmatory test recommended by Clinical and Laboratory Standards Institute and imipenem (IMP) /EDTA combined disc method was performed to detect metallo-beta-lactamase mediated resistance mechanism. RESULTS: We found high level of beta lactamase mediated resistance mechanism as part of multidrug resistance. Among 250 MDR isolates, 60% isolates were extended-spectrum-beta-lactamase producers and 17.2% isolates were metallo-beta-lactamase producers. Co-existence of extended-spectrum-beta-lactamase and metallo-beta-lactamase identified in 6.8% isolates. CONCLUSIONS: Beta-lactamase mediated resistance mechanisms are accounting very high in the multidrug resistant isolates of E. coli. Therefore, early detection of beta lactamase mediated resistant strains and their current antibiotic susceptibility pattern is necessary to avoid treatment failure and prevent the spread of MDR.

Inadequate Empirical Antibiotic Therapy in Hospital Acquired Pneumonia.

INTRODUCTION: Inadequate empirical antibiotic therapy for HAP is a common phenomena and one of the indicators of the poor stewardship. This study intended to analyze the efficacy of empirical antibiotics in the light of microbiological data in HAP cases. METHODS: Suspected cases of HAP were followed for clinico-bacterial evidence, antimicrobial resistance and pre and post culture antibiotic use. The study was taken from February,2014 to July 2014 in department of Microbiology and department of Respiratory medicine prospectively. Data was analyzed by Microsoft Office Excel 2007. RESULTS: Out of 758 cases investigated, 77(10 %) cases were HAP, 65(84%) of them were culture positive and 48(74 %) were late in onset. In early onset cases, isolates were Acinetobacter 10(42%), Escherichia coli 5(21%), S.aureus 4(17%), Klebsiella 1(4%) and Pseudomonas 1(4%). From the late onset cases Acinetobacter 15(28%), Klebsiella 17(32%) and Pseudomonas 13(24%) were isolated. All Acinetobacter, 78% Klebsiella and 36% Pseudomonas isolates were multi drug resistant. Empirical therapies were inadequate in 12(70%) of early onset cases and 44(92%) of late onset type. Cephalosporins were used in 7(41%) of early onset infections but found to be adequate only in 2(12%) cases. Polymyxins were avoided empirically but after cultures were used in 9(19%) cases. CONCLUSIONS: Empirical antibiotics were vastly inadequate, more frequently so in late onset infections. Use of cephalosporins empirically in early onset infections and avoiding empirical use of polymyxin antibiotics in late onset infections contributed largely to the findings. Inadequate empirical regimen is a real time feedback for a practitioner to update his knowledge on the local microbiological trends.

Outbreaks of cholera in Nepal.

This paper presents the study of the etiological agents of diarrhea in children below 14 years of age, this study was conducted from May 1995 to April 1996. One thousand one hundred seven (1,107) children with acute diarrhea receiving Oral Rehydration Therapy (ORT) at National Kanti Children's Hospital were included in this study. Stool samples of these patients were investigated at the Microbiology Laboratory, Department of Microbiology, Institute of Medicine. None of the stool samples showed the growth of Vibrio cholerae 0139 synonym Bengal. In Nepal, V.cholerae could be isolated from June to November. From December to May, no cases of V. cholerae were detected. Therefore, we address to this incidence as outbreaks rather than endemic. Mixed infections along with V. cholerae were also seen in 29% of cholera patients. V. cholerae 01, Hikojima types were the major isolates in our study followed by Ogawa type. V. cholerae, Hikojima and Ogawa serotypes were associated with mixed infection in 16.1% and 12.9% of patients, respectively. These isolates were associated with Shigella, Salmonella and pathogenic E. coli.

Comparative bacterial study of oral rehydration solution (ORS) prepared in plain unboiled and boiled drinking water of Kathmandu valley.

Result of bacterial study on Oral Rehydration Solution (ORS) prepared in plain unboiled and boiled drinking water of Kathmandu valley is reported. Of the total 100 water samples collected from different sources and area all the samples, as a base line study, were subjected for the examination of bacterial presence. Eighty eight percent of the water samples studied were found to be unsatisfactory for drinking. Thirty five percent of the ORS prepared in unboiled water and kept for 24 hours at room temperature showed increased bacterial count whereas none of the ORS prepared in 5 minute boiled water and kept for 24 hour at room temperature showed any bacterial growth. Decreased bacterial count was not found in any of the ORS prepared in unboiled water. Typical coliform bacilli were found grown in 57.0% of the ORS prepared in unboiled water samples.

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.

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.

Metallo-beta-lactamase producing gram-negative bacterial isolates.

BACKGROUND: The global emergence of metallo-ß-lactamase (MBL) producing bacterial isolates causing lower respiratory tract infection (LRTI) has resulted in fewer therapeutic options in treatment modalities. However, to our knowledge no studies regarding MBLs had been done so far in Nepal. Therefore, this study was carried out to assess the current level of MBL producing bacterial isolates in our setup. METHODS: This was a cross-sectional study conducted over a period of six months (June to November 2008) at Bacteriology laboratory of a teaching hospital. A total of 1120 specimens representing lower respiratory tract (sputum, endotracheal secretion and bronchial washing) were processed from outpatients and inpatients, with suspected LRTI, at TUTH. The specimens were collected and processed according to the standard methodology. Combination disk method and Double disk synergy test methods were used for the detection of MBL producing isolates. RESULTS: Respiratory pathogens were recovered from 497 (44.4%) of suspected cases. Among these, gram-negative bacteria were observed in 448 (84.0%). Multidrug resistance (MDR) was found in 286 (53.7%) of the total bacterial isolates. MBL was present in 6 (1.3%) of the total 448 gram-negative isolates. MBL was detected by both DDST and CD methods in 3 isolates each of Pseudomonas aeruginosa and Acinetobacter spp. from inpatients. All MBL producers were MDR. CONCLUSIONS: MBL-producing gram negative bacteria were detected from LRTI isolates in this study and this data can be used as base-line information of this novel type of ß-lactamase in our setup.

Onychomycosis: clinical pattern and prevailing fungi in Kathmandu.

Onychomycosis is a chronic mycotic infection of finger and toe nails that affects the quality of life in a significant proportion. The study was aimed to identify the clinical patterns and etiological agents of onychomycosis. The study population comprised of the suspected cases of onychomycosis, attending the outpatients department of Dermatology of Tribhuvan University Teaching Hospital, Kathmandu, during November 2006 to March 2008. Detailed history was taken and clinical pattern was noted. Nail sample was obtained from patients with suspected onychomycosis and was subjected for direct microscopy and fungal culture. After first inoculation, the culture tubes were examined every day for one week and thereafter weekly and the fungus was identified. A total of 218 patients were included in the study. The age of the patients ranged from 4 to 88 years with mean of 32.8 +/- 15.4. Maximum of the patients were in the age group of 21-30 years. M: F ratio was 1.05:1. Duration of the disease varied from 1 month to 15 years. Direct microscopy was positive in 64.22% and culture was positive in 41.7%. Dermatophytes were isolated in 54.9%, yeasts in 39.6% and non-dermatophyte molds in 5.5%. As a whole, Trichophyton rubrum was the most common fungal isolate (82%). Yeast infection was more common in females. Yeast was significantly more commonly implicated as a pathogen in finger nail onychomycosis. Dermatophytes were more frequently isolated from toe nail onychomycosis. In conclusion, T rubrum was the most common fungal isolate.

Opportunistic infections in relation to CD4 level among HIV seropositive patients from central Nepal.

CD4 cells status of HIV patients provides one of the benchmarks against the progression of HIV/AIDS. Regular investigation of opportunistic infection in HIV patients is one of the major components of HIV/AIDS care and support service. Between October 2007 and May 2008, a cross-sectional analytical study was carried out in Tribhuvan University Teaching Hospital with an objective to find the relationship between CD4 level and opportunistic infections. After taking informed consent pre-structured questionnaire was filled and specimens were collected to investigate major opportunistic infections (OIs) as per standard microbiological procedure. All the information were entered into SPSS 11.5 system and analyzed. Of the 150 patients, 100 (66.7%) were males and 50 (33.3%) were females. The age group 21-30 years was predominant (42.7%) followed by 31-40 years (42%). Oral candidiasis was found to be the predominant OIs (32.0%) followed by streptococcal pneumonia (28.7%), Salmonella infection (20.7%), cryptosporidial infection (19.3%) and tuberculosis (10.0%). Significant relationship could be established between low CD4 count (<200) and the appearance of oral candidiasis (/2=9.16, p<0.05) but no such relationship could be established regarding other OIs. So, it can be concluded that appearance of oral candidiasis is the strong evidence of advanced stage of HIV infection.

Antibiotic susceptibility pattern of nosocomial isolates of staphylococcus aureus in a tertiary care hospital, Nepal.

INTRODUCTION: Methicillin resistant Staphylococcus aureus (MRSA), the most common cause of nosocomial infection has been a major cause of morbidity and mortality around the world. They are normally resistant to most of the antibiotics used in clinical practice. This study has been carried out to find out the resistance pattern among S. aureus. METHODS: During November 2007 to June 2008, clinical samples from patients with nosocomial infection were processed for culture and sensitivity following standard methodology in microbiology laboratory, Tribhuvan University teaching hospital, Kathmandu, Nepal. RESULTS: Among 149 Staphylococcus aureus isolates, highest resistance was observed against Penicillin (91.94%) followed by Fluoroquinolone (61.74%), Erythromycin (52.94%), Gentamicin (46.98%), Cotrimoxazole (42.95%), Tetracycline (40.94%) and others, whereas susceptibility was observed maximum against Chloramphenicol (94.85%) followed by Rifampicin (92.61%), Tetracycline (59.06%), Cotrimoxazole (57.04%), and others. None of the isolates were resistant to Vancomycin and Teicoplanin. Of these isolates 44.96 % of the isolates were Methicillin resistant S. aureus (MRSA). Resistance to Penicillin, Fluoroquinolone, Erythromycin, Gentamicin, Co-trimoxazole and Tetracycline were associated significantly with MRSA isolates (chi2= 8.779, p<0.05, chi2= 74.233, p<0.05, chi2= 84.2842, p<0.05, chi2= 108.2032, p<0.05, chi2= 88.1512, p<0.05 and chi2= 79.1876, p<0.05 respectively). Although most of the Methicillin sensitive S. aureus (MSSA) isolates were susceptible to both Rifampicin and Chloramphenicol, only Rifampicin susceptibility was significantly associated with them (chi2= 10.1299, p<0.05). Among three Biochemical tests for the detection of beta lactamase detection namely chromogenic, iodometric and acidimetric test, chromogenic test method had highest sensitivity and specificity. CONCLUSIONS: Since MRSA comprised a greater part of S. aureus isolates and were multi-resistant, patients infected by such strains should be identified and kept in isolation for hospital infection control and treated with second line of drug like vancomycin.

Onychomycosis: a clinico-epidemiological study.

Onychomycosis is a common nail disorder. Far more than being a simple cosmetic problem, infected nail serves as a chronic reservoir, which can give rise to repeated mycotic infections. The study was undertaken to determine the various clinical patterns of onychomycosis. This prospective cross sectional study was conducted in clinically suspected patients of onychomycosis attending out patients department of dermatology, T.U. Teaching hospital between August 2006 and July 2007. Various data were obtained and clinical patterns were noted. Out of 182 clinically suspected patients of onychomycosis, 52.7% were males with male: female ratio of 1.1:1. Onychomycosis was predominant among the younger patients with slight male preponderance. Fingernails were more frequently involved in females whereas toenails in males. The most common clinical type was distal and lateral subungual onychomycosis. 58.2% had other concomitant fungal infections apart from onychomycosis. Onychomycosis could serve as a good reservoir for recurrent cutaneous superficial fungal infections. Hence, adequate treatment of onychomycosis can prevent from these recurrent cutaneous superficial fungal infections.

Carriage of antibiotic-resistant fecal bacteria in Nepal reflects proximity to Kathmandu.

Within Nepal, geographic, social, and economic barriers greatly limit access to allopathic health care. The country therefore offered the opportunity to evaluate the effect of antibiotic accessibility (as measured by allopathic medicine consumption) on antibiotic resistance in the normal intestinal flora. The aerobic gram-negative fecal flora of 33-34 healthy adults from each of 3 villages with different access to health care facilities in Kathmandu were examined for antibiotic susceptibility. The frequency of antibiotic resistance decreased significantly with increasing distance from Kathmandu and decreasing population density but did not reflect contact with health care providers or individual medicine consumption. The findings suggest that an individual's overall exposure to antibiotics and antibiotic-resistant bacteria (resulting from close proximity to other community members and to sources of accessible allopathic health care, such as in the vicinity of Kathmandu), has an equal or greater impact on an individual's carriage of antibiotic-resistant bacteria than does direct consumption of antibiotics.

The glutaraldehyde test as a rapid screening method for pulmonary tuberculosis: a preliminary report.

When equal parts of EDTA-blood and 2.5% glutaraldehyde solution were mixed, a shorter gelification time was observed in blood from patients with pulmonary tuberculosis--diagnosed by clinical and X-ray examination--than in blood from patients with other respiratory diseases or with non-respiratory diseases, or from healthy volunteers. Of a total of 539 persons tested, 267 had pulmonary tuberculosis as judged by clinical and X-ray examination and 238 of these reacted positively to the glutaraldehyde test with a gelification time of less than 10 minutes. When clinical examination and X-ray evaluation are used as bases for the diagnosis of pulmonary tuberculosis, the glutaraldehyde test gives a sensitivity of 89% and a specificity of 95%. It is suggested that this simple and cheap method, together with clinical examination, might be a useful screening tool for identifying cases of pulmonary tuberculosis in peripheral health posts in developing countries where X-ray and other facilities are lacking.