Evaluation of an immunochromatographic test for discrimination between Mycobacterium tuberculosis complex & non tuberculous mycobacteria in clinical isolates from extra-pulmonary tuberculosis.

Background & objectives: Accurate diagnosis of tuberculosis (TB) is crucial to facilitate early treatment of the patients, and to reduce its spread. Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non tuberculous mycobacteria (NTM) may or may not be the same, but the treatment regimen is always different for both the infections. Differentiation between MTBC and NTM by routine laboratory methods is time consuming and cumbersome. This study was aimed to evaluate an immunochromatographic test (ICT), based on mouse monoclonal anti-MPT64, for simple and rapid discrimination between MTBC and NTM in clinical isolates from extra-pulmonary tuberculosis cases. Methods: A total of 800 clinical samples were collected from patients suspected to have extra-pulmonary tuberculosis. Preliminary diagnosis has been done by direct Ziehl–Neelsen (ZN) staining followed by culture in BACTEC system. A total of 150 clinical isolates, which were found positive in BD 460 TB system during September 2009 to September 2010 were selected for the screening by ICT test. p-nitro-α-acetylamino- β-hydroxy propiophenone (NAP) test was performed for differentiation of MTBC and NTM. M. tuberculosis complex was further confirmed by IS6110 PCR of BACTEC culture positive isolates, this served as the reference method for MTBC identification and comparative evaluation of the ICT kit. Results: Of the 150 BACTEC culture positive isolates tested by ICT kit, 101 (67.3%) were found positive for MTBC and remaining 49 (32.7%) were considered as NTM. These results were further confirmed by IS6110 PCR that served as the reference method for detection of MTBC. H37Rv reference strain was taken as a control for ICT test and IS6110 PCR. The reference strain showed the presence of MPT64 antigen band in the ICT test. Similar bands were formed in 101 of 102 MTBC isolates tested, proving 99.1 per cent sensitivity and no bands were detected in 48 (100%) NTM isolates tested, proving 100 per cent specificity of the ICT kit. Interpretation & conclusions: Our findings show that ICT test can be used on direct culture positive specimens. It does not require any special equipment, is simple and less time consuming. It can easily discriminate between MTBC and NTM and thus can help in appropriate management of tuberculosis.

Detection of Mycobacterium tuberculosis directly from sputum specimens & phenotypic drug resistance pattern of M. tuberculosis isolates from suspected tuberculosis patients in Chennai.

Background & objectives: mRNA is more rapidly destroyed in cells than rRNA or genomic DNA, an assay targeting bacterial mRNA would provide a better guide to mycobacterial viability than amplification tests directed at DNA or rRNA targets. This study was carried out to standardize reverse transcriptase PCR (RT-PCR) targeting 85B gene for the rapid detection of viable Mycobacterium tuberculosis from sputum specimens of suspected TB patients at Chennai, South India and to detect MDR-TB circulating in this population. Methods: Sputum samples from clinically suspected tuberculosis patients (n=301) and 78 controls were included in the study. The sputum samples were collected in sterile diethyl pyrocarbonate (DEPC) treated containers and transported in ice to the laboratory within 2 h to prevent degradation of RNA. RT-PCR targeting 85B gene, mycobacterial culture and phenotypic drug susceptibility testing for the first line drugs streptomycin (S), isoniazid (H), rifampicin (R), ethambutol (E) and pyrazinamide (Z) were performed by BACTEC microMGIT culture system for all the sputum specimens. Results: All the 78 controls were negative for culture and RT-PCR. Among the 301 sputum specimens from patients, 231 (76.8%) were RT-PCR positive and 70 (23.2%) were negative. There were 166 M. tuberculosis isolates, of which 11 (2.9%) were MDR-TB, 33 (8.7%) were polyresistant, 31 (8.2%) were monoresistant and 91 (30.2%) were sensitive to all five first line anti-tuberculous drugs by phenotypic drug susceptibility testing. Monoresistance was higher with Z [20 (20.8%)], followed by S [6 (3%)]. Interpretation & conclusions: RT-PCR targeting 85B gene of M. tuberculosis was a specific, rapid, reliable technique to detect the M. tuberculosis directly from sputum specimens. Our results showed that 2.9 per cent of M. tuberculosis isolates in the study population of Chennai were MDR.

comparison of two-step tuberculin skin test between health-care workers and nonhospital employees

The tuberculin test is widely used to identify tuberculosis infection. Some individuals infected with Mycobacte-rium tuberculosis may have an initial negative skin test reaction to tuberculin. The two step purified protein derivative skin test can decrease misinterpretation of tuberculin test .This study was aimed at comparing the two- step tuberculin skin test and booster phenomenon in health care workers and non hospital employees. One hundred and eighty five health service employees from Razi University Hospital and 181 non hospital employees were subjected to an initial tuberculin skin test. Those who were negative on the first test underwent a second one 2 weeks later. The reactions to the tests were measured 72 hours later. Tests with an induration of > 10 mm was considered positive. Moreover, second tests with an induration of at least 6 mm increase relative to the relevant first tests were considered positive. Compared to non hospital employees [n=79, 43.6%], a significantly higher number of health care workers [n=113, 61.1%] were positive on the first tuberculin test. 18.5% of health care workers and 31.5% of non hospital employees demonstrated a boosted reaction after the second tuberculin test. The occurrence of boosted reaction was significantly associated with age both groups. There was no association between the presence of BCG scars and the occurrence of boosted reaction. the present study shows that the prevalence of TB was higher among health care worker than non hospital employees. Moreover, it demonstrated that non hospital employees had a higher age-associated booster reaction to the second tuberculin skin test than health care workers. Therefore, to avoid missing false negative cases, it would be necessary to do a second tuberculin test for subjects with a negative reaction to the initial test, especially in subjects older than 40 years

Identification of mycobacterium tuberculosis from meningitis cases using API-ZYM based scheme

A biochemical identification scheme for Mycobacterium spp. [Moinard et al., 1989] consisting of the API-ZYM strip plus five additional tests was evaluated on 50 strains of acid-fast bacilli isolated from cerebrospinal fluid of meningitis cases in Egypt. All isolates were identified as Mycobacterium tuberculosis. This five-hour assay was easy to run and interpret when freshly grown isolates [20-25 days old] served as the test inoculum