Rapid and Simple Approach for Identification of Mycobacterium tuberculosis Complex Isolates by PCR-Based Genomic Deletion Analysis

Abstract

Although the virulences and host ranges differ among members of theafricanum, but further characterization resulted in profiles specific for all members. Although six RD regions were used in the analyses with the original 88 isolates, it was found that the use of RD 1, RD 9, and RD 10 was sufficient for initial screenings, followed by the use of RD 3, RD 5, and RD 11 if the results for any of the first three regions were negative. When 605 sequential clinical isolates were screened, 578 (96%) were identified as M. tuberculosis, 6 (1%) were identified as M. africanum, 8 (1%) were identified as M. bovis, and 13 (2%) were identified as M. bovis BCG. Since PCR-based assays can be implemented in most clinical mycobacteriology laboratories, this approach provides a rapid and simple means for the differentiation of members of TBC, especially M. bovis and M. tuberculosis, when it is important to distinguish between zoonotic sources (i.e., cattle and unpasteurized dairy products) and human sources of tuberculosis disease.The Mycobacterium tuberculosis complex (TBC) (4, 34) comprises the closely related organisms M. tuberculosis, M. africanum, M. bovis, the M. bovis BCG vaccine strain, and two rarely seen members, M. microti and M. canettii (35). Differentiation of the members of the TBC is necessary for the treatment of individual patients and for epidemiological purposes, especially in areas of the world where tuberculosis has reached epidemic proportions or wherever the transmission of M. bovis between animals or animal products and humans is a problem. In addition, it can be important to rapidly identify isolates of M. bovis BCG recovered from immunocompromised patients.Although no clear-cut means of differentiation of the members of the TBC was found in the past by using numerical classification (34), a few conventional methods have been useful. Those methods include assays for the ability to metabolize glycerol or pyruvate in Loewenstein-Jensen medium, oxygen preference (aerophilic versus microaerophilic), niacin accumulation, nitrate reductase activity, colony morphology, and resistance to two compounds, thiophen-2-carboxylic acid hydrazide (TCH) and pyrazinamide (PZA) (12,19,38). Partially due to the slow growth of the TBC, interpretation of the results of these assays can be highly subjective, especially interpretation of differences in colony morphology (19), which can be due to the loss of virulence or to mutations associated with drug resistance. An alternative approach is the use of high-performance liquid chromatography; however, only the profile for M. bovis BCG differs from those for the other members of the complex (10).Testing for resistance to TCH has been reported to be the only single test that assigned isolates to any specific member of the TBC; classical M. tuberculosis isolates are resistant to TCH, irrespective of their resistance to isoniazid (8). Alternatively, the Asian strain of M. tuberculosis and all other members of the TBC are TCH susceptible (39, 40). However, cross-resistance to TCH has ...

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

“…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.…”

“…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.…”

“…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.…”

Diagnosis of gastrointestinal tuberculosis: Using cytomorphological, microbiological, immunological and molecular techniques — A study from Central India

“…This abdominal form of TB has an insidious course like any other chronic infectious disease without any specific laboratory, radiological or clinical findings. Due to this non-specificity and great difficulties in its diagnosis a number of rapid investigative methods have been surfacing out to aid in the diagnosis of GITB employing a diverse MTB genomic targets including the IS 6110 insertion sequences (16)(17)(18)(19). Undeniably, the PCR systems developed so far have shown good levels of sensitivity (90 to 100%) only on AFB smear -positive samples (20).…”

Distinct Clinical Features in Nontuberculous Mycobacterial Disease with or without Latent Tuberculosis Infection

“…DNA was isolated by 80% ethanol precipitation, dissolved in 50-µl sterile distilled water and stored in −20°C until assay. DNA samples were amplified for the RD1 region (150 bp) by PCR analysis using the same primers described previously (Parsons et al 2002). DNA from the H37RV strain of M. tuberculosis was used as a positive control.…”