Evaluation of a Rapid Differentiation Test for Mycobacterium Tuberculosis from other Mycobacteria by Selective Inhibition with p-nitrobenzoic Acid using MGIT 960

Department of Microbiology, SMS Medical College, Jaipur, India.
Journal of laboratory physicians 07/2010; 2(2):89-92. DOI: 10.4103/0974-2727.72157
Source: PubMed


Tuberculosis is caused by Mycobacterium tuberculosis (M.tb) as well as Non-tubercular mycobacterium (NTM) with similar clinical presentation. Infections due to NTM are reported to have increased in the past few years. Growth of M.tb is inhibited by p-Nitrobenzoic acid (PNB), whereas, NTM are resistant. One hundred and nine isolates from various clinical samples were identified up to species level by their growth rate, pigmentation, and a battery of biochemical tests, including niacin accumulation, nitrate reduction, and heat-stable catalase (68°C) reactions. Para-nitrobenzoic acid (PNB) inhibition test was performed to differentiate between M.tb and NTM. PNB was added to the Lowenstein-Jensen (LJ) medium and BACTEC™ MIGIT (Mycobacteria Growth Indicator Tube)960 medium to a final concentration of 500 μg/ml. All the M.tb isolates, including Mycobacterium tuberculosis H37Rv (standard strain), were inhibited by PNB on both LJ and MGIT 960. Of the NTM isolates, all were resistant to PNB on MGIT 960 and on LJ PNB, except one isolate of Mycobacterium marinum that was resistant to MGIT 960 PNB, but was susceptible to LJ PNB. The reporting time for M.tb ranged from 4-11 days (median 5.9 days) by MGIT 960 and for NTM it was 2-10 days with an average of 4.5 days. This study was carried out to establish the accuracy and efficiency of MGIT 960 PNB and to differentiate between M.tb and NTM.

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    • "For BACTEC MGIT-960T M culture, cerebrospinal fluid (CSF), lymph node tissue extract, endometrial tissue extract and pleural fluid were directly inoculated into MGIT-960 tubes and remaining samples were carried out for DNA isolation. All samples were confirmed for acid fast bacilli (AFB) by ZN staining and further subjected for identification of MTB complex using p-nitro benzoic acid (PNB) assay [15]. "
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    ABSTRACT: Background: To date, the advancements in polymerase chain reaction (PCR) assures accurate, fast identification and mycobacterial speciation in clinical settings, which promotes a better tuberculosis (TB) treatment regimen. Methods: In this study, a total of 78 clinically suspected cases of TB were processed for the detection of Mycobacterial infections by standard Ziehl Neelsen (ZN) staining, conventional Lowenstein-Jensen (LJ) and BACTEC MGIT-960™ liquid culture. Strain typing was performed by using Double Repetitive Element PCR (DRE-PCR) and Duplex PCR (DPCR) to differentiate Mycobacteriumtuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM), respectively. Results: Of 78 clinical isolates, 25 (32%) were drug-susceptible, and 53 (68%) were resistant to at least one drug. The BACTEC MGIT-960™ showed the highest (88.5%) positivity rate, compared with conventional LJ (82%) and ZN smear (61.5%). The mean time detection and drug susceptibility for MTB was 28 and 40days in LJ culture, and 10 and 13days in BACTEC MGIT-960™ culture. Using DPCR, Mycobacteriumavium infection was identified in HIV-positive (2.56%) and MTB in HIV-negative patients (97.4%), and the DRE-PCR system divulged 15 unique genotype patterns, and an institutional-based epidemiology database was created. Conclusions: The combination of an in-house DRE-DPCR system could possibly identify and differentiate MTB from other mycobacterial species in a single reaction. In addition, restriction polymorphism analysis and DNA sequencing of NTM could assist in species identification directly from clinical isolates.
    Full-text · Article · Jan 2015 · International Journal of Mycobacteriology
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    • "The indirect DST was performed on the MGIT 960 system using INH and RIF following the procedure recommended by the manufacturer (Becton Dickinson and Company, 2005). OFL and KAN were assessed following the protocol provided by Rüsch-Gerdes et al. with modifications (Morcillo et al., 2010; Rüsch-Gerdes et al., 2006) and PNB was used as previously described (Sharma et al., 2010). The tested drug concentrations are shown in Table 1. "
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    ABSTRACT: Conventional culture and drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and time consuming. For this reason alternative rapid culture and DST techniques are urgently needed to shorten the time for drug-resistance detection. A total of 222 smear-positive sputum samples were evaluated by the direct nitrate reductase assay (D-NRA) on Lowenstein-Jensen medium, for the rapid and simultaneous detection of resistance to isoniazid (INH), rifampicin (RIF), kanamycin (KAN) and ofloxacin (OFL). The p-nitrobenzoic acid was also included for identification of the M. tuberculosis complex. Results were compared with the BACTEC MGIT 960 as gold standard. A 100% of concordance was obtained between both methods for all tested drugs. D-NRA showed a turnaround time of 16.9 days to obtain results while that of the indirect MGIT 960 system was 29 days. D-NRA is a low-cost technology, easy to set up in clinical laboratories and suitable to be used for DST of M. tuberculosis in all smear-positive samples.
    Full-text · Article · Jan 2014 · Journal of Medical Microbiology
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    • "Many of these methods are technically demanding, time-consuming and require specialized reagents. In addition, ambiguous results have been reported when using these tests [8]. Other methods such as molecular probes and high performance liquid chromatography (HPLC) have been proposed for mycobacterial species differentiation but these methods are technically laborious and expensive which prevents them from being applied in laboratories with limited resources [9]. "
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    ABSTRACT: The timely differentiation of Mycobacterium tuberculosis complex (MTC) and non-tubercular mycobacterium (NTM) species is urgently needed in patient care since the routine laboratory method is time consuming and cumbersome. An easy and cheap method which can successfully distinguish MTC from NTM was established and evaluated. 38 mycobacterial type and reference strains and 65 clinical isolates representing 10 species of mycobacterium were included in this study. Metabolites of p-nitrobenzoic acid (PNB) reduction were identified using liquid chromatography and tandem mass spectrometry (LC/MS/MS). A spectrophotometric method was developed to detect these metabolites, which was evaluated on a number of MTC and NTM species. All of the tested NTM species and strains reduced PNB to p-aminobenzoic acid (PABA), while none of the MTC strains showed a similar activity. Spectrophotometric detection of PABA had 100% sensitivity and specificity for MTC and NTM differentiation among the type strains and the clinical isolates tested. PABA was identified as one of the metabolites of PNB reduction. All the tested NTM species metabolized PNB to PABA whereas the MTC members lacked this activity. A simple, specific and cost-effective method based on PABA production was established in order to discriminate MTC from NTM from cultured organisms.
    Full-text · Article · Nov 2013 · PLoS ONE
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