Evaluation of variants of carbol fuchsin solution to stain acid-fast bacilli in-situ by the pot method.

BACKGROUND: Ziehl-Neelsen (ZN) staining requires heating, and pre-stained smears contain viable bacilli. OBJECTIVE: To evaluate four variants of carbol fuchsin solution by the pot method and compare the results with ZN staining, taking culture as gold standard. METHOD: Five hundred sputum samples from presumptive tuberculosis cases were homogenised and divided into two parts. One part was subjected to routine ZN staining and culture on solid medium, the other was equally distributed into four pots. Equal quantities of the basic fuchsin (BF) variant were added to each pot. Variant I contained 2% BF with 10% phenol and 4% ammonium sulphate (PhAS), while Variant II had 0.6% BF with PhAS; Variants III and IV contained respectively 2% and 0.6% BF with 10% phenol only. After 1 h, smears were made from each pot and culture was performed on Löwenstein-Jensen medium. Smear results were compared with the ZN results and evaluated against culture. RESULTS AND CONCLUSION: Variant III gave excellent results compared to ZN (κ = 0.97), with sensitivity, specificity, and positive and negative predictive values similar to those of ZN, taking culture as gold standard. Pot contents were negative for Mycobacterium tuberculosis culture.

Comparision of Three Laboratory Tests for Detection of AmpC ß Lactamases in Klebsiella Species and E. Coli.

BACKGROUND AND OBJECTIVE: AmpC ß lactamases are one of the important causes of drug resistance in gram negative bacteria. Failure to detect these enzymes in the laboratory has contributed to therapeutic failures but there are till date no standard guideline available. This study was therefore undertaken to evaluate three phenotypic laboratory tests and the inhibitors used in two of the tests to detect AmpC ß lactamases produced by E. coli and Klebsiella species as they are most commonly isolated organisms. METHODS: E. coli and Klebsiella isolates from different clinical samples were tested for ESBLs production as per CLSI guidelines and excluded from the study. The non-ESBLs isolates were then screened for AmpC ß lactamases production, by cefoxitin and then confirmed by three different methods, i.e., Disc Potentiation Test (DPT) , Double Disc Synergy Test (DDST) and Modified Three Dimensional Test (M3DT) which in the absence of molecular methods, was taken as the gold standard. Boronic acid and cloxacillin were used as inhibitory agents in the Disc Potentiation and Double Disc synergy Tests. RESULTS: A total of 2,933 isolates were tested out of which 165 isolates were detected as non ESBLs producers,135 (81.82%) when screened for AmpC ß lactamases based on resistance to cefoxitin were labelled as positive. 30 (18.18%) cefoxitin sensitive isolates were labelled as probably non AmpC producers . M3DT, in addition to detecting all the 135 (100%) cefoxitin resistant isolates, also detected 5 (16.67%) cefoxitin sensitive isolates as AmpC producers. Other phenotypic tests, DPT and DDST with different inhibitors like boronic acid and cloxacillin in different potencies were all found to be less sensitive. The best results among these two methods were obtained with DDST using cloxacillin 500µg. CONCLUSION: In the absence of recommended guidelines for AmpC detection, the study reports, among the tests performed, M3DT as the best phenotypic method for AmpC confirmation, as it is not only the most sensitive but also specific test for AmpC as it rules out the resistance due to other mechanisms like the porin channel.

Detection of metallo-ß-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-ß-lactamases (MBLs), which hydrolyze a variety of ß-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, ß-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-ß-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.

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.

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.

Characteristics of Vibrio cholera 0139 strains isolated in Sevagram (Maharashtra) during April-August 1993.

A total of 44 strains of Vibrio 0139 serotype isolated between April and August 1993 at Sevagram (Wardha) were examined for expression of a number of biochemical and physiological characteristics. All strains fermented lactose within 24 h and belonged to Heiberg group III. Salt tolerance to 8 per cent NaCl was seen in 22.72 per cent strains. Haemolysis of sheep RBCs and haemagglutination of human 'O', chicken and rabbit RBCs was consistently positive. All the strains were sensitive to tetracycline and resistant to polymyxin B and cotrimoxazole.