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.

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.

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.

Clinical features of HIV/AIDS and various opportunistic infections in relation to antiretroviral status among HIV seropositive individuals from Central Nepal.

BACKGROUND: So far, antiretroviral therapy is the only effective treatment available to HIV/AIDS patients. Provision of combined package of treatment, care and support service as well as regular assessment of the therapy increases its effectiveness. OBJECTIVE: The aim of this study was to establish the relationship between antiretroviral therapy status and clinical features/opportunistic infections among HIV seropositive individuals. MATERIALS AND METHOD: This is a cross-sectional study. Study was carried out between October 2007 and May 2008 in 150 HIV patients of Kathmandu, Central Nepal. After taking informed consent pre-structured questionnaire was filled to assess clinical features and specimen were collected to investigate major OIs as per standard microbiological procedure. All the information were entered into SPSS 11.5 system and analysed. RESULT: Of the 150 patients, 100 (66.7%) were males and 50 (33.3%) were females. The age group 21-30 years was predominant followed by 31-40 years (42%). Significant relationship could be established between intake of ART and cardinal symptoms of HIV/AIDS (chi2 value ranging from 4.11 to 9.34). However, no significant relationship could be established between the intake of ART and distribution of different OIs (chi2 values ranging from 0.15 to 1.6). CONCLUSION: Antiretroviral therapy was found to effective enough to reduce the clinical features of AIDS. Diagnosis and treatment of opportunistic infections should be routinely done for both groups of patients.

Tuberculosis co-infection in HIV infected persons of Kathmandu.

Tuberculosis is itself a major Public health problem in Nepal and the emergence HIV further complicated the issue. A cross-sectional analytical study was conducted between January 2004 and August 2005, with a general objective to determine the Tuberculosis co-infection status in HIV/AIDS cases of Nepal. Altogether 100 HIV infected persons visiting different Voluntary counseling and testing centers (VCT) and HIV/AIDS care centers located in Kathmandu valley were enrolled in the study. Investigation of tuberculosis was done by standard method prescribed by WHO using sputum specimen. Among 100 HIV infected cases, 66 (66.0%) were males and 34 (34.0%) were females. Majority of the HIV cases were in the age group 21-30 (60.0%) followed by 31-40 (31.0%). Tuberculosis was detected in 23 cases with highest prevalence in the age group 21-30 years (65.2%). No significant relationship could be established between gender and TB (c2 = 0.83, p > 0.01).Significant relationship was established between smoking/alcoholic habit and the subsequent development of tuberculosis (c2 = 7.24, p < 0.05 for smoking habit; c2 = 4.39, p < 0.05 for alcoholic habit at 1 degree of freedom). Among 22 culture positive isolates the predominant was Mycobacterium avium complex (40.9%) followed by M. tuberculosis (27.3%), M. kansasii (18.2%), M. fortuitum (9.1%) and M. chelonae (4.5%). Among the 23 cases of tuberculosis, 22 cases were diagnosed by cultural technique of which 4 cases were smear positive while the remaining one case was diagnosed by direct microscopy although it was culture negative. Smear negative Tuberculosis is found to be alarmingly higher in HIV positive individuals of productive age group. The disease significantly higher in smokers and alcoholics.

Characterization of mycobacteria in HIV/AIDS patients of Nepal.

Besides Mycobacterium tuberculosis, a number of other Mycobacterium species are also occasional human pathogens. Tuberculosis due to Mycobacterium avium complex (MAC) and Mycobacterium kansasii is particularly prevalent in AIDS patients as compared to the normal population. A cross-sectional study was carried out during January 2004 to August 2005 in 100 HIV-infected persons visiting Tribhuvan University, Teaching Hospital, and about a dozen of HIV/AIDS care centers of Kathmandu with the objectives to characterize the different mycobacterial species in HIV/AIDS patients. Three sputum specimens from each person were used to investigate tuberculosis by Ziehl-Neelsen staining, culture and identification tests. Among the 100 HIV-infected cases, 66 (66%) were males and 34 (34%) were females. Sixty percent of the cases were in the age group of 21-30 years. Mycobacteria were detected in 23 (23%) HIV cases of which 15 (65.2%) were in the age group of 21-30 years ; 17(74%) were males and 6 (26 %) were females. Among 23 co-infected cases, 22 were culture positive for mycobacteria. Among these, the predominant one was Mycobacterium avium complex (MAC), 9 (41%), followed by M. tuberculosis, 6 (27%), M .kansasii, 4 (18%), M. fortuitum, 2 (10%) and M. chelonae 1 (4%). Significant relationship was established between smoking/alcoholism and the subsequent development of tuberculosis (chi(2)=7.24, p<0.05 for smoking habit and chi(2)=4.39, p<0.05 for alcoholism). Fourteen (61%) co-infected cases presented with weight loss and cough whereas diarrhea was presented only by those patients with atypical mycobacterial co-infection, which was as high as 5 (56%) in patients with MAC co-infection. This study demonstrated the predominance of atypical mycobacteria, mainly MAC, in HIV/AIDS cases and most of them were from sputum smear-negative cases.

Tuberculosis and other clinical presentation of HIV/AIDS in patients with or without undergoing antiretroviral therapy in Kathmandu.

OBJECTIVES: To screen tuberculosis (TB) and examine the clinical presentation of AIDS in HIV sero-positive persons. METHODS: A Cross-sectional study was designed. One hundred HIV infected persons were randomly selected from different parts of the country visiting Tribhuvan University, Teaching Hospital, Kathmandu and different HIV/AIDS care centres. After taking informed consent, questionnaires were filled and three sputum specimens from each person were collected to investigate tuberculosis by Ziehl-Neelsen staining and culture. Data generated were entered into SPSS 11.5 and relevant statistical tools were applied. RESULTS: Among 100 HIV infected cases, 66 (66%) were males and 34 (34%) were females. Sixty percent of the cases were in the age group of 21-30 years. Majority of them were Smokers (41%), alcoholics (34%), illiterates (54%) and unemployed (59%). Heterosexual activity (51%) was found to be the major risk factor for HIV infection. Of the 100 HIV cases, 23 (23%) were co-infected with tuberculosis of which 18(78%) were sputum smear negative tuberculosis, mostly developed in late stage of HIV infection. Weight loss (54%) and diarrhoea (43%) were the major clinical presentations of AIDS. Antiretro-viral therapy non-receiver were more likely to suffer with various clinical disorders/TB as compared to ARV therapy receiver but the values were statistically insignificant, 2 values ranging from 0.003 to 2.24, p>0.05. CONCLUSION: Prevalence of tuberculosis was still high in HIV/AIDS patients, and specifically, sputum smear negative tuberculosis cases constituted the significant proportion, particularly in late stage of HIV infection. Weight loss and diarrhoea were found to be the major clinical presentation of AIDS. Illiterate and unemployed young adults involved in unsafe sexual practice and drug addiction were high risk of acquiring HIV infection. Key words: Clinical features, HIV/AIDS, Kathmandu; Smear Negative Tuberculosis.