Multicenter Evaluation of a Transcription-Reverse Transcription Concerted Assay for Rapid Detection of Mycobacterium tuberculosis Complex in Clinical Specimens

Abstract

A European multicenter study was performed to evaluate the performance of a new method, based on the transcription-reverse transcription concerted reaction (TRC-2), which enabled one-step amplification and real-time detection of the Mycobacterium tuberculosis 16S rRNA target directly in clinical specimens. A total of 633 respiratory and nonrespiratory specimens were tested, and the results were compared with those from smears and cultures. A total of 129 patients (Paris center) were followed up in order to evaluate the clinical performance of TRC-2. By using M. tuberculosis complex strains to inoculate sterile sputa, the detection limit of TRC-2 was found to be 30 to 50 CFU/ml. A total of 548 respiratory specimens and 59 extrapulmonary specimens were assessable. For pulmonary specimens, the sensitivities of TRC-2 and acid-fast smear were 86.8% and 50.4%, respectively (P ‫؍‬ 0.002). The specificities were 97.5% and 100%, respectively. For extrapulmonary specimens, the sensitivities of TRC-2 and acid-fast smear were 83.3% and 8.3% (P < 0.0001), and the specificities were 95.8% and 100%, respectively. Fifteen of 129 patients were diagnosed with pulmonary tuberculosis (TB). The sensitivities of culture and TRC-2 were 80% (12/15) and 86.7% (13/15) (P ‫؍‬ 0.16), and the specificities were 100% and 93.9%, respectively. Based on an 11.6% incidence of TB in our population, the positive predictive values of TRC-2 and culture were 81.3% and 100%, respectively, and the negative predictive values were 98.2% and 97.4%, respectively. These results demonstrated that detection of M. tuberculosis complex in clinical specimens by TRC-2 with ready-to-use reagents was an efficient and rapid method for the diagnosis of pulmonary and extrapulmonary TB.Accurate and early diagnosis of tuberculosis (TB) remains a critical step in the management and control of TB (22). To this day, the primary rapid tool in case detection remains the microscopic examination of clinical specimens in order to detect acid-fast bacilli. The advantages and disadvantages of the acidfast smear (AFS) are well defined in the literature. Although culture is considered the gold standard, it has the major disadvantage of requiring 2 to 6 weeks to obtain a result, since mycobacteria are slow-growing organisms (5). The use of an approach combining smear microscopy and specific identification at the same time will enhance predictive values for the rapid diagnosis of TB.Nucleic acid amplification technology (NAAT) tests represent an important technical advance for microbiology laboratories. However, NAAT developed for the rapid diagnosis of TB has never achieved the same sensitivity as culture (2,5,11,18,19). In addition, NAAT tests have been time-consuming, despite the fact that some approaches are now partially automated (Cobas Amplicor) (1, 9). These systems are more rapid than culture-based methods, but they can still require up to 12 h and a series of complicated procedures to get the results. They need a high level of expertise from experienced and dedicated techni...

The Biology of Mycobacterium Tuberculosis Infection.

“…These assays form the basis for the microbiological diagnosis of TB and the clinicians may require detection of Mtb in one or more specimens depending on the clinical symptoms, if any 51. High sensitivity and specificity has been observed in the detection of Mtb in specimens such as sputum, bronchoalveolar lavage or induced sputum for the diagnosis of pulmonary TB 52. The introduction of new, highly sensitive, fully automated molecular assays for the detection of Mtb has been recognized as a major achievement of the last decades,53 though it is important to remind that molecular diagnosis should not be ordered routinely when the clinical suspicion of TB is too low 54–56.…”

“…51 High sensitivity and specificity has been observed in the detection of Mtb in specimens such as sputum, bronchoalveolar lavage or induced sputum for the diagnosis of pulmonary TB. 52 The introduction of new, highly sensitive, fully automated molecular assays for the detection of Mtb has been recognized as a major achievement of the last decades, 53 though it is important to remind that molecular diagnosis should not be ordered routinely when the clinical suspicion of TB is too low. 54 – 56 Non-pulmonary forms of TB may be more problematic to diagnose because of the difficulties in identifying the proper specimens and the lower sensitivity of the microbiological assays in the non-pulmonary specimens, probably resulting from a lower bacterial concentration.…”

Comprehensive Multicenter Evaluation of a New Line Probe Assay Kit for Identification of Mycobacterium Species and Detection of Drug-Resistant Mycobacterium tuberculosis

“…M. tuberculosis was identified at hospitals A and B by use of TRCRapid M.TB kits (Tosoh Bioscience, Tokyo, Japan), based on the transcription-reverse transcription concerted reaction (13,44), and at hospitals C to F and RIT by use of the Cobas Amplicor MTB test. M. avium and M. intracellulare were identified at hospitals C to F and RIT by use of the Cobas Amplicor M. avium and M. intracellulare tests, respectively.…”

Gargle sample is an effective option in a novel fully automated molecular point-of-care test for influenza: a multicenter study

“…It has been used to diagnose tuberculosis, nontuberculous mycobacterial infections, mycoplasma pneumonia, chlamydial infections, and gonorrhea. (2, 3) Recently, a novel rapid TRC that can detect influenza A and B within 15 min of nasopharyngeal swabs and gargle samples without purification was developed by simplifying the sample preparation step. (4) In a previous single-center study, the novel TRC method was of comparable sensitivity to the conventional reverse transcription-polymerase chain reaction (RT-PCR) method for detecting influenza viruses in both nasopharyngeal swabs and gargle samples.…”