Rapid Identification Of Mycobacteria Research Articles

GenoType CM Direct® and VisionArray Myco® for the Rapid Identification of Mycobacteria from Clinical Specimens.

M. tuberculosis is the single infectious agent responsible for most deaths worldwide outside of pandemics. Diseases due to non-tuberculous mycobacteria (NTM) are increasing in many regions of the world. The two molecular assays GenoType CM direct® (GTCMd) (Bruker, Billerica, MA, USA) and VisionArray Myco® (VAM) (ZytoVision, Bremerhaven, Germany) are based on the DNA/DNA hybridization technique, and allow for the identification of tuberculous and the most clinically relevant non-tuberculous mycobacterial species from clinical specimens. We evaluated the performance of both assays for the identification of mycobacteria from 65 clinical specimens of 65 patients and compared it with the results of conventional culture. Based on conventional culture that recovered 37 mycobacterial isolates including 11 tuberculous and 26 NTM isolates, sensitivity, specificity, positive predictive value and negative predictive value were 89.2%, 81.5%, 86.8% and 84.6% for GTCMd and 73.0%, 96.3%, 96.4% and 72.2% for VAM. Additionally, GTCMd identified mycobacteria from five and VAM from one culture-negative sample. Both assays identified a mycobacterium in one sample overgrown by other microorganisms. Two M. abscessus subsp. abscessus isolates grown from culture were identified as M. chelonae by GTCMd assay. In conclusion, both assays improve the rapid identification of mycobacteria directly from clinical specimens.

Multicenter evaluation of the biochip assay for rapid detection of mycobacterial isolates in smear-positive specimens

ObjectivesThe objective of this study was to conduct a multicentre evaluation of the performance of the biochip assay in the rapid identification of mycobacteria in smear-positive sputum specimens. MethodsA total of 1751 sputum specimens were obtained from 7 cities in Zhejiang, China. All of the specimens were used for the discrimination of Mycobacterium species using the biochip assay, and the results were compared to the golden standard method of culture, hsp65, 16S rRNA and rpoB sequence analysis. ResultsIn the 1751 sputum specimens, 1685 samples were cultured successfully; among these samples, 1361 were Mycobacterium tuberculosis, 323 were NTM and 1 was Nocadia farcinica. Of the 323 NTM, most of them were Mycobacterium intracellulare(52.5%) followed by Mycobacterium abscessus (20.7%), Mycobacterium avium (11.7%), Mycobacterium kansasii (9.6%) and Mycobacterium fortuitum (1.9%). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the biochip assay to differentiate TB and NTM from AFB positive specimens were 99.8%, 99.7%, 99.9%, 99.1%, 98.8%, 1, 1, and 99.7%, respectively. The concordance between the biochip assay and mycobacterial culture for the identification of NTM species was 95.4%. ConclusionsThe biochip assay is a reliable tool for the rapid identification of most mycobacteria in clinical sputum specimens. This assay can be helpful for physicians in the early diagnosis and treatment of mycobacterium infections.

Rapid identification of mycobacteria from positive MGIT broths of primary cultures by MALDI-TOF mass spectrometry.

BackgroundRapid identification of mycobacteria is important for timely treatment and the implementation of public health measures. The MGIT system ensures rapid detection of mycobacteria, but identification is usually delayed by days to weeks due to further subculture on solid medium. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to effectively identify mycobacteria isolates subcultured from solid or liquid media. Reports of identification directly from MGIT broths of both sterile and non-sterile clinical specimens, omitting the subculture step, were limited and not satisfactory before. Our identification method dramatically shortened delay from detection to identification of mycobacteria.MethodologyWe assessed the performance of the Vitek MS IVD version 3.0 for direct identification of NTM and M.tuberculosis from primary MGIT cultures, and assessed two sample preparation methods.ResultsDirect identification of NTM from positive MGIT broths, using MALDI-TOF VITEK MS with IVD v.3.0, generated high rates of acceptable results reaching 96.4% (80/83), and up to 100% (83/83) for sample preparations including a 0.1% SDS washing step. The sensitivity of VITEK MS to identify M.tuberculosis from MGIT tubes was 58/72 (80.6%), when using immunochromatography (ICA) test as gold standard. A characteristic colony clumping, wool-like appearance was observed in 48, and all 58 (100%) were correctly identified as M.tuberculosis using MALDI-TOF. The detection rate of M.tuberculosis complex was low (10/24, 41.6%) in the 24 MGIT tubes that was polymicrobial. Our method significantly reduced both the reagent cost and turnaround time.ConclusionsBased on a simplified protocol, we showed that MALDI-TOF MS can be used for rapid identification of NTM directly from primary MGIT cultures within the routine clinical laboratory workflow. However, we recommend an initial ICA test to screen for M.tuberculosis complex, due to a low identification rate of M. tuberculosis in the presence of polymicrobial cultures using MALDI-TOF.

Mycobacterial inactivation protein extraction protocol for matrix-assisted laser desorption ionization time-of-flight characterization of clinical isolates.

Rapid identification of mycobacteria has been made possible with matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) in recent years. Working with high concentrations of mycobacteria in a PC-3 containment facility makes MALDI-TOF cumbersome and costly. Therefore removing the inactivated isolate's protein extract from the PC-3 facility is needed for efficient identification in a routine PC-2 laboratory. This work describes a novel chemical and mechanical disruption protein extraction method, which provides reliable MALDI-TOF results from solid and liquid media, while ensuring laboratory safety. When compared to sequencing results, 93.9% of the clinical isolates were identified in LJ media and 89% of the clinical isolates were identified in MGIT media. The MIPE protocol produces a high quality protein extract with improved isolate identification without compromising result turn-around-times or laboratory safety.

A novel liquid media mycobacteria extraction method for MALDI-TOF MS identification using VITEK® MS

A low-cost identification method that can be performed directly from a positive liquid medium culture is needed for the diagnosis of mycobacterial infections. Here, we describe a novel, cost-effective, and validated method that allows for direct and rapid identification of mycobacteria from a positive liquid culture using VITEK® MS with a total process duration under 45min. From a liquid mycobacteria culture a 3.0mL aliquot is removed 24–72h post positivity and centrifuged to create a pellet. After decanting, the tube is blotted dry, the pellet is re-suspended in 0.5mL of 70% ethanol and then transferred into a 2.0mL tube containing glass beads. Mycobacteria are disrupted mechanically followed by a 10min. incubation at room temperature to complete inactivation. Inactivated material is pelleted by centrifugation and then re-suspended in 10μL of 70% formic acid and 10μL of acetonitrile. After centrifugation, 1μL of supernatant (protein extract) is deposited onto target slide, allowed to dry, and then 1μL CHCA matrix is added.A seeded study was conducted to demonstrate the reliability of the method, a total of 251 culture samples obtained from automated culture systems (BacT/ALERT® MP bottles, BACTEC MGIT™ 960 tubes, and VersaTREK® Myco bottles), were tested and resulted in 98.8% correct identification. Reproducibility was shown by testing three organisms across three reagent lots, between four laboratory technicians, over the course of five days for three liquid media systems resulting in a total of 180 deposits with an overall correct identification of 98.9% with the remaining results giving no identification. Additional studies were performed including comparison of different mechanical disruption techniques, stability of frozen extracts, and stability of slide deposits to allow for flexibility in a routine clinical workflow.The described method proved to be safe while providing consistent and reproducible results for different species of mycobacteria and is compatible with the three most widely used liquid media medium detection systems.

Utility of MALDI-TOF MS in the rapid identification of mycobacteria

Utility of MALDI-TOF MS in the rapid identification of mycobacteria

Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.

Direct identification of mycobacteria from smear-positive sputum samples using an improved multiplex polymerase chain reaction assay

The rapid identification of mycobacteria from smear-positive sputum samples is very important. To identify the Mycobacterium tuberculosis complex (MTBC) and frequently isolated nontuberculous mycobacteria strains directly from smear-positive sputum samples, an improved multiplex polymerase chain reaction (PCR) assay was developed. Nine pairs of primers targeting the 16S–23S rDNA internal transcribed spacer-1, hsp65, and the early secretory antigen (ESAT-6) gene sequences were developed, and their efficacy was evaluated in comparison to traditional culturing and 16S rRNA gene sequencing methods. A total of 200 smear- and culture-positive sputum specimens collected between November 2005 and May 2006 were used for the analysis. The results of the assay showed an accurate identification rate for acid-fast bacilli (AFB) 3+, AFB 2+, and AFB rare/1+ samples of 98%, 95%, and 53%, respectively. The improved multiplex PCR method saves time and has advantages for identifying mycobacteria from AFB 2+ and 3+ sputum samples. The method is suitable for use in countries with a high MTBC prevalence rate.

Fast identification of Mycobacteria from positive Mb/Bact bottles using a multiplex PCR

Background: The rapid identification of mycobacteria from culture-positive Mb/Bact bottles is an important clinical issue. Furthermore, the availability of a non-expensive, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In this study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from positive Mb/Bact (Organon-Teknika) bottles. A multiplex PCR focusing the hsp65 gene, and the IS6110 was done and evaluated for its efficiency compared to PRA identfication, and PNB inhibition. Methods: Mycobacterial strains were grown from clinical samples and preserved in solid media (LJ). A panel of 91 M. tuberculosis strains, and 26 Non-TuberculousMycobacteria were submitted to the following identification techniques: a) hsp65 gene amplification followed by restriction (PRA); b) p-Nitrobenzoic acid (PNB) growth inhibition in liquid media (Mb/Bact); 3) Multiplex-PCR, using two sets of primers: TB284/TB850 (IS6110 MTC spcific primers), and TB11/TB12 (65 kD antigen, Mycobacterium genus specific primers). The mycobacteria was inactivated being heated at 80oC for 20min before DNA extraction. DNA was extracted by the freezing and thawing method. 50 l of the the supernatant was used for amplification. Identification using hsp65 restriction was done according to international protocols (Telenti et al, 1993), and considered the gold-standard. PNB was added to liquid Mb/Bact bottles to a final concentration of 500 g/ml. p-aminobenzoic acid, and the Mycobacterial concentration was adjusted to the MacFarland 1 standard. Results: The hsp65 restriction analysis identified all Mycobacteria tested as: M.tuberculosis (61 strains), M.avium, M.intracellulare, M.fortuitum, M.abscessus, M.gordonae, M.szulgai. There was 100% agreement between the tests performed Conclusion: A simple and inexpensive Multiplex-PCR as described here, can readly distinguish among M. tuberculosis Complex strains, and Non-Tuberculous Mycobacteria.

Rapid Identification of Mycobacteria by Raman Spectroscopy

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.

Application of the hsp65 PRA method for the rapid identification of mycobacteria isolated from clinical samples in Belgium

Biochemical identification of mycobacteria is slow and many times fail to produce correct results. We compared PCR-restriction fragment length polymorphism analysis (PRA) of hsp65 and biochemical methods for the identification of mycobacteria from human samples in Belgium. PRA was found useful in the identification of mycobacteria and simple to implement as a quick method in the laboratory.

Rapid identification of mycobacteria from AIDS patients by capillary electrophoretic profiling of amplified SOD gene

Aim-Rapid differentiation of mycobacterial species at the genomic level.Methods-The manganese superoxide dismutase (SOD) gene (464 bp) and 16SrRNA (353 bp) from 104 isolates (18 species) of mycobacteria were amplified using polymerase chain reaction (PCR). Products were sequenced and a phenogram of SOD sequences derived. PCR products of SOD gene were digested with HaeIII, and restriction fragment profiles visualised using capillary electrophoresis.Results-Novel SOD sequences were found for M szulgai, M marinum, M phlei, M smegmatis, M chelonei, M paratuberculosis, M malmoense, M intracellulare serotype 7, M intracellulare serotype 18, and M celatum types 1, 2, and 3. Phylogenetic analysis indicated that 18 of 19 species studied had 8-29% interspecies and <6% intraspecies sequence diversity in the SOD gene. No consistent differences were detected between AIDS and non-AIDS isolates. M paratuberculosis showed a unique SOD sequence with a 1.1% (SD 0.5%) diversity from M avium. Capillary electrophoresis profiles were able to differentiate 16 of 18 species within 24 hours.Conclusions-A phenogram of SOD sequences clearly delineated all mycobacterial species and showed two distinct clusters, fast growing species, and the M avium complex (MAC). Within the MAC, M avium (five types), M intracellulare (five types), M scrofulaceum (two types), and M paratuberculosis (one type) could be demonstrated. Phylogenetic diversity of M celatum from MAC, previously suggested by 16SrRNA data, was confirmed. This simple and rapid method for DNA extraction, in conjunction with capillary electrophoresis of SOD restriction fragments, allows rapid identification of mycobacterial isolates.

Rapid identification of mycobacteria by the gen-probe accuprobesystem

Chemiluminescent acridinium ester-labelled (AE)-DNA probes (Gen-Probe, Inc., San Diego, Calif.) for the identification of Mycobacterium tuberculosis complex (MTBC) and the M. avium-M. intracellulare complex (MAC) were evaluated using 184 mycobacterial isolates cultured in BACTEC 12B vials (Becton Dickinson and Co., Towson, Md.). A 1.5 mL aliquot from BACTEC 12B vials containing acid-fast bacilli and a Growth Index of > 200 was concentrated 15-fold using a centrifugation step prior to performing the test procedure. When 184 mycobacterial isolates were tested (42MTBC/142 nontuberculous mycobacteria) using the AE-MTBC probe, there was 100% sensitivity and specificity when compared with conventional identification procedures. Criteria for using the AE-MAC probe were defined to optimize results whilst minimizing laboratory costs. Ninety-one (64%) of AE-MTBC probe negative isolates failed to meet selection criteria and were not tested. When 51 (36%) of the AE-MTBC-probe negative mycobacterial isolates were tested, the AE-MAC probe was found to be 88% sensitive and 100% specific. The non-isotopic Gen-Probe test is a rapid and practical alternative to current procedures for differentiation of mycobacteria.

The BACTEC Radiometric System for Detection and Rapid Identification of Mycobacteria

We have found the current BACTEC system using 12B medium to be a rapid, reliable method for the detection and differentiation of mycobacteria species. We evaluated the results of 1,081 mycobacterial cultures using a combination of BACTEC andhowenstein-Jensen medium. Twenty-nine cultures (2.68%) were positive for clinically significant isolates with either BACTEC, Lowenstein-]ensen, or both. BACTEC had an overall sensitivity of 89.6% for both Mycobacterium tuberculosis and other mycobacteria compared to 69% for recovery on Lowenstein-Jensen medium. The combined mean time to detection was greatly reduced with BACTEC compared to Lowenstein-] ensen medium, 12.4 vs24.3 days. Rapid differentiation between M tuberculosis complex and other mycobacteria was achieved with an overall mean time of4.4 days. Preliminary identification using the NAP differential test was confirmed with conventional tests in 22 of 24 cases. Two preliminary identifications were incorrect—one was M tuberculosis read as mycobacteria other than tuberculosis and one was M kansasii read as M tuberculosis complex. Suggestions are made for stricter interpretationof the NAP differential growth pattern to avoid such incorrect preliminary identification.