Shubhada Shenai

Unrecognized cross-contamination has been known to occur in laboratories frequently, especially with sensitive recovery system like BACTEC 460 TB. In March 2001, we investigated a pseudo-outbreak of Mycobacterium tuberculosis isolates in three smear negative clinical specimens and would like to present our experience in this communication. Methods : All suspected cases were confirmed by checking the drug susceptibility and DNA fingerprints using spoligotyping as well as restriction fragment length polymorphism. Results: On investigation, the most likely cause was found to be the use of common decontamination reagents and phosphate buffer. Conclusions: To avoid erroneous diagnosis, we have devised a dedicated decontamination procedure, which includes separate aliquoting of phosphate buffer and decontamination reagents per patient. Timely molecular analysis and appropriate changes to specimen processing have been identified as useful measures for limiting laboratory cross contamination.

To evaluate Pyrazinamide (PZA) susceptibility results obtained by phenotypic MGIT 960 TB system against enzymatic Pyrazinamidase assay and genotypic pncA gene sequencing. To find the prevalence of infections caused by M. bovis in PZA resistant M. tuberculosis complex isolates. 33 consecutive PZA resistant and 30 consecutive PZA susceptible isolates reported for PZA susceptibility testing by MGIT 960 TB system were included in this study. Presence of active pyrazinamidase enzyme was sought by using the Wayne assay. The pncA gene was amplified by PCR and then sequenced to screen mutations. All the PZA resistant isolates were further spoligotyped to identify M. bovis, if present. Of 33 PZA resistant strains by MGIT 960, 31 were Wayne assay negative and two were positive. Of the 30 susceptible PZA strains six were Wayne assay negative reporting false resistance. PncA gene sequencing revealed that 32 of the 33 MGIT PZA resistant isolates had diverse nucleotide changes scattered throughou...

To establish the critical test concentrations for seven second-line anti-tuberculosis drugs in the Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 TB system and to evaluate its efficacy compared to the Bactec 460 TB system. This study was carried out in three phases. In Phase I, pan-susceptible strains were tested to establish the minimum inhibitory concentration; in Phase II, mostly resistant strains were tested to determine a critical test concentration; and in Phase III, actual clinical isolates were tested to validate the optimal critical concentrations established in Phases I and II. The critical concentrations established for seven second-line drugs with the Bactec MGIT 960 system are amikacin 1.0 microg/ml, capreomycin 2.5 microg/ml, kanamycin 2.5 microg/ml, ofloxacin 2.0 microg/ml, moxifloxacin 1.0 microg/ml, ethionamide 5.0 microg/ml and para-amino salicylic acid 4.0 microg/ml. The Bactec MGIT 960 System is an accurate and reliable method for rapid drug susceptibility...

Rapid diagnosis of tuberculosis is essential to initiate timely and appropriate treatment to curb the spread of this potentially life threatening disease. The purpose of this study was to evaluate a phage amplification technology viz., FASTPlaque TB, for the diagnosis of tuberculosis. We evaluated the clinical utility of this new assay by analyzing 50 respiratory and 40 non-respiratory specimens, using FASTPlaque TB kit (Biotec Laboratories, UK) and the performance was compared with TB Bactec 460 semi-automated liquid culture system and conventional LJ culture method. In case of respiratory specimens phage assay gave good specificity (100%) compared with TB Bactec whereas with respect to LJ method the sensitivity and specificity were 93.1% and 88.2% respectively. In case of non-respiratory specimens comparison of results obtained by phage assay showed sensitivity of 90.9% and specificity of 88.8% with respect to TB Bactec and 87.5% and 93.8% with respect to LJ method. We believe tha...

Serpentine cord formation in BACTEC MGIT 960 medium was evaluated as a rapid method for the presumptive identification of M. tuberculosis complex (MTBC). Total 2527 samples were processed for AFB culture using MGIT 960 TB system over a period of three months. AFB smears were prepared from 1000 MGIT tubes flagged positive by the MGIT instrument and stained by ZN method to examine presence or absence of serpentine cording. The cord formation was compared with PNBA [p-nitro benzoic acid] test on MGIT system and all controversial cases were further evaluated by NAP [p-nitro-a-acetylamino-phydroxypropiophenone] test on BACTEC 460 TB system. Of the 1000 culture positives, 904 (90.4%) were identified as mycobacteria, of which 869 (96%) showed cording by smear microscopy. One (0.1%) was identified as nocardia. In the remaining 95 (9.5%) cases, primary smear made from MGIT vial was negative. Of 869 cultures showing serpentine cord formation, 842 were confirmed as MTBC and 27 as NTM by PNBA a...

We conducted a study of osteoarticular tuberculosis in patients from private and public settings in a disease endemic area. Our objective was to assess the role of mycobacterial culture and polymerase chain reaction (PCR) in the diagnosis of osteoarticular tuberculosis (TB) in settings where only clinical and imaging diagnosis form the basis for treatment. Ninety-three consecutive specimens collected from clinically suspected patients of osteoarticular TB were screened for bacterial culture, mycobacterial culture and in-house nested PCR. In addition, specimens were examined by imaging and histopathology. Ten specimens collected from patients suffering from other bone diseases were included as negative controls. Of the 93 clinically suspected TB patients, mycobacterial culture was positive for Mycobacterium tuberculosis (MTB) in 47 (51%) patients who were confirmed as definite TB cases. Of the remaining patients, 16 (17%) were diagnosed as probable, 19 (20%) as possible, and 11 (12%) as only clinically suspected TB cases. In-house nested PCR was positive in 65 (70%) cases. Fifteen patients were resistant to one or more anti-tuberculous drugs; twelve patients were multi-drug resistant, two of whom were extensively drug resistant. Mycobacterial cultures using liquid media with susceptibility should form the backbone of management of osteoarticular TB. Nested PCR enhances the sensitivity if performed in addition to culture.