Evaluation of a Simple in-House Test to Presumptively Differentiate Mycobacterium tuberculosis Complex from Nontuberculous Mycobacteria by Detection of p-Nitrobenzoic Acid Metabolites

Guirong Wang,Suting Chen,Stuart Wilson,Qian Liang,Xia Yu,Hairong Huang,Olivier Neyrolles

doi:10.1371/journal.pone.0080877

Guirong Wang, Suting Chen + Show 5 more

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https://doi.org/10.1371/journal.pone.0080877

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Journal: PLoS ONE	Publication Date: Nov 18, 2013
Citations: 21	License type: CC BY 3.0

Affiliation: Beijing Chest Hospital, Capital Medical University

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.

Highlights

Tuberculosis (TB) is one of the major causes of morbidity and mortality worldwide
Conventional tests to differentiate Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM) are mostly based on using different inhibitors such as hydroxylamine hydrochloride (HA), 8-azaguanine [3], sodium salicylate, pnitro-α acetylamino β hydroxypropiophenone (NAP) [4,5], nitroxoline [6], and propylene glycol [7]
Microorganisms In total 38 type and reference strains of the genus mycobacterium, including 5 MTC members and 33 NTM species, 65 clinical isolates representing 32 M .tuberculosis strains, 5 M. abscessus strains, 5 M. avium strains, 5 M. fortuitum strains, 5 M. gordonae strains, 5 M. intracellulare strains, 5 M. kansassi strains, 1 M. malmoense strains, 1 M. parascrofulaceum strains, 1 M. scrofulaceum strains were investigated in this study

Summary

Introduction

Tuberculosis in humans is primarily caused by bacterial special of the Mycobacterium tuberculosis complex (MTC), infections due to a range of nontuberculous mycobacteria (NTM) have been reported to be increasing [1]. Conventional tests to differentiate MTC from NTM are mostly based on using different inhibitors such as hydroxylamine hydrochloride (HA), 8-azaguanine [3], sodium salicylate, pnitro-α acetylamino β hydroxypropiophenone (NAP) [4,5], nitroxoline [6], and propylene glycol [7]. Many of these methods are technically demanding, time-consuming and require specialized reagents. 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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