Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India

Purpose: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. Materials and Methods: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. Results: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. Conclusion: MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.     

Validation of use of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and application of the method to the investigation of a hospital outbreak.

Acinetobacter spp. are being reported with increasing frequency as causes of nosocomial infection. In order to identify reservoirs of infection as quickly as possible, a rapid typing method that can differentiate epidemic strains from environmental and nonepidemic strains is needed. In 1993, a cluster of Acinetobacter baumannii isolates from five patients in the adult intensive therapy unit of our tertiary-care teaching hospital led us to develop and optimize a rapid repetitive extragenic palindromic sequence-based PCR (REP-PCR) typing protocol for members of the Acinetobacter calcoaceticus-A. baumannii complex that uses boiled colonies and consensus primers aimed at repetitive extragenic palindromic sequences. Four of the five patient isolates gave the same REP-PCR typing pattern as isolates of A. baumannii obtained from the temperature probe of a Bennett humidifier; the fifth isolate had a unique profile. Disinfection of the probe with 70% ethanol, as recommended by the manufacturer, proved ineffective, as A. baumannii with the same REP-PCR pattern was isolated from it 10 days after cleaning, necessitating a change in our decontamination procedure. Results obtained with REP-PCR were subsequently confirmed by ribotyping. To evaluate the discriminatory power (D) of REP-PCR for typing members of the A. calcoaceticus-A. baumannii complex, compared with that of ribotyping, we have applied both methods to a collection of 85 strains that included representatives of six DNA groups within the complex. Ribotyping using EcoRI digests yielded 53 patterns (D = 0.98), whereas 68 different REP-PCR patterns were observed (D = 0.99). By computer-assisted analysis of gel images, 74 patterns were observed with REP-PCR (D = 1.0). Overall, REP-PCR typing proved to be slightly more discriminatory than ribotyping. Our results indicate that REP-PCR typing used boiled colonies is a simple, rapid, and effective means of typing members of the A. calcoaceticus-A. baumannii complex.