Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex, Real-Time PCR

Abstract

The rapid identification of mycobacteria from culture is of primary importance for the administration of empirical antibiotic therapy and for the implementation of public health measures, yet there are few commercially available assays that can easily and accurately identify the mycobacteria in culture in a timely manner. Here we report on the development of a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacteria in our laboratory in two parallel reactions. The mycobacteria identified by this assay include the Mycobacterium tuberculosis complex (MTC), the M. avium complex (MAC), the M. chelonae-M. abscessus group (MCAG), the M. fortuitum group (MFG), and M. mucogenicum. The primer targets included the 16S rRNA gene and the internal transcribed spacer. The assay was initially validated with a repository of reference strains and was subsequently tested with 314 clinical cultures identified by the AccuProbe assay or high-performance liquid chromatography. Of the 314 cultures tested, multiplex, real-time PCR produced congruent results for 99.8% of the 1,559 targets evaluated. The sensitivity and the specificity were each 99% or greater for MTC (n ‫؍‬ 96), MAC (n ‫؍‬ 97), MCAG (n ‫؍‬ 68), and M. mucogenicum (n ‫؍‬ 9) and 95% and 100%, respectively, for MFG (n ‫؍‬ 19). We conclude that this multiplex, real-time PCR assay is a useful diagnostic tool for the rapid and accurate identification of MTC and clinically relevant nontuberculous mycobacteria.Mycobacterium tuberculosis, the causative agent of tuberculosis, is among the leading infectious causes of death in developing nations (23). In resource-rich countries, where tuberculosis is not endemic, nontuberculous mycobacteria (NTM) are responsible for the majority of mycobacterial infections in both immunocompromised and immunocompetent individuals (14). The clinical gravity of mycobacterial infections necessitates rapid isolation by culture and timely identification by the appropriate diagnostic assays. Rapid identification not only serves to focus empirical antibiotic therapy and thus avoid unnecessary drug exposure but also may aid with the appropriate respiratory isolation and prevention of secondary cases (15).Few molecular methods commercially available in the United States easily and rapidly identify mycobacteria in culture. Although the AccuProbe assay by Gen-Probe (San Diego, CA) is sufficient for the identification of isolates of the Mycobacterium tuberculosis complex (MTC) and the M. avium complex (MAC) (37), it lacks probe sets specific for rapidly growing mycobacteria, such as the M. M. peregrinum, and M. alvei (11). For these isolates, other methods, such as biochemical reactions (6, 33), high-performance liquid chromatography (HPLC) (13), and DNA sequencing (17), are required for identification. To address the need for a simple and rapid molecular assay with a broader identification scope, we developed a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacterial isolates (both slowly growing and ra...

Identification of Mycobacterium tuberculosis in the cerebrospinal fluid of patients with meningitis using nested PCR

“…The PCR system also has been used as an epidemiological tool in strain classification (23,24). The most widely used target sequence for the diagnosis of tuberculosis has been the IS6110 insertion element, present in a different number of copies in the genome of species of the M. tuberculosis complex (12,35) and PCR techniques based on this sequence have shown to be useful for diagnosis.…”

“…The analysis of the genomic sequence of M. tuberculosis (19) and studies on comparative genomics (20,21) have identified major deletions that specifically characterize different species of the M. tuberculosis complex, and are useful in differentiating these from non-tuberculous mycobacteria (22)(23)(24)(25). Some studies have reported the use of PCR and quantitative PCR to identify M. tuberculosis in the CSF of patients with TBM, with varying sensitivity and specificity (25,26).…”

Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system

“…Primers targeted to the 16S rRNA gene, RNA polymerase gene (rpoB), hsp65 gene or 16-23S internal transcribed spacer (ITS) region have been reported in relation to detecting mycobacteria by PCR in previous publications. As the HRMA assay could differentiate gene fragments of up to 300 bp, four pairs of genus-specific primers flanking gene fragments of different mycobacteria were selected from five previous studies (Foongladda et al, 2009;Kim et al, 2010;Richardson et al, 2009;Roth et al, 2000;Shrestha et al, 2003). The primer sequences are listed in Table S1 (available in JMM Online).…”

Comparison of Different DNA Extraction Protocols for Molecular Diagnosis of Mycobacterium Tuberculosis

“…In the last decade, PCR became the most commonly used diagnostic techniques in TB specialized labs worldwide due to its potential to detect MTB directly from clinical samples (Richardson et al, 2009). The sensitivity of PCR depends to a large extend on the quality of extracted DNA as the lysis of mycobacterial cell wall is difficult.…”