Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting

Abstract

Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting.

Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor

“…However, traditional detection methods (i.e., AFB smear, L-J culture, and biochemical tests) usually cannot meet the requirements necessary for rapid detection in terms of time and sensitivity. PCR and PCR-based assays (e.g., real-time quantitative PCR and GeneXpert techniques) and immunologic tests (e.g., T-SOPT TB and enzyme-linked immunosorbent assays) can achieve rapid detection for MTB, but the special instrument requirements and/or expensive reagents hinder their application in the field and in resource-poor areas ( Gallo et al, 2016 ; Lekhak et al, 2016 ; Rao et al, 2021 ). Among a wide variety of rapid examination methods, LAMP, as a low-cost and valuable technique, has been widely used in the detection of various pathogens, including viruses, bacteria, and fungi ( Han et al, 2007 ; Hsu et al, 2017 ).…”

“…The reasons for this may be due to (i) the limitation of sample quality (e.g., impurities and bacterial number) and/or the proficiency of the testing, (ii) the presence of dead and/or nonculturable bacilli in samples, and (iii) false positives in mLAMP-LFB assays; however, the probability of false positives in mLAMP-LFB is low because the experiments involved the specific labeled primer and LFB verification ( Agrawal et al, 2016 ; Wang et al, 2018 ). Interestingly, two samples (collected from TB cases) tested negative by four detection methods, which may be caused by incorrect collection of specimens and/or the lack of IS6110 (including three examination-positive specimens) and mtp40 elements in the bacilli genome ( Kathirvel et al, 2014 ; Gallo et al, 2016 ). Although culture methods are often regarded as the “standard” for MTB detection/identification, they are time-consuming (4–8 weeks) and inefficient and easily delay the optimal opportunity of the patient for treatment.…”

“…In recent years, various molecular techniques have been widely applied to rapidly recognize MTBC strains ( Nghiem et al, 2015 ; Agrawal et al, 2016 ). Polymerase chain reaction (PCR) and PCR-based assays (e.g., multiplex PCR, real-time quantitative PCR, and GeneXpert methods) have also been reported as the most commonly performed methods in past studies ( Nghiem et al, 2015 ; Gallo et al, 2016 ; Lekhak et al, 2016 ). Among them, GeneXpert, which utilizes the DNA-PCR technique to simultaneously detect MTB and rifampicin resistance-related mutations, is widely used in the diagnosis of TB ( Agrawal et al, 2016 ).…”

“…Among them, GeneXpert, which utilizes the DNA-PCR technique to simultaneously detect MTB and rifampicin resistance-related mutations, is widely used in the diagnosis of TB ( Agrawal et al, 2016 ). Although these methods have outstanding analytical capabilities, the requirements of special apparatuses (thermal cycling instruments), poor availability (reactions require expensive reagents), and long detection procedures (2–4 h) restrict their application in point-of-care and field laboratories ( Gallo et al, 2016 ; Lekhak et al, 2016 ).…”

Speeding up the diagnosis of multidrug-resistant tuberculosis in a high-burden region with the use of a commercial line probe assay

“…Primary mycobacterial cultures referred to IAL in liquid mycobacteria growth indicator tube (MGIT) or on solid media were presumptively identified by observing growth and microscopic characteristics to differentiate MTBC from nontuberculous mycobacteria (NTM). Subsequent identification by phenotypic tests, including MPT64 protein detection, was carried out whenever needed, as already described 12 13 …”