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 Mycobacterium tuberculosis 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 40. days in LJ culture, and 10 and 13. days in BACTEC MGIT-960™ culture. Using DPCR, Mycobacterium avium 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.
    International Journal of Mycobacteriology 01/2015; 4(1). DOI:10.1016/j.ijmyco.2014.11.061
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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.
    PLoS ONE 11/2013; 8(11):e80877. DOI:10.1371/journal.pone.0080877 · 3.23 Impact Factor
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    • "This test was performed by adding Superoxol to a positive culture of mycobacteria at 37°C. 5 min after addition of the Superoxol, the strength of the catalase reaction was determined by measuring the height of the bubbles, characteristic of the catalase reaction, in the tube. MTBC has low catalase activity.[18] "
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    ABSTRACT: Mycobacterium tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) may or may not have same clinical presentations, but the treatment regimens are always different. Laboratory differentiation between MTBC and NTM by routine methods are time consuming and cumbersome to perform. We have evaluated the role of GenoType(®) Mycobacterium common mycobacteria/additional species (CM/AS) assay for differentiation between MTBC and different species of NTM in clinical isolates from tuberculosis (TB) cases. A total of 1080 clinical specimens were collected from January 2010 to June 2012. Diagnosis was performed by Ziehl-Neelsen staining followed by culture in BacT/ALERT 3D system (bioMerieux, France). A total of 219 culture positive clinical isolates (BacT/ALERT(®) MP cultures) were selected for differentiation by p-nitrobenzoic acid (PNB) sensitivity test as and BIO-LINE SD Ag MPT64 TB test considering as the gold standard test. Final identification and differentiation between MTBC and different species of NTM were further confirmed by GenoType(®) Mycobacterium CM/AS assay (Hain Lifescience, Nehren, Germany). Out of 219 BacT/ALERT(®) MP culture positive isolates tested by PNB as 153 MTBC (69.9%) and by GenoType(®) Mycobacterium CM/AS assay as 159 (72.6%) MTBC and remaining 60 (27.4%) were considered as NTM species. The GenoType(®) Mycobacterium CM/AS assay was proved 99.3% sensitive and 98.3% specific for rapid differentiation of MTBC and NTM. The most common NTM species were; Mycobacterium fortuitum 20 (33.3%) among rapid growing mycobacteria and Mycobacterium intracellulare 11 (18.3%) among slow growing mycobacteria. The GenoType(®) Mycobacterium assay makes rapid and accurate identification of NTM species as compared with different phenotypic and molecular diagnostic tool and helps in management of infections caused by different mycobacteria.
    Journal of laboratory physicians 07/2013; 5(2):83-9. DOI:10.4103/0974-2727.119847
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