Identification Of Mycobacterium Tuberculosis Complex Research Articles

Evaluation of three real-time PCR assays for differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria species in liquid culture media

We evaluated the analytical performance of M. tuberculosis complex (MTBC)/nontuberculous mycobacteria (NTM) PCR assays for differential identification of MTBC and NTM using culture-positive liquid media. Eighty-five type strains and 100 consecutive mycobacterial liquid media cultures (MGIT 960 system) were analyzed by a conventional PCR assay (MTB-ID® V3) and three real-time PCR assays (AdvanSure™ TB/NTM real-time PCR, AdvanSure; GENEDIA® MTB/NTM Detection Kit, Genedia; Real-Q MTB & NTM kit, Real-Q). The accuracy rates for reference strains were 89.4%, 100%, 98.8%, and 98.8% for the MTB-ID V3, AdvanSure, Genedia, and Real-Q assays, respectively. Cross-reactivity in the MTB-ID V3 assay was mainly attributable to non-mycobacterium Corynebacterineae species. The diagnostic performance was determined using clinical isolates grown in liquid media, and the overall sensitivities for all PCR assays were higher than 95%. In conclusion, the three real-time PCR assays showed better performance in discriminating mycobacterium species and non-mycobacterium Corynebacterineae species than the conventional PCR assay.

Cord formation in BACTEC<SUP>TM</SUP> medium aids rapid identification of <I>Mycobacterium tuberculosis</I> complex

Mycobacterium tuberculosis complex (MTC) organisms form serpentine cords in fluid culture medium. Reporting of a presumptive identification of MTC based on cording allows rapid identification of patients with tuberculosis. A total of 612 positive mycobacterial cultures from 316 patients over 3 years (2008-2010) were evaluated for the presence of cord formation. Cording was identified in 426 (69.6%) specimens, while the reference laboratory confirmed M. tuberculosis in 424 specimens (69.3%). Sensitivity of the test in our laboratory was 99.1% (95%CI 97.4-99.7) and specificity was 96.8% (95%CI 92.8-98.7). Presumptive identification of M. tuberculosis by the presence of cording formation is both sensitive and specific.

Rapid identification of Mycobacterium tuberculosis complex in clinical isolates by combining presumptive cord formation and MPT64 Antigen Immunochromatographic Assay

PurposeCombining the results of presumptive cord formation in smear and MPT64 Antigen Immunochromatographic Assay has been suggested to reduce the false negative and positive rates for identification of Mycobacterium tuberculosis (MTB) complex in liquid culture. This study was done to evaluate the clinical utility of combining the results of the two tests for rapid identification MTB complex in mycobacterial isolates. Methods484 isolates of mycobacteria obtained in MGIT culture were identified using presumptive cord formation in smear and further by MPT64 Antigen ICT assay. Result obtained were analyzed taking PNB inhibition test as the reference standard. ResultsCombining the results of the two tests, 464 (95.9%) isolates were correctly identified while discrepant results were obtained in 20 (4.1%) isolates. When the results of the two tests were intersected, the specificity and PPV was 100%, but the sensitivity decreased to 96.4% and the NPV to 68.6%. On the other hand, when the results of the two methods were combined, the sensitivity and NPV was 100%, but the specificity decreased to 88.6% and the PPV to 99.1%. ConclusionPresumptive cord formation and MPT64 antigen ICT assay can be used in combination for identification of MTB complex. When both the test are positive, the culture can be reported to contain MTB complex. If both the tests are negative, the culture should be reported to contain NTM. Only when discrepant results are obtained by the two tests, further evaluation is necessary to ensure an accurate diagnosis.

Mycobacterium tuberculosis complex identification by polymerase chain reaction from positive culture in patients from Jamot and Mbalmayo district hospitals

mycobacteria (NTM) such as Para nitro benzoic acid (PNB) inhibition tests are often time consuming. This study aim at using PCR amplification of specific markers (hupB, IS6110, IS1081, oxyR and rpoB) for more rapid detection of MTBC in positive cultures. The study was conducted in Jamot Hospital, the largest urban treatment center for tuberculosis in Cameroon and in Mbalmayo District Hospital, a small rural district hospital. Mycobacterial culture was performed on all smear positive sputa. All positive cultures were subjected to drug susceptibility testing (DST) using the indirect proportion method. On the same way, MTBC were differentiated from other mycobacreia using the PNP inhibition test. DNA extracted from positive cultures was subjected to PCR amplification using specific primers (hupB, IS6110, IS1081, oxyR and rpoB). Analysis of PCR products was done by agarose gel electrophoresis. A total of 79 smear positive pulmonary tuberculosis patients were enrolled at the two sites. Drug susceptibility carried out showed that among the samples analyzed, 68 (86.08%) were susceptible to all TB drugs tested, while 11 (13.92%) were resistant to at least one of them. Resistance to streptomycin was the most frequent (8.86%), followed by resistance to isoniazid (5.06%). Identification by PCR using specific markers as hupB, IS6110, IS1081, oxyR and rpoB revealed that the mycobacterium strains belonged to the MTBC.In short, identification by PCR using these specific makers revealed that mycobacterium species responsible for pulmonary tuberculosis in patients from Jamot and Mbalmayo District Hospital belonged to the MTBC. Also PCR technique is more rapid compared to the PNP inhibition test. Key words : Mycobacterium tuberculosis complex, polymerase chain reaction (PCR), Cameroon.

Les nouveaux outils de diagnostic microbiologique de la tuberculose maladie

This review focuses on the role of new tools in the “modern” microbiological diagnosis of tuberculosis. Traditional techniques of microscopy and culture remain essential to diagnostic certainty, but some innovations replace daily the older techniques such as the identification of Mycobacterium tuberculosis complex by immunochromatography or mass spectrometry MALDI-TOF type from positive cultures, or susceptibility testing in liquid medium. New tools that use molecular techniques have become important. They all have in common to optimize the fight against tuberculosis by reducing diagnostic delay. They also allow rapid detection of drug resistance. However, the techniques of gene amplification directly from clinical samples are still less sensitive than culture. Bacteriological diagnosis of tuberculosis disease therefore still relies on the complementarities of different phenotypic and molecular techniques.

Identification of Mycobacterium tuberculosis Complex by Culture and Duplex Polymerase Chain Reaction in Bovines

Identification of Mycobacterium tuberculosis Complex by Culture and Duplex Polymerase Chain Reaction in Bovines

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria.

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Morphologic assessment of cord factor for rapid and presumptive identification of Mycobacterium tuberculosis complex

Morphologic assessment of cord factor for rapid and presumptive identification of Mycobacterium tuberculosis complex

Direct identification of mycobacteria from culture media using a multiplex real-time PCR assay: report on its application in a clinical laboratory in a region of high tuberculosis endemicity

There are few commercial assays that easily and correctly identify the mycobacteria from culture in a clinical laboratory with a high workload. Thus, we developed and evaluated a scheme for the identification of mycobacteria using a multiplex real-time PCR assay and report on its application in our laboratory. The scheme consisted of 3 stepwise PCRs. Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) were differentially detected in the step 1 PCR, and the NTM species were identified in the step 2 and 3 PCRs. Over the 1.5-year study period, 1136 isolates of MTC and 618 isolates of NTM were detected, and the species of 608 (98.4%) of the 618 NTM isolates were identified. We conclude that the established scheme is a very useful diagnostic approach for the rapid and accurate identification of MTC and clinically relevant NTM in a clinical laboratory in a region where tuberculosis is endemic.

Molekularna identifikacija vrsta Mycobacterium tuberculosis kompleksa i netuberkuloznih mikobakterija

Conclusion: Identification of mycobacteria isolated from respiratory specimens over a three-month period by using molecular techniques revealed that the most frequently isolated mycobacterial species in Serbia is M. tuberculosis, while nontuberculous mycobacteria (NTM) species isolated from respiratory specimens of local patients are M. gordonae, M. xenopi and M. kansasii. Introduction: Fast and accurate identification of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria to the species level is of great importance. Highly sensitive and specific molecular techniques are methods of choice for identification of mycobacteria to the level of species. The Aim: The aim of the study was identification of mycobacteria recovered from pulmonary specimens in Serbia during a three-month period, to the level of species. Material and methods: Identification of 112 mycobacterial cultures was performed using the Genotype MTBC and CM (HAIN, Lifescience, Germany) assays. Results: In total, 88 (78,57%) isolates were identified as species M. tuberculosis, while 24 (21,43%) isolates were identified as NTM. The identification to the species level was achieved in 17 (70,83%) NTM isolates, while 7 (29,17%) were identified as Mycobacterium sp. The most frequently isolated NTM species was M. gordonae (10; 58,82%), while the isolation rates of the remaining species were as follows: M. xenopi (5; 29,41%) and M. kansasii (2; 11,77%).

Simultaneous detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria in respiratory specimens

Many nontuberculous mycobacteria (NTM) species have clinical significance, and the rapid and reliable identification of Mycobacterium tuberculosis complex (MTBC) and NTM species is important. We evaluated the simultaneous detection of MTBC and NTM in respiratory specimens. MTBC and NTM were simultaneously detected and identified by laboratory-developed (LDT) real-time PCR, multiplex real-time PCR/melting curve analysis, rpoB PCR restriction fragment length polymorphisms and the AdvanSure Mycobacteria GenoBlot assay (LG Life Sciences). Eighty-five respiratory specimens from 69 patients showed simultaneous detection of MTBC and NTM. A line probe assay showed 70.6% concordance with LDT. Ten patients (14.5%) had a history of tuberculosis, and eight patients (11.6%) had been previously diagnosed with bronchiectasis. Mixed cultures were present one time in 57 patients (82.6%) and repeatedly in 12 patients (17.4%). MTBC was more frequent in 44 patients (63.8%), and NTM was isolated in seven patients (10.1%). The commonly detected NTM species in the mixed cultures were Mycobacterium intracellulare (29.0%) and Mycobacterium abscessus (29.0%). Co-isolation caused a failure of antitubercular drug susceptibility testing in 2 patients (2.9%). Molecular methods allow MTBC and NTM species to be simultaneously identified in respiratory specimens. NTM isolated with MTBC has clinical significance in some patients and should not be ignored.

Selecting criteria for improving results of an immunochromatographic assay for Mycobacterium tuberculosis complex identification from BacT/ALERT cultures

We describe a combined macro and microscopic criteria for rapid presumptive differentiation between Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) evaluated by two independent observers. This strategy achieved rapid MTC identification in most cases (95.8% expert observer and 91.6% novice observer) with significant savings compared to more expensive and unnecessary tests.

Performance of Cobas TaqMan MTB for Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens

Objective: Because tuberculosis is a major global health problem, rapid and accurate identification of Mycobacterium tuberculosis complex (MTBC) in clinical specimens is essential for the control of the spread of tuberculosis. One of real-time polymerase chain reaction assays for direct detection of MTBC, Cobas TaqMan MTB (Roche Diagnostics) assay has been widely used in clinical setting in Korea. The aim of study was to evaluate of the performance of Cobas TaqMan MTB assay in respiratory specimens. Methods: Total of 619 respiratory specimens (572 sputum specimens and 47 bronchoalveolar washing fluid specimens) was collected in August and September, 2011. All specimens were analyzed for the detection of MTBC by direct smear examination, mycobacterial culture, and Cobas TaqMan MTB assay. Results: When the culture method was used as the gold standard test for comparison, Cobas TaqMan MTB assay for detection of MTBC had a sensitivity and specificity of 80.9% (38/47) and 100% (572/572), respectively. Positive predictive value and negative predictive value of Cobas TaqMan MTB assay was 100% and 98.5%, respectively. The sensitivity of Cobas TaqMan MTB assay was 100% in AFB smear-positive samples and 59.1% in smear-negative samples. Conclusion: Cobas TaqMan MTB assay has an excellent performance in smear-positive respiratory specimens. But performance of Cobas TaqMan MTB assay in smear-negative respiratory samples was not good. For detection of paucibacillary tuberculosis, conventional mycobacterial culture must not be replaced by Cobas TaqMan MTB assay.

Applicability of In-House Loop-Mediated Isothermal Amplification for Rapid Identification of Mycobacterium tuberculosis Complex Grown on Solid Media

A simple, rapid, and low-cost identification method is required in tuberculosis high-burden countries. We report the applicability of in-house loop-mediated isothermal amplification (LAMP) targeting 16S ribosomal RNA for the rapid identification of Mycobacterium tuberculosis complex grown on Lowenstein-Jensen media. Eighty acid-fast staining-positive clinical isolates were selected and used to evaluate the LAMP assay in comparison with polymerase chain reaction and conventional culture-based tests. The LAMP assay identified 60 M. tuberculosis isolates from 80 clinical isolates using simple heat-extracted DNA directly from the colony suspension. The results were in complete agreement with those obtained using the other methods, and the utility of the direct LAMP assay from a colony was demonstrated. The LAMP assay appears to be a practical and low-cost method that can be used for the rapid identification of M. tuberculosis isolates and suitable for endemic low-resource settings.

Evaluation of (SD) MPT64 antigen rapid test, for fast and accurate identification of Mycobacterium tuberculosis complex

To evaluate the SD MPT64 assay for rapid, preliminary identification of Mycobacterium tuberculosis complex (MTBC). All specimens were processed using a standard methodology and inoculated into an automated liquid culture system (BD MGIT960). All signal positive cultures had a smear prepared and tested using a commercial molecular assay. From a carefully mixed MGIT960 vial, 100 μL of broth was loaded into the sample well, and the result recorded after 15 min. Repeat isolates from patients were excluded as were positive cultures contaminated with non-mycobacteria. Fifty MTBC and 150 non-tuberculous mycobacteria were isolated during the study period. Test sensitivity was 98.04%, specificity (98.68%), positive predictive value (96.15%), and a negative predictive value (99.34%). There were two false positive results: Mycobacterium gastri and Mycobacterium fortuitum which were both identified by 16S rDNA and rpoB sequence analysis. The SD MPT64 assay showed excellent performance. The major advantages are: (1) simplicity of test procedure, (2) rapid turnaround time, and (3) relatively inexpensive. When used in conjunction with the presence of serpentine cording in a stained smear from culture, a preliminary identification of MTBC may be made with high confidence.

Comparison of the MGIT TBc immunochromatographic assay with the Accuprobe Gen-Probe TB assay for identification of Mycobacterium tuberculosis complex: results from a low-burden tuberculosis setting

We compared the BD MPT64 TBc Antigen assay with the Gen-Probe TB assay for identifying Mycobacterium tuberculosis (MTB) from liquid culture vials. The BD TBc Antigen assay was more sensitive than and as specific as the Gen-Probe TB assay, making it a useful alternative for the rapid detection of MTB.

Detonation Nanodiamonds for Rapid Detection of Clinical Isolates of Mycobacterium tuberculosis Complex in Broth Culture Media

Routinely used molecular diagnostic methods for mycobacterium identification are expensive and time-consuming. To tackle this problem, we develop a method to streamline identification of Mycobacterium tuberculosis complex (MTBC) in broth culture media by using detonation nanodiamonds (DNDs) as a platform to effectively capture the antigen secreted by MTBC which is cultured in BACTEC MGIT 960, followed by the analysis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). The 5 nm DNDs can capture the MTBC secretory antigen without albumin interference. With on diamond digestion, we confirm the DND captured antigen is cell filtrate protein 10 (CFP-10) because its Mascot analysis shows a score of 68. The dot blotting method further verifies a positive reaction with anti-CFP-10, indicating that CFP-10 is secreted in the medium of mycobacterium growth indicator tube (MGIT) and captured by DNDs. The minimal CFP-10 protein detection limit was 0.09 μg/mL. Furthermore, our approach can avoid the false-positive identification of MTBC by immunological methods due to cross-reactivity. Five hundred consecutive clinical specimens subjected to routine mycobacteria identification in hospital were used in this study, and the sensitivity of our method is 100% and the specificity is 98%. The analysis of each MTBC sample from culture solution can be finished within 1 h and thus shortens the turnaround time of MTBC identification of gold standard culture methods. In sum, DND MALDI-TOF MS for the detection of MTBC is rapid, specific, safe, reliable, and inexpensive.

Identification of Mycobacterium tuberculosis complex by polymerase chain reaction of Exact Tandem Repeat-D fragment from mycobacterial cultures

This study evaluated an in-house polymerase chain reaction (PCR) for rapid identification of the Mycobacterium tuberculosis complex (MTBC) using the MTBC-specific Exact Tandem Repeat D (ETR-D) as the amplification target. In a prospective study, 801 clinical isolates identified as MTBC and 15 nontuberculous mycobacteria were analyzed. Mycobacterial DNA was extracted from automated broth cultures or from egg-based media. The amplification of the ETR-D showed to a sensitivity of 99.6% and a specificity of 100% for the correct identification of MTBC; improved extractions protocols led to 100% sensitivity. The main utility of this technique is the simplicity, rapidity, low cost and accuracy.

Rapid and Accurate Identification of Mycobacterium tuberculosis Complex and Common Non-Tuberculous Mycobacteria by Multiplex Real-Time PCR Targeting Different Housekeeping Genes

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100% for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100%, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

Comparison of two in-house real-time PCR assays with MTB Q-PCR Alert and GenoType MTBDRplus for the rapid detection of mycobacteria in clinical specimens

An in-house IS6110 real-time PCR (IH IS6110), MTB Q-PCR Alert (Q-PCR) and GenoType MTBDRplus (MTBDR; Hain Lifescience) were compared for the direct detection of Mycobacterium tuberculosis complex (MTBC) in 87 specimens following automated NucliSENS easyMAG DNA extraction. This included 82 first smear-positive specimens and three smear-negative specimens. Another in-house real-time PCR with a Mycobacterium genus-specific probe for the internal transcribed spacer (ITS) region (IH ITS) was used to allow a full comparison with culture results. The sensitivities of IH IS6110, Q-PCR, MTBDR and IH ITS for MTBC detection were 100, 92, 87 and 87 %, respectively, compared with culture. Both IS6110-based real-time PCRs (in-house and Q-PCR) were similar in performance, with 91.2 % concordant results for MTBC detection. Inhibition rates were low, with zero to three specimens producing uninterpretable results. However, the Q-PCR failed to detect MTBC in five samples that were smear negative or had few acid-fast bacilli (one to 10 bacilli in 10 microscopic fields) detected by IH IS6110. IH ITS was the least sensitive assay but may be useful when used in conjunction with IS6110 PCR results to determine the presence of non-tuberculous mycobacteria in smear-negative specimens. None of the real-time PCR assays tested provided drug-resistance data. It was concluded that an IH IS6110 assay could easily be incorporated into the workflow of a diagnostic laboratory for rapid and accurate identification of MTBC from clinical specimens. The inclusion of an internal control and amplification of an ITS target enhance the diagnostic utility of the test.