Simultaneous detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacteria coexisting in patients with pulmonary tuberculosis by single-tube multiplex PCR

Leila Heidari; Gholamreza Rafiei Dehbidi; Ali Farhadi; Golnar Sami Kashkooli; Farzaneh Zarghampoor; Sepide Namdari; Noorossadat Seyyedi; Saeid Amirzadh Fard; Abbas Behzad-Behbahani

doi:10.18502/ijm.v15i3.12900

Leila Heidari Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Gholamreza Rafiei Dehbidi Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Ali Farhadi Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Golnar Sami Kashkooli Shiraz Referral Mycobacteriology Laboratory, Shiraz University of Medical Sciences, Shiraz, Iran
Farzaneh Zarghampoor Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Sepide Namdari Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Noorossadat Seyyedi Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Saeid Amirzadh Fard Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Abbas Behzad-Behbahani Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

DOI: https://doi.org/10.18502/ijm.v15i3.12900

Keywords: Isoniazid; Multiplex polymerase chain reaction; Mycobacterium tuberculosis; Nontuberculous mycobacteria; Rifampin; Sputum

Abstract

Background and Objectives: In clinical diagnostics, molecular methods are used to detect Mycobacterium tuberculosis bacilli (MTB) and to distinguish them from non-tuberculous mycobacteria (NTM). They are also used to make the right treatment decision for the patient as soon as possible. The aim of this study was to establish a rapid and novel multiplex PCR (mPCR) assay for the detection and differentiation of MTB and NTM in a single tube.

Materials and Methods: 100 sputum samples positive for acid-fast bacilli (AFB) were included in this study. Mycobacterial culture, biochemical tests, and antibiotic susceptibility testing were performed on samples. After alkaline decontamination, total DNA was extracted from the samples. A primer pair targeting the rpoB gene, encoding the beta-subunit of RNA poly- merase, was used to detect MTB and NTM, amplifying a 235-bp fragment of MTB and a 136-bp sequence of NTM. A pair of primers targeting a 190-bp fragment of the IS6110 region of MTB was also used to confirm the results. The sensitivity and specificity of the mPCR assay were evaluated using DNA extracted from standard strains. The amplified products were then analyzed by conventional agarose gel electrophoresis.

Results: Of 100 AFB smear-positive sputum samples, 92 MTB DNA, 7 NTM DNA, and one mixed-infection sample were identified in a single tube using mPCR assay. There was no correlation between the AFB degree of smear positivity and PCR results. Of seven NTM isolates, 6 (86%) were resistant to rifampin, isoniazid, and ethambutol, the three first-line anti-tuber- culosis drugs.

Conclusion: A single-tube mPCR assay based on the rpoB gene provides a rapid and reliable means of detecting and differentiating MTB and NTM in sputum specimens.