Mycobacterium Tuberculosis In Clinical Specimens Research Articles

Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens.

Tuberculosis diagnosis requires rapid, simple and highly sensitive methods. Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated protein (Cas) systems are increasingly being used for clinical diagnostic applications, due to their high flexibility, sensitivity and specificity. We developed a sensitive Mycobacterium tuberculosis (MTB) complex polymerase chain reaction (PCR)-CRISPR/Cas13a detection method (CRISPR-MTB) and then evaluated its performance in detecting MTB in clinical specimens. The conserved MTB IS1081 sequence was used to design CRISPR-derived RNAs (crRNAs) and T7 promoter sequencing-containing PCR primers for use in the CRISPR-MTB assay, then assay performance was evaluated using 401 clinical specimens. The CRISPR-MTB assay provided a low limit of detection of 1 target sequence copy/μL and excellent specificity. Furthermore, use of the assay to detect MTB in bronchoalveolar lavage fluid (BALF), sputum and pus samples provided superior sensitivity (261/268, 97.4%) as compared to sensitivities of acid-fast bacilli (130/268, 48.5%) and mycobacterial culture (192/268, 71.6%) assays, and comparable or greater sensitivity to that of GeneXpert MTB/RIF (260/268, 97.0%). The CRISPR-MTB assay, which provides excellent sensitivity and specificity for MTB detection in sputum, BALF and pus samples, is a viable alternative to conventional tests used to diagnose TB in resource-limited settings.

Occurrence of disputed rpoB mutations among Mycobacterium tuberculosis isolates phenotypically susceptible to rifampicin in a country with a low incidence of multidrug-resistant tuberculosis

BackgroundAccurate drug susceptibility testing (DST) of Mycobacterium tuberculosis in clinical specimens and culture isolates to first-line drugs is crucial for diagnosis and management of multidrug-resistant tuberculosis (MDR-TB). Resistance of M. tuberculosis to rifampicin is mainly due to mutations in hot-spot region of rpoB gene (HSR-rpoB). The prevalence of disputed (generally missed by rapid phenotypic DST methods) rpoB mutations, which mainly include L511P, D516Y, H526N, H526L, H526S, and L533P in HSR-rpoB and I572F in cluster II region of rpoB gene, is largely unknown. This study determined the occurrence of all disputed mutations in HSR-rpoB and at rpoB codon 572 in M. tuberculosis strains phenotypically susceptible to rifampicin in Kuwait.MethodsA total of 242 M. tuberculosis isolates phenotypically susceptible to rifampicin were used. The DST against first-line drugs was performed by Mycobacteria growth indicator tube (MGIT) 960 system. Mutations in HSR-rpoB (and katG codon 315 and inhA-regulatory region for isoniazid resistance) were detected by GenoType MDBDRplus assay. The I572F mutation in cluster II region of rpoB was detected by developing a multiplex allele-specific (MAS)-PCR assay. Results were confirmed by PCR-sequencing of respective loci. Molecular detection of resistance for ethambutol and pyrazinamide and fingerprinting by spoligotyping were also performed for isolates with an rpoB mutation.ResultsAmong 242 rifampicin-susceptible isolates, 0 of 130 pansusceptible/monodrug-resistant isolates but 4 of 112 polydrug-resistant isolates contained a disputed rpoB mutation. All 4 isolates were also resistant to isoniazid and molecular screening identified additional resistance to pyrazinamide and ethambutol in one isolate each. In final analysis, 2 of 4 isolates were resistant to all 4 first-line drugs. Spoligotyping showed that the isolates belonged to different M. tuberculosis lineages.ConclusionsFour of 242 (1.7%) rifampicin-susceptible M. tuberculosis isolates contained a disputed rpoB mutation including 2 isolates resistant to all four first-line drugs. The occurrence of a disputed rpoB mutation in polydrug-resistant M. tuberculosis isolates resistant at least to isoniazid (MDR-TB) suggests that polydrug-resistant strains should be checked for genotypic rifampicin resistance for optimal patient management since the failure/relapse rates are nearly same in isolates with a canonical or disputed rpoB mutation.

Direct Detection of Mycobacterium tuberculosis in Clinical Specimens Using Nucleic Acid Amplification Tests

Tuberculosis continues to be a significant cause of morbidity and mortality worldwide. Rapid detection of Mycobacterium tuberculosis directly in clinical specimens using nucleic acid amplification tests enables infected patients to be placed on appropriate therapy much sooner than when results of conventional culture methods are used. The availability of rapid results also facilitates infection control measures to interrupt transmission of tuberculosis in healthcare settings. The era of commercially available molecular diagnostics for detection of M. tuberculosis began 25 years ago and now encompasses multiple assays that can detect organisms in the M. tuberculosis complex. Several assays also detect chromosomal mutations associated with antimicrobial resistance to further refine therapeutic strategies early in the course of disease. NAATs have revolutionized the diagnosis of TB across the globe and have become the standard of care in many high-burden developing countries.

Rapid Detection of Ethambutol-Resistant Mycobacterium tuberculosis in Clinical Specimens by Real-Time Polymerase Chain Reaction Hybridisation Probe Method

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein–Jenson (L–J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L–J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L–J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.

The effectiveness of GenoType MTBDRplus using in the diagnosis of tuberculosis in Zaporizhzhia region

The method GenoType MTBDRplus, v.2 allows to study clinical material from positive and negative smears, to examine patients with new and previously treated cases of tuberculosis pulmonary and extrapulmonary localization. But now there is a small amount of researches concerning the use of this method in the diagnosis of tuberculosis. Aim. To evaluate the effectiveness of GenoType MTBDRplus, v.2 method using in clinical material research due to the diagnosis of tuberculosis in the Zaporizhzhia region in comparison with standard methods of investigation. Materials and methods. The analysis of 52 results of studies of clinical material using the GenoType MTBDRplus test system, v.2 from patients who were examined and treated in dispensaries of the Zaporizhzhia region in 2016 was carried out. There were 67.3 % of men and 32.7 % of women among the patients. The mean age of the patients was 46.0 ± 1.9 years. The study of the clinical material using the GenoType MTBDRplus test, v.2 was performed according to the standard instructions. Results. The results were positive in 12.5 % of the cases when tested by GenoType MTBDRplus, v.2 (p ˂ 0.05) among negative results of the microscopy of the clinical material. The proportion of coincidence between the cultural and molecular genetic methods was 90 % for sputum and 100 % for other clinical material. In the presence of multiresistance, both methods of investigation coincided in the number of detected cases (50 % for the GenoType MTBDRplus method, v.2 and 42.9 % for the cultural method, respectively, p ˂ 0.05). In comparison with the results of the cultural method, the GenoType MTBDRplus test, v.2 made it possible to establish resistance to rifampicin in 35.7 % (p < 0.05) of patients whose resistance was not determined. False-positive and false-negative results are due, perhaps, to the heterogeneity of strains of mycobacterium tuberculosis in clinical specimens, the absence of viable mycobacteria, the absence of phenotypic manifestations of mycobacterium tuberculosis genetic mutations, and errors in the investigations. The sensitivity of the GenoType MTBDRplus test, v.2 is about 78 %, but the specificity reaches 97 %. At the same time, the sensitivity of the test for multidrug resistance determining reaches 100 %, the specificity is about 89 %. The greater positive predictive value for cases in which the DNA of mycobacteria is determined and the negative predictive value is for cases in which multidrug resistance is determined. Conclusions. This indicates that the using of the GenoType MTBDRplus test, v.2, together with the cultural method of investigation, has a high diagnostic value, high sensitivity (100 %) and specificity (89 %) for the definition of multiresistance, and makes it possible to obtain a result in a short time compared to culture.

Early tuberculosis treatment monitoring by Xpert® MTB/RIF: Figure 1–

To the Editors: Progress made to improve laboratory capacity for tuberculosis (TB) diagnosis led to the development of molecular assays that are now replacing conventional microscopy and culture-based methods on a large scale [1, 2]. Unfortunately, current molecular techniques detect both live and dead bacteria, and a positive result does not imply the viability of the pathogen. Indeed, DNA can persist for a long period after bacterial death and nucleic acid from dead bacteria is equally amplifiable. Therefore, molecular assays are unsuitable for treatment monitoring and/or for infection control purposes. We report an innovative approach to selectively amplify DNA derived from viable Mycobacterium tuberculosis in clinical specimens, which is useful for monitoring mycobacterial load in pulmonary TB patients during anti-TB treatment. The protocol is based on pre-treatment of samples with propidium monoazide (PMA; Biotium Inc., Hayward, CA, USA), a chemical compound that can intercalate the DNA of non-viable (or membrane-damaged) organisms but is excluded from viable bacteria. After light activation, PMA binds covalently to the DNA, preventing its amplification by PCR [3]. After light exposure, unbound PMA is not able to interact further with DNA molecules. The assay was first optimised using acid-fast bacilli (AFB)-negative sputum samples spiked with dead or live mycobacteria at different concentrations. In brief, live Mycobacterium fortuitum cells were added to N -acetyl-cysteine decontaminated sputum specimens negative for AFB by smear microscopy at a final concentration of 106 bacteria·mL−1. An aliquot of this laboratory-made sample was treated to heat kill the M. fortuitum cells. PMA stock solution was prepared and stored at -20°C and protected from light exposure, until use, as recommended by the manufacturers. PMA was added as a pre-treatment at a final concentration of 500 μM and incubated for 30 min at 4°C in the …

Evaluation of conventional molecular diagnosis of Mycobacterium tuberculosis in clinical specimens from Morocco

Tuberculosis is a major public health threat, annually affecting new individuals worldwide, especially those in developing countries. Rapid detection of the agent and effective treatment are two important factors in controlling this disease. The present study aimed to evaluate polymerase chain reaction (PCR) as a rapid and direct molecular method for the diagnosis of Mycobacterium tuberculosis (MTB) in 70 clinical specimens (62 sputum samples, six cerebrospinal fluids, and two biopsies) using heat shock protein (hsp65) as the gene target. Automated sequencing of the same gene was used for the identification of MTB to the species level. The sensitivity of PCR was 81.13%, with specificity of 88.24%; the positive and negative predictive values were 95.56% and 60%, respectively. Based on these results, the hsp65 gene sequence can be used to differentiate the members of MTB complex from non-tuberculosis mycobacteria (NTM).

Use of loop-mediated isothermal amplification of DNA for the rapid detection of Mycobacterium tuberculosis in clinical specimens

Loop-mediated isothermal amplification (LAMP) is a recently developed molecular method that has been successfully implemented in the detection of Mycobacterium tuberculosis in clinical specimens. LAMP has several advantages, such as rapidity, high sensitivity, ease of application and cost-effectiveness. As a result, it is anticipated that its use for the detection of M. tuberculosis is likely to become widespread, especially in low-resource countries. The present review aimed to present this method and all of the available information on its implementation in the detection of M. tuberculosis in clinical specimens.

The clonal composition of Mycobacterium tuberculosis in clinical specimens could be modified by culture

The application of molecular tools has revealed that infection by Mycobacterium tuberculosis (MTB) is more complex than initially assumed. Genotyping is generally performed on cultures. However, there is no information about bacterial clonal complexity in clinical specimens or whether standard culture procedures can modify this complexity. An in vitro assay was performed to determine whether culture can modify the clonal complexity of the MTB population in clinical specimens. Pairs of MTB strains (10 pairs) or stain-positive sputa (4 pairs) were mixed in different volumetric proportions. The DNA extracted from the mixtures before and after culture was genotyped using mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis to detect potential changes in the proportion of the mixed strains. In 6/10 pairs of MTB strains and 2/4 pairs of sputa, marked changes were observed in clonal composition after culture, even in mixtures of strains differing in their drug-susceptibility patterns. In some cases, only one of the mixed strains was detected after culture. The initial clonal composition in bacteriologically complex clinical specimens could be underestimated if genotyping analysis is performed after culture. Genotyping strategies aimed at analyzing clinical samples must be optimized to reveal the real dimension of clonal complexity in infection by MTB.

Routine Using Pattern and Performance of Diagnostic Tests for Tuberculosis on a University Hospital

Introduction: Nucleic acid amplification tests to detect Mycobacterium tuberculosis in clinical specimens are used increasingly as a laboratory tool. We aimed to investigate the routine using pattern and the effects on therapeutic decision of diagnostic tests for tuberculosis in our hospital. MethodsIn this descriptive study, we investigated retrospectively the routine using pattern and the effects on therapeutic decision of diagnostic tests for tuberculosis. Patients with discordant results were clinically evaluated retrospectively by a chest physician. Samples were tested for the presence of M. tuberculosis by a smear technique, M. tuberculosis culture growth technique (Löwenstein-Jensen and/or BACTEC-960), and IS6110 polymerase chain reaction (PCR). ResultsCulture positivity was 7.2% (83 of 1159 patients). In total, 198 (62.4%) were tested with PCR, acid-fast bacilli, and culture. On the basis of culture results as a gold standard, sensitivity, specificity, positive predictive value, and negative predictive value of PCR were 46%, 89%, 23%, and 93.5%, respectively. ConclusionsSelection of appropriate patients for further testing and exclusion of low-risk patients from microbiologic testing by experienced clinicians may help to optimize the positive predictive value of PCR.

Evaluation of the COBAS TaqMan MTB PCR test for the detection of mycobacterium tuberculosis in clinical specimens with manual and automated DNA extraction

Introduction The Northern Territory records one of the highest rates of new Mycobacterium tuberculosis (MTB) infections in Australia, especially among the indigenous population. PCR techniques for the detection of MTB are being increasingly used in addition to routine methods. Aim To assess the COBAS TaqMan MTB PCR test (Roche) as a rapid diagnostic tool for clinical specimens and compare the results to standard culture diagnosis. Also, to evaluate MagNA Pure LC (Roche) automated DNA extraction as an alternative to manual extraction for specimen preparation. Methods From 83 patients, 158 respiratory specimens were tested for MTB by routine microscopy and culture, and aliquots from each sample were tested by polymerase chain reaction (PCR) after manual and automated DNA extraction. Results Overall, 12 samples from three patients were MTB PCR positive. These were 100% concordant with culture results or with previously confirmed MTB infection in patients on treatment at the time of testing. Results from manual and automated DNA extraction were identical. Conclusions MTB PCR improves patient care, infection control and cost-effective management, especially for patients from remote indigenous communities. Automated DNA extraction can optimise the benefits of this rapid diagnostic tool by improving turnaround time and laboratory workload.

Clinical Evaluation of the Microscopic-Observation Drug-Susceptibility Assay for Detection of Tuberculosis

There is an urgent need for low-cost methods for rapid, accurate detection of Mycobacterium tuberculosis in clinical specimens. The microscopic-observation drug-susceptibility (MODS) assay is a relatively low-cost and simple liquid culture method that has been proposed for use in resource-limited environments. This prospective study evaluated the performance of the MODS assay for detection of M. tuberculosis in persons undergoing evaluation for pulmonary tuberculosis in Brazil and Honduras. Respiratory specimens were evaluated using smear microscopy, culture on Lowenstein-Jensen medium, and culture using the MODS assay. A subset of specimens was also cultured using the Mycobacterial Growth Indicator Tube (MGIT) 960 automated system (Becton Dickinson). A study subject was considered to have tuberculosis if at least 1 culture on Lowenstein-Jensen medium was positive for M. tuberculosis. A total of 1639 respiratory specimens obtained from 854 study subjects were analyzed. On a per-subject basis, MODS sensitivity was 97.5% (95% confidence interval [CI], 95.7-98.6), and specificity was 94.4% (95% CI, 93.1-95.2). Median times to detection were 21 days (interquartile range [IQR], 17-25 days) and 7 days (IQR, 5-10) for culture on Lowenstein-Jensen medium and for the MODS assay, respectively (P<.01). For 64 specimens cultured using the MGIT 960 automated system, median time to growth was similar for the MODS assay (7 days; IQR, 7-10 days) and the MGIT 960 automated system (8 days; IQR, 6-11.5 days; P=.16). The percentage of contaminated cultures was lower for the MODS assay than for culture on Lowenstein-Jensen medium (3.8% vs. 5.8%; P<.01). The MODS assay is a relatively simple test whose good performance characteristics for detection of pulmonary tuberculosis may make it suitable for resource-limited environments.

PCR amplification of shorter fragments from thedevR(Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis

This study was designed to assess the vital issue of gene target length and PCR assay performance in relation to the detection of Mycobacterium tuberculosis in clinical specimens. Two PCR assays that amplify fragments of varying lengths from the devR gene of M. tuberculosis were evaluated. Using M. tuberculosis DNA the 'short-length' PCR assay detected 250-500 genome equivalents vs. 500-1,000 genome equivalents by the 'long-length' assay. In comparison to a highly sensitive smear microscopy test (universal sample processing smear), the sensitivity of the 'short-length' assay was 97.8% vs. 69.9% of the 'long-length' assay in sputum specimens (n=506) from patients being evaluated for a possible diagnosis of tuberculosis. The 27.9% absolute increase in sensitivity was statistically significant (P<0.001). Our results indicate that in a clinical setting when all other conditions are equal, the amplification of a shorter gene fragment of devR increases the sensitivity and efficiency of the PCR assay in spite of using a single copy gene as target.

Bacteriophage- based tests for the detection of Mycobacterium tuberculosis in clinical specimens: a systematic review and meta- analysis

BackgroundSputum microscopy, the most important conventional test for tuberculosis, is specific in settings with high burden of tuberculosis and low prevalence of non tuberculous mycobacteria. However, the test lacks sensitivity. Although bacteriophage-based tests for tuberculosis have shown promising results, their overall accuracy has not been systematically evaluated.MethodsWe did a systematic review and meta-analysis of published studies to evaluate the accuracy of phage-based tests for the direct detection of M. tuberculosis in clinical specimens. To identify studies, we searched Medline, EMBASE, Web of science and BIOSIS, and contacted authors, experts and test manufacturers. Thirteen studies, all based on phage amplification method, met our inclusion criteria. Overall accuracy was evaluated using forest plots, summary receiver operating (SROC) curves, and subgroup analyses.ResultsThe data suggest that phage-based assays have high specificity (range 0.83 to 1.00), but modest and variable sensitivity (range 0.21 to 0.88). The sensitivity ranged between 0.29 and 0.87 among smear-positive, and 0.13 to 0.78 among smear-negative specimens. The specificity ranged between 0.60 and 0.88 among smear-positive and 0.89 to 0.99 among smear-negative specimens. SROC analyses suggest that overall accuracy of phage-based assays is slightly higher than smear microscopy in direct head-to-head comparisons.ConclusionPhage-based assays have high specificity but lower and variable sensitivity. Their performance characteristics are similar to sputum microscopy. Phage assays cannot replace conventional diagnostic tests such as microscopy and culture at this time. Further research is required to identify methods that can enhance the sensitivity of phage-based assays without compromising the high specificity.

Direct detection of Mycobacterium tuberculosis in clinical specimens using single-tube biotinylated nested polymerase chain reaction-enzyme linked immunoassay (PCR-ELISA)

A biotinylated single-tube nested polymerase chain reaction (PCR) assay with microwell hybridization assay (bPCR-ELISA) was developed for detection of Mycobacterium tuberculosis in clinical specimens. A total of 659 specimens (601 respiratory specimens and 58 nonrespiratory specimens) were collected for evaluation using three DNA amplification techniques: newly designed bPCR-ELISA, in-house single-tube nested PCR for IS 6110 gene sequence (nPCR), and commercial automated assays, the Cobas Amplicor System from Roche Diagnostic Systems (aPCR). Sixty-four (9.7%) specimens were culture-positive for M. tuberculosis. Eleven (1.7%) specimens culture-positive for nontuberculosis mycobacteria were negative by all three PCR assays. The resolved performance of bPCR-ELISA, nPCR, and aPCR was found at sensitivities of 97%, 94%, and 97%, respectively. All three PCR assays exhibited a 100% specificity. In evaluation of bPCR-ELISA, a clear distinction between PCR-positive and PCR-negative specimens when an OD 405 value of 0.6 was chosen as cut-off. With serial dilutions of M. tuberculosis H37Rv DNA, the detection limit of bPCR-ELISA was found to be 0.75 cfu per reaction at OD 405 value of 0.6. Our developed bPCR-ELISA provides a highly sensitive and low-costing molecular diagnosis suitable for developing countries with high prevalence of tuberculosis.

Diagnosis of Mycobacterium tuberculosis in cases of pulmonary infections from Jordanian hospitals.

The aim of this study is to use the diagnostic utility of smear technique for the detection of Mycobacterium tuberculosis in clinical specimens obtained from patients suspected of having pulmonary tuberculosis. A total of 305 respiratory specimens (broncoalveolar lavage, sputum and pleural fluid) were collected from 298 patients having pulmonary infections as bronchitis, tuberculosis, pneumonia and other respiratory diseases. Results revealed that 25 specimens collected from 22 patients were positive (8.2%) by acid fast (AF) smear. Data indicated that the specificity and positive predictive value of the conventional smear assay were 100%, however, the sensitivity of the smear examination was 73.5%. Conventional smear technique actually detected M. tuberculosis in respiratory specimens and it could be applied for early and specific diagnosis of tuberculosis in such patients.

The evaluation of FAST PlaqueTB TM test for the rapid diagnosis of tuberculosis

FAST PlaqueTB TM (Biotec Laboratories Ltd., Ipswich, UK) is a rapid test which utilizes bacteriophage amplification technology for the detection of viable Mycobacterium tuberculosis in clinical specimens. We evaluated performance of the FAST PlaqueTB TM test by comparing with BACTEC 460 TB culture system (Becton Dickinson Co., Maryland, USA), polymerase chain reaction (PCR) and acid fast bacilli (AFB) smear methods. We investigated 192 sputum specimens collected from the patients suspected of having pulmonary TB by AFB smear, BACTEC 460 TB culture system, PCR and FAST PlaqueTB TM test. The sensitivity of AFB smear, PCR and FAST PlaqueTB TM test were 57.8%, 84.4% and 87.5% respectively when we accepted BACTEC 460 TB culture system as gold standard. We conclude that FAST PlaqueTB TM test has a good potential for rapid diagnosis of Mycobacterium tuberculosis as a result of the evaluation of these three tests by comparison to the BACTEC 460 TB culture system.

Routine use of the gen-probe MTD2 amplification test for detection of Mycobacterium tuberculosis in clinical specimens in a large public health mycobacteriology laboratory

The new version of the Amplified Mycobacterium Tuberculosis Direct Test, MTD2 (Gen-Probe Inc., San Diego, CA) has been implemented as part of the regular testing algorithm for detecting Mycobacterium tuberculosis (MTB) in selected respiratory and non-respiratory specimens in our laboratory. At the Central Public Health Laboratory, Etobicoke, Ontario, we receive specimens for the detection of mycobacteria from all areas of the Province of Ontario. The laboratory processes approximately 25,000 specimens per year, and receives approximately 2000 reference cultures for identification. There are 600 to 700 new cases of tuberculosis detected yearly. Over the 1-year period (1997–98), 823 specimens were tested by MTD2 and the results were compared with radiometric culture (Bactec, Becton Dickinson, Sparks, MD) and clinical diagnosis, giving an overall sensitivity, specificity, positive predictive value and negative predictive values of 100%, 99.6%, 97.4% and 100%, respectively. Two hundred and two cases of respiratory TB and 56 cases of extrapulmonary TB were detected by MTD2 within 0–4 days of specimen arrival in the laboratory. By appropriate selection of specimens for testing, the MTD2 can provide a fast, accurate, and cost-effective method for the detection of MTB in clinical specimens.

Molecular diagnosis of tuberculosis: current clinical validity and future perspectives.

The rapid development and availability of a variety of new molecular genetic technologies present the clinician with an array of options for the accurate diagnosis of infectious diseases. This is particularly the case for tuberculosis, since molecular methods have been rapidly introduced into all working areas of the mycobacteriology laboratory. Nucleic acid amplification methods to detect Mycobacterium tuberculosis in clinical specimens are increasingly used as a tool to diagnose tuberculosis. The bulk of recently available data from clinical evaluations performed under routine laboratory conditions indicate that these molecular methods are rapid and sensitive, but yet inferior, to culture with regard to sensitivity and specificity. Therefore, until gene amplification tests have proved to be reliable and quality control procedures exist, their clinical validity remains controversial. Consequently, definition of selected clinical applications of gene amplification to routine diagnosis of tuberculosis is important and need to be discussed. This review will focus on the clinical role of molecular methods in the direct detection and diagnosis of M. tuberculosis in clinical samples. In addition, molecular genetic approaches designed to determine drug susceptibility and to discriminate strains below the species level will be outlined and discussed in terms of their current and future clinical applicability.

Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories.

Nucleic acid amplification to detect Mycobacterium tuberculosis in clinical specimens is increasingly used as a laboratory tool for the diagnosis of tuberculosis. However, the specificity and sensitivity of these tests may be questioned, and no standardized reagents for quality control assessment are available. To estimate the performance of amplification tests for routine diagnosis, we initiated an interlaboratory study involving 30 laboratories in 18 countries. We prepared blinded panels of 20 sputum samples containing no, 100, or 1,000 mycobacterial cells. Each laboratory was asked to detect M. tuberculosis by their routine method of nucleic acid amplification. Only five laboratories correctly identified the presence or absence of mycobacterial DNA in all 20 samples. Seven laboratories detected mycobacterial DNA in all positive samples, and 13 laboratories correctly reported the absence of DNA in the negative samples. Lack of specificity was more of a problem than lack of sensitivity. Reliability was not found to be associated with the use of any particular method. Reliable detection of M. tuberculosis in clinical samples by nucleic acid amplification techniques is possible, but many laboratories do not use adequate quality controls. This study underlines the need for good laboratory practice and reference reagents to monitor the performance of the whole assay, including pretreatment of clinical samples.