[Application of rapid detection for rifampin resistance in clinical isolates of Mycobacterium tuberculosis by phage amplified biologically assay].

OBJECTIVE: To set up a rapid detection method for rifampin susceptibility with phage amplified biologically (PhaB) assay and to evaluates its value in the detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis. METHODS: The assay was established to detect rifampin resistance in 524 clinical isolates of Mycobacterium tuberculosis, and the result was compared to that of the absolute concentration method. The minimum inhibitory concentration (MIC) was detected by BACTEC MGIT 960 method for the discrepant isolates. RESULTS: Rifampin susceptibility results were available for 524 strains of Mycobacterium tuberculosis. A total of 223 strains were found to be rifampin resistant and 301 strains were rifampin susceptible detected by PhaB assay, but 211 and 313 strains were respectively found to be rifampin resistant and susceptible by conventional methods. There were 198 and 288 rifampin resistant and susceptible strains both detected by the two methods. The drug susceptibility of 35 strains was the same in 38 discrepant isolates by the PhaB assay and absolute concentration method. The sensitivity, specificity, positive and negative predictive values as well as the overall accuracy for the PhaB assay was 93.8%, 92.0%, 88.8%, 95.7% and 92.7% respectively if the judgment standard was adopted by conventional methods. CONCLUSION: The result of PhaB assay was available within 2 days. This method, which is simple and does not need special equipment, can be used for rapid screening for rifampin resistance from Mycobacterium tuberculosis.

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study.

BACKGROUND: Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients. METHODS AND FINDINGS: The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001). CONCLUSIONS: The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.

Nontuberculous mycobacteria: susceptibility pattern and prevalence rate in Shanghai from 2005 to 2008.

BACKGROUND: An increasing incidence of disease caused by nontuberculous mycobacteria (NTM) is being reported. The purpose of this study was to determine the isolation rates of NTM from various clinical specimens, and their antimicrobial susceptibility patterns, over a 4-year period in Shanghai. METHODS: All NTM isolated between 2005 and 2008 at Shanghai Pulmonary Hospital, a key laboratory of mycobacteria tuberculosis in Shanghai, China, were identified with conventional biochemical tests and 16S rRNA gene sequencing. Antimicrobial susceptibility for all NTM was determined using the BACTEC MGIT 960 system. RESULTS: A total of 21,221 specimens were cultured, of which 4868 (22.94%) grew acid fast bacilli (AFB), and 248 (5.09%) of the AFB were NTM. The prevalence rate of NTM was determined as 4.26%, 4.70%, 4.96% and 6.38% among mycobacteria culture positive samples in years 2005, 2006, 2007 and 2008 respectively. These data indicated that the prevalence rate has continuously increased. Sixteen different species of NTM were identified, the most commonly encountered NTM in Shanghai were M. chelonae (26.7%), followed by M. fortuitum (15.4%), M. kansasii (14.2%), M. avium-intracellulare complex (13.1%) and M. terrae (6.9%). The rare species identified were M. marinum, M. gastri, M. triviale, M. ulcerans, M. smegmatis, M. phlci, M. gordonae, M. szulgai, M. simiae, M. scrofulaceum and M. xenopi. The five most commonly identified NTM species showed high drug resistance to general anti-tuberculosis drugs, particularly, M. chelonae and M. fortuitum appear to be multi-drug resistance. CONCLUSIONS: The prevalence of NTM in Shanghai showed a tendency to increase over the course of the study. The five most commonly isolated NTM species showed high drug resistance to first line anti-tuberculosis drugs.