Drug susceptibility of rapid and slow growing non‑tuberculous mycobacteria isolated from symptomatics for pulmonary tuberculosis, Central India.

Background: Non‑tuberculous mycobacteria (NTM) are emerging as important pathogens. Their treatment also differs from that of Mycobacterium tuberculosis. In India, any datum on them is scarce as species identification and drug susceptibility are not performed in most laboratories. Susceptibility also differs from one geographic area to another, and in our country, there are no data even to guide the clinicians to start treatment empirically. Methodology: The present study endeavours to generate drug susceptibility data on NTM isolated from sputum samples collected and stored from 6445 symptomatics for pulmonary tuberculosis during a prevalence survey and from specimens received from the hospital. Isolates were not necessarily associated with the disease. Species were identified and antibiotic susceptibility was performed using micro‑broth dilution technique as per the standard Clinical and Laboratory Standards Institute guidelines. Results: A total of 65 NTM with 11 species were identified, of which 27 belonged to Mycobacterium fortuitum complex, 14 Mycobacterium gordonae, 9 Mycobacterium avium, 7 Mycobacterium flavescens, 4 Mycobacterium scrofulaceum and one each of others. Sensitivity to amikacin for M. fortuitum was 95.22% (20 out of 21), followed by ciprofloxacin (76.19%) and clarithromycin (71.42%). All the 9 M. avium isolates, 11 of M. gordonae (78.57%), 5 of M. flavescens and 2 of M. scrofulaceum were sensitive to clarithromycin. All NTM were resistant to first‑line antitubercular drugs except 8, which were sensitive to streptomycin. Conclusions: Drug sensitivity of NTM varies from species to species. While amikacin was the best for rapidly growing mycobacteria, clarithromycin was the most active drug against M. avium and other slow growers.

Detection of metallo-β-lactamases producing Acinetobacter baumannii using microbiological assay, disc synergy test and PCR.

Background: One leading factor responsible for resistance in Acinetobacter baumannii, an important opportunist in health care institutions globally, is the production of carbapenamases like metallo-β-lactamases (MBLs), which hydrolyze a variety of β-lactams including penicillin, cephalosporins and carbapenems. However, neither any standard guidelines are available nor any method has been found to be perfect for their detection. Various methods have shown discordant results, depending upon the employed methodology, β-lactamase substrate and MBL inhibitor used. This study aims to evaluate two phenotypic methods against PCR as gold standard among carbapenem resistant A. baumannii for identifying MBL producers. Materials and Methods: A total of 130 A. baumannii were screened for imipenem and meropenem resistance by Kirby-Bauer disc diffusion method. Phenotypic expression of MBL was detected by EDTA-imipenem-microbiological (EIM) assay and extended EDTA disc synergy (eEDS) test and presence of bla-IMP and bla-VIM was detected by PCR in all the carbapenem resistant isolates. Results: Of the 43 imipenem and/or meropenem resistant A. baumannii isolates, 4 (9.3%) were found to be MBL producers by EIM and 3 (6.97%) by eEDS. Only bla-VIM gene was detected in 7 (16.28%) by PCR. In addition EIM detected 14 (32.56%) carbapenem resistant non-metallo enzyme producers. Conclusion: Of the two MBL genes targeted, bla-VIM was only detected and that too in isolates resistant to both imipenem and meropenem. Further, EIM was useful in differentiating MBL from non-metalloenzymes producers.

Comparison of disc and MIC reduction methods with polymerase chain reaction for the detection of metallo-β-lactamase in Pseudomonas aeruginosa.

Purpose: The present study was undertaken to evaluate the screening antibiotic, confirmatory phenotypic test and agent against PCR as gold standard and to detect the prevalent MBL gene. Materials and Methods: Three hundred and twenty-six Pseudomonas aeruginosa isolates were screened for resistance to Imipenem (IPM), Meropemem (MEM) and Ceftazidime (CAZ) by disc diffusion. Isolates resistant to any of these were considered screen test-positive for MBL and were subjected to Double disc synergy test (DDST) and Disc potentiation test (DPT: Using IPM, MEM and CAZ alone and with EDTA), Minimum inhibitory concentration (MIC) reduction [four-fold or more reduction in MIC of IPM and MEM in presence of chelators: EDTA and 1,10-phenanthroline (EPI/EPM: EDTA-phenanthroline- Imipenem/Meropenem Broth Microdilution method)] and polymerase chain reaction (PCR) for blaIMP and blaVIM . Results: Screen test-positives by MEM and CAZ were 19.3% as against 17.8% by IPM. MEMDDST, DPT and EPM confirmed 100% screen-test positives as against 93.7% by CAZ DDST and DPT-2, 76.2% by CAZ DPT-1, 88.9% by IPM DDST, 85.7% by IPM DPT-1 and 92.1% by EPI. IPMand CAZ DDST together confirmed 100% while IPM and CAZ DPT-2 confirmed 96.8%. All 63 screen-test positives showed the presence of blaVIM . Conclusions: MEM was found to be the best screening and confirmatory agent for MBL detection and blaVIM was found to be the prevalent MBL gene in this part of the country.

Comparison of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide tube method with the conventional method and real-time polymerase chain reaction for the detection of rifampicin resistance in Mycobacterium tuberculosis.

Colorimetric methods are cheap, reproducible, and rapid methods of detecting drug resistance in Mycobacterium tuberculosis. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) method is one such technique that has been established in our laboratory to detect rifampicin resistance. The present study compared the results of the MTT method with those of the proportion method and real-time polymerase chain reaction (RTPCR) in order to establish sensitivity and specificity of MTT. The mutations for rifampicin resistance occur in rpoB gene, and the commonest reported are in codons 526 and 531. Therefore, RTPCR was targeted at these two codons. The concordance of MTT with the proportion method and RTPCR was 94 and 72.77%, respectively, and that of RTPCR with the proportion method was 77.77%. While the study confirmed that the MTT method is a good method for detecting rifampicin resistance, it also brought out the fact that RTPCR when targeted for limited mutations is not a good tool. Either the genotypic method used should target the total 81-bp rpoB genome or methods such as DNA sequencing should be used. For resource-constraint laboratories, the MTT method can be considered as a better choice.

Mycobacterium avium bacteremia and dual infection with mycobacterium avium and mycobacterium wolinskyi in the gut of an aids patient – First case report.

An AIDS patient was admitted to a tertiary care hospital in central India with fever, weight loss, breathlessness, night sweats, diarrhoea, BMI 14kg/m2, Hemoglobin 8gm% and CD4 counts 120 cells/cumm. His blood culture by BACTEC 460 TB system revealed Mycobacterium avium bacteremia and stool culture grew Mycobacterium avium and mycobacterium wolinskyi.

Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples.

Purpose: Clindamycin is commonly used in the treatment of erythromycin resistant Staphylococcus aureus causing skin and soft tissue infections. In vitro routine tests for clindamycin susceptibility may fail to detect inducible clindamycin resistance due to erm genes resulting in treatment failure, thus necessitating the need to detect such resistance by a simple D test on routine basis. Materials and Method: 247 Staphylococcus aureus isolates were subjected to routine antibiotic susceptibility testing including oxacillin (1ìg) by Kirby Bauer disc diffusion method. Inducible clindamycin resistance was detected by D test, as per CLSI guidelines on erythromycin resistant isolates. Results: 36 (14.5%) isolates showed inducible clindamycin resistance, nine (3.6%) showed constitutive resistance while remaining 35 (14.1%) showed MS phenotype. Inducible resistance and MS phenotype were found to be higher in MRSA as compared to MSSA (27.6%, 24.3% and 1.6%, 4% respectively). Conclusion: Study showed that D test should be used as a mandatory method in routine disc diffusion testing to detect inducible clindamycin resistance.

Bacteriological Profile of Epidural Catheters.

The bacteriological profile of epidural catheters was studied in 88 patients. Skin swabs before catheterization and before removal of catheter with their controls were cultured in TSB Medium. The catheter hub, the portion at the skin puncture site and at the tip were cultured in TSB Medium. The 1cm of the catheter bit just before the tip was cultured in TGB medium for anaerobes.Both, the skin controls swabs and the anaerobic culture, were negative. From the remaining, 56 positive cultures were obtained. Staphylococcus epidermidis was the predominant organism in 52% followed by staphylococcus aureus 25%. The remaining 23% was shared by Acinetobacter, Pseudomonas, Klebsiella, and E. coli. All the positive cultures from skin prior to epidural catheterization had turned sterile by 48 hours, indicating continued bactericidal action of the disinfectant. The likely source of positive skin cultures at 48 hours is hair follicles.The catheter tip culture was positive in 9 specimen, none of which resulted in the formation of epidural abscess. In 3 cases the cultures of skin puncture site and the tip were identical indicating tracking-in of the organisms.

Isolation and identification of nontuberculous mycobacteria from water and soil in central India.

Nontuberculous mycobacteria (NTM), important organisms in the Genus Mycobacterium and commonly present in the environment, are known to cause disseminated disease in AIDS patients. In this study, NTM were isolated from environment (soil and water) of the AIDS patients with disseminated NTM disease to know the prevalence of environmental NTM species and their correlation with clinical isolates from patients of the same area. Paraffin baiting technique was used to isolate NTM from environmental samples. Once isolated, subcultures were made on Lowenstein Jensen and Middlebrook 7H10 media and the species were identified using phenotypic and genotypic techniques. A total of 26 NTM isolates belonging to seven different species could be identified. Mycobacterium avium was the only species isolated from both clinical and environmental samples of the same patient; but the isolates did not match using PCR for IS 1311 and IS 1245 spacer sequences.

Mycobacterial ES-31 Serine Protease - A Biomarker for Mycobacterium tuberculosis - A preliminary Report.

There is a need for simple and reliable method to identify Mycobacterium tuberculosis from AFB smear positive cases. Utility of mycobacterial ES-31 serine protease as a marker to detect Mycobacterium tuberculosis bacilli was explored using Fluorescein isothiocyanate conjugated anti-ES-31 serine protease antibody. The presence of ES-31 serine protease in bacilli was indicated by green fluorescence on the cell surface. Green fluorescence was observed with M.tb. H37Ra bacilli and M.tb. H37Rv bacilli while no Fluorescence was observed with M. chelonae, Nocardia farcinicum as well as in E. coli showing the usefulness of ES-31 serine protease as a marker for identification of mycobacterium tubercle bacilli in cultures.

Status of high level aminoglycoside resistant Enterococcus faecium and Enterococcus faecalis in a rural hospital of central India.

Considering the emergence of high level aminoglycoside resistance (HLAR) in enterococci this study was undertaken to determine their status in a rural setting. HLAR by disc diffusion and agar dilution, beta lactamase by nitrocefin disc and vancomycin resistance by agar dilution was determined in 150 enterococcal isolates, as per NCCLS guidelines. Only two species, Enterococcus faecalis (85.5%) and Enterococcus faecium (14.7%) were recovered, mostly from blood. Forty six percent showed HLAR. Multi drug resistance and concomitant resistance of HLAR strains to beta lactams were quite high. None showed beta lactamase activity or vancomycin resistance.

Evaluation of rapid MTT tube method for detection of drug susceptibility of Mycobacterium tuberculosis to rifampicin and isoniazid.

PURPOSE: To evaluate MTT method for detection of drug resistance to rifampicin and isoniazid in M.tuberculosis . This method utilises the ability of viable mycobacterial cells to reduce MTT( 3-4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide). METHODS: The method was standardised with known resistant and sensitive strains of M.tuberculosis and was then extended to 50 clinical isolates. An inoculum of 10 7 cfu/mL was prepared in Middlebrook 7H9 medium supplemented with oleic acid, albumin, dextrose and catalase. For each drug three tubes were used, one with INH(0.2microg/mL) or RIF(1microg/mL), another as inoculum control and third as blank control. These were incubated at 37 degrees C for four and seven days respectively for RIF and INH after which MTT assay was performed. Results were read visually and by colorimeter at 570 nm. Relative optical density unit (RODU) of 0.2 was taken as cut off. Results were compared with drug sensitivity obtained by proportion method using LJ medium. RESULTS: For rifampicin, concordance with proportion method was 90% by visual and 94% by RODU. Sensitivity and specificity was 86.8% and 100% respectively by visual method and 95.2% and 87.5% respectively by RODU. For Isoniazid, concordance was 94% and sensitivity and specificity was 94.7 and 91.7% respectively by both visual and RODU. CONCLUSIONS: MTT assay proved to be rapid and cheap method for performing drug sensitivity of M.tuberculosis.

Changing patterns of Vibrio cholerae in sevagram between 1990 and 2005.

PURPOSE: A retrospective analysis was done to note changes in prevalence, distribution of biotypes, serotypes, antibiotic susceptibility patterns and phage types of Vibrio cholerae isolated in Mahatma Gandhi Institute of Medical Sciences, Sevagram over a period of 16 years. METHODS: A total of 535 strains of V. cholerae were isolated from 10,406 stool samples and rectal swabs from January 1990 to December 2005. These comprised of serogroups O1 - 427 (79.89%), O139 - 86 (16.07%) and non O1, non O139 - 22 (4.11%). No classical V. cholerae was isolated. RESULTS: Vibrio cholerae serogroup O1 serotype Ogawa was the predominant isolate till 1992. During 1993, serogroup O139 became the main isolate; however, it completely disappeared during 1995-1996 only to reappear in 1997. Serotype Inaba in our area was conspicuous by its absence with only two strains being isolated till June 1999, but during July-December 1999, 11 out of 15 V. cholerae O1 isolates were El Tor Inaba. T4 was the predominant phage type till 1990, T2 during 1991-1994 and T27 (as per the new scheme) thereafter. Resistance to tetracycline varied between 2 and 17% for V. cholerae O1. CONCLUSIONS: The paper reports on the changing epidemiological markers of V. cholerae isolated from a rural hospital over a period of 16 years.

Candida colonization in preterm babies admitted to neonatal intensive care unit in the rural setting.

PURPOSE: Candida colonization in neonates results in significant morbidity and mortality. The purpose of this study was to determine colonization of Candida spp. in preterm babies and identify the risk factors. METHODS: Swabs from oral, rectum, groin and umblicus of 103 preterm and 100 term neonates were obtained within 24 hours of birth, day three, day five, day seven and thereafter every week till the neonate was admitted in the neonatal intensive care unit (NICU). Swabs were also collected from the mother's vagina prior to delivery. Twice every month, air of the NICU was sampled by settle plate and swabs were collected from the hands of health care workers and inanimate objects of NICU. Identification and speciation was done by standard methods. Antibiotic sensitivity was studied against amphotericin B, ketoconazole and fluconazole by disk diffusion method. RESULTS: Colonization with Candida was significantly higher in preterms. Earliest colonization was of oral mucosa and 77.1% of the preterms had colonised at various sites by the first week of life. Significant risk factors in colonized versus non-colonized preterms were male sex, longer duration of rupture of membranes (DROM), administration of steroids and antibiotics and vaginal colonization of mothers, whereas those in preterms versus terms were low birth weight and gestational age. C. albicans was the commonest species, both in the colonized preterms (45.9%) and vagina of mothers. Resistance was seen to fluconazole and ketoconazole only. No Candida spp. was isolated from health care personnel or environment. CONCLUSIONS: Colonization of preterms by Candida is a significant problem in NICU and the significant risk factors observed in colonized preterms were male sex, longer DROM, administration of steroids and antibiotics and vaginal colonization of mothers.

Evaluation of different methods for diagnosis of P. falciparum malaria.

Rapid diagnosis is a prerequisite for institution of effective treatment and reducing the mortality and morbidity of falciparum malaria. This study was taken up to compare the efficacy of various rapid methods viz, acridine orange, Plasmodium falciparum histidine rich protein II antigen detection and Field's stain with traditional microscopy i.e, Leishman stain for diagnosing falciparum malaria. Thick and thin blood films of 443 consecutive patients with history of fever with chills and rigors were examined by Leishman and Field's method. Acridine orange stained wet mounts of blood were examined under fluorescence microscopy. All films were examined by two independent microbiologists. Plasmodium falciparum histidine rich protein II antigen was detected using commercially available kit, Paracheck Pf. Out of the 443 subjects examined for P.falciparum 18.28% were detected by Leishman stain, 6.32% by Field's stain, 18.28% by acridine orange and 18.1% by antigen based technique. Field's stain missed 53 (65.4%), while Paracheck Pf was negative in 6(7.4%) of the Leishman positive samples. All Field's stain and acridine orange positives were positive by Leishman, but five Paracheck Pf positives were negative. Leishman stain is cost effective but if facilities are available one should use acridine orange for screening. The antigen detection kits are rapid, simple and are useful but to rule out false negatives in clinically suspected cases, Leishman stain is reliable.

Keratitis due to Colletotrichum dematium--a case report.

Colletotrichum dematium has been rarely reported from India before. The present case, a farmer, developed peripheral corneal ulcer five days following trauma with plant. At presentation his visual acuity was 6/60 (unaided) and 6/24P with pinhole. Slit lamp and fluorescent stain examination revealed paracentral corneal ulcer with irregular margins, stromal infiltration and multiple epithelial defects. Microbiological examination of corneal samples confirmed the initial diagnosis of fungal corneal ulcer and the fungus was identified as C.dematium. Patient was treated with topical natamycin and ciprofloxacin. Patient left against medical advice and was lost to follow up. This report emphasizes that Colletotrichum keratitis may not be rare. Early diagnosis may help in institution of specific therapy early in the disease.