Evaluation of a Single-Tube Multiplex Real-Time PCR for Differentiation of Members of the Mycobacterium tuberculosis Complex in Clinical Specimens

Abstract

Members of the M. microti, M. canettii, M. caprae, M. pinnipedii, and M. mungi (1). Differentiation between the species within this complex is important for public health surveillance and reference testing, as most species within the MTBC have been reported to infect humans (6). Additionally, it may be important information for physicians to rapidly identify severe side effects of M. bovis BCG in patients with bladder cancer or in vaccinated individuals or to assess transmission of M. bovis from animals or animal products. It is also helpful to direct patient treatment, as M. bovis is intrinsically resistant to pyrazinamide (PZA) (14). A recent report on the incidence of M. bovis in San Diego, CA, highlighted the importance of routine species-level identification in U.S. tuberculosis (TB) surveillance. That investigation reported the growing incidence of TB caused by M. bovis (45% of all culture-positive TB cases in children between 1994 and 2005), which may be representative of other communities in the United States (17). In New York, 3 to 7% of the MTBC specimens that our laboratory receives each year are identified as species other than M. tuberculosis.Currently there are few methods available to rapidly differentiate species within the MTBC. The existing methods are not always suitable; as conventional (biochemical) methods are highly subjective, high-pressure liquid chromatography (HPLC) can identify only M. bovis BCG (3), and DNA probe and amplification assays based on 16S rRNA gene sequences can identify specimens only as MTBC. Published molecular assays to differentiate members of the MTBC are not in realtime PCR formats (6,14,20), do not identify members other than M. tuberculosis, M. bovis, and M. bovis BCG (10, 15), and are not validated for use on clinical specimens (10,15,16). To date no single assay to perform this diagnostic test exists for probe-based differentiation of members of the MTBC directly from clinical specimens.Our laboratory has historically relied on a well-validated conventional PCR based on comparative genomics (14), specifically, regions of difference (RD), that is utilized on cultured material to differentiate the MTBC species present. This assay is generally reliable but has limitations because culture may take 2 to 8 weeks and the conventional PCR assay involves two to five separate reactions requiring postamplification processing, thereby increasing the risk of contamination. For these reasons, a single-tube, multiplex, real-time PCR assay (MTBC-RD real-time PCR) was developed for rapid differentiation of members of the MTBC from both clinical specimens and cultured material, based on the same comparative genomics. This new assay was evaluated on 919 patient samples received by the Wadsworth Center (WC), New York State Department of Health, over a 16-month period.

Region of Difference 4 in Alpine Mycobacterium caprae Isolates Indicates Three Variants

“…The primers and the probe are listed in Table 2. Nucleotide sequence data for the primers and the probe were deduced from various reports (14,16,18,26,33) or were generated in this study using the NCBI/Primer-BLAST program (http://www.ncbi.nlm.nih .gov/tools/primer-blast/index.cgi?LINK_LOCϭBlastHome) based on the sequences derived from whole-genome sequencing analysis.…”

“…Rodriguez et al (8) confirmed the existence of 545-bp RD4-specific sequences in 62 of 63 M. caprae isolates. Notably, the detection of an RD4 deletion has been used as a major criterion for distinguishing M. bovis from the other members of the MTC, especially from M. caprae (16)(17)(18)(19)(20)(21).One M. caprae-specific T-to-G substitution at position 1311 in the gyrB sequence has been described in several M. caprae isolates from central Europe (12,22,23) and Spain (3,8). However, Kubica et al (24) reported that 3 of 55 German M. caprae isolates from human patients did not show this M. caprae-specific SNP.…”

“…Rodriguez et al (8) confirmed the existence of 545-bp RD4-specific sequences in 62 of 63 M. caprae isolates. Notably, the detection of an RD4 deletion has been used as a major criterion for distinguishing M. bovis from the other members of the MTC, especially from M. caprae (16)(17)(18)(19)(20)(21).…”

Espondilodiscitis con absceso intrarraquídeo y paraespinal causada por bacilo de Calmette-Guérin (BCG)

“…Desde el punto de vista bioquímico, lo inhibe la hidracida del ácido tiofeno-2-carboxílico, muestra fuerte actividad ureasa y es resistente a pirazinamida y a cicloserina (esto último lo diferencia del M. bovis común). Aunque ya hay descritas en la literatura especializada algunas técnicas para desmembrar el complejo [18][19][20] , no hay muchas pruebas comercializadas, siendo éste un aspecto de una gran importancia clínica y epidemiológica. Para la identificación de la especie causante de la infección del presente caso se realizó una técnica de amplificación de ácidos nucleicos casera.…”

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

“…Unlike PCR assays previously mentioned for differentiation of members of Mtb complex, a real-time PCR assay, developed by Halse and colleagues (Halse et al, 2011) …”