Mycobacterium Tuberculosis Direct Research Articles

Evaluation of two commercial nucleic acid amplification kits for detecting Mycobacterium tuberculosis in saliva samples.

The objective of this study was to evaluate the efficacy of two commercial nucleic acid amplification techniques to identify Mycobacterium tuberculosis in saliva. Fifty-two participants were recruited, 32 patients with a clinical and microbiological diagnosis of pulmonary tuberculosis and 20 healthy volunteers as controls. Three sputum samples were collected from each participant and were examined by direct bacilloscopy, cultured in liquid and solid media, and processed using the Mycobacterium tuberculosis direct (MTD) test for rRNA detection. One saliva sample was collected from each participant using conventional methods and was examined by direct bacilloscopy, cultured, and processed using the MTD test for rRNA detection and the FluoroType Mycobacterium tuberculosis assay for DNA detection. In saliva samples, the sensitivity, specificity, and positive and negative predictive values of the MTD test were 71.8%, 95%, 95.8%, and 67.8%, respectively. The values obtained with the FluoroType assay were 56.2%, 90%, 90%, and 56.2%, respectively. Our results indicate that when a sufficient volume of sputum cannot be obtained, saliva could be an alternative biological sample for the rapid diagnosis of pulmonary tuberculosis using commercial nucleic acid amplification techniques.

Diagnosis of tuberculosis by using a nucleic acid amplification test in an urban population with high HIV prevalence in the United States.

BackgroundUse of nucleic acid amplification tests (NAAT) for the diagnosis of Mycobacterium tuberculosis (TB) has been recommended on respiratory specimens submitted for acid-fast bacilli (AFB) testing. It also helps distinguish between TB and non-tuberculous mycobacteria (NTM) species in a setting where NTM rates are relatively high. The purposes of this study are to describe the trend and characteristics of all AFB smear-positive respiratory samples that underwent amplified Mycobacterium tuberculosis direct (MTD) testing, a type of NAAT, and to evaluate the clinical utility and necessity of the test for diagnosis of TB in a population with high-HIV prevalence.MethodsProspective diagnostic testing and retrospective data analyses were conducted on all AFB smear-positive respiratory samples that underwent MTD testing from 2001 to 2011 at Grady Memorial Hospital (GMH), Atlanta, USA. The test performance was compared to culture.ResultsA total of 2,240 AFB smear-positive specimens from 1,412 patients were tested and analyzed in the study. The proportion of specimens that were culture-positive for TB was 28.5%. Sensitivity, specificity, positive predictive value, and negative predictive value of the MTD were 99.0%, 98.0%, 95.3% and 99.6%, respectively. A downward trend was observed in the yearly numbers as well as the proportions of MTD-positive specimens during the study period (p<0.01). There were 2,027 (90.5%) specimens from patients with known HIV status, of which 70.6% was HIV positive and the majority of them (81.8%) had CD4 counts of less than 200 cells/µL. HIV-positives were more likely to have NTM compared to HIV negatives (67.7% vs. 35.4%, p<0.01).ConclusionDespite the decrease in the incidence of TB, NAAT continues to be an accurate and important diagnostic test in a population with high HIV prevalence, and it differentiates TB and NTM organisms.

Evaluation of three rapid assays for Mycobacterium tuberculosis complex detection in a comprehensive hospital from West China

ObjectivesTo assess the capacity of rapid and accurate confirmation of the Mycobacterium tuberculosis complex (MTBC) in a Chinese clinical laboratory. Design and methodsThis prospective study investigated three rapid assays, the Amplified Mycobacterium Tuberculosis Direct (MTD) test, real-time PCR, and acid-fast bacilli (AFB) smear, for direct detection of MTBC in a large consecutive series of different clinical specimens. Performance parameters were estimated and compared overall and for separate specimen categories using a combined reference gold standard. ResultsThe overall sensitivities were similar for MTD and real-time PCR (62.26% vs. 58.49%), significantly higher than those of AFB smear (31.13%). Among three assays, MTD had a satisfactory sensitivity in respiratory specimen (73.33%) and a nearly perfect detection for smear-positive samples (96.97%). Real-time PCR showed a high positive rate (58.97%) in regard to nonrespiratory specimen. A combination of molecular assays with conventional methods reached marked additive diagnostic values (sensitivity up to 76.42%), higher than each method individually. All detection systems showed excellent specificities (>96.00%). ConclusionsThe present study indicated that our lab had a moderate diagnostic performance for tuberculosis. Quality guarantee for specimen pretreatment, as well as combination analysis, will enable these assays to better incorporate into the routine laboratory workflow in China.

Initial experience with GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.

Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. Rapid and accurate diagnosis of pulmonary and extra-pulmonary tuberculosis (TB) is still a great challenge. The GeneXpert MTB/RIF assay is a novel integrated diagnostic system for the diagnosis of tuberculosis and rapid detection of Rifampin (RIF) resistance in clinical specimens. In 2012, the Arkansas Tuberculosis Control Program introduced GeneXpert MTB/RIF assay to replace the labour-intensive Mycobacterium Tuberculosis Direct (MTD) assay. To rapidly diagnose TB within two hours and to simultaneously detect RIF resistance. Describe the procedure used to introduce GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.Characterise the current gap in rapid M. tuberculosis diagnosis in Arkansas.Assess factors that predict acid fast bacilli (AFB) smearnegative but culture-positive cases in Arkansas.Illustrate, with two case reports, the role of GeneXpert MTB/RIF assay in reduction of time to confirmation of M. tuberculosis diagnosis in the first year of implementation. Between June 2012 and June 2013, all AFB sputum smearpositive cases and any others, on request by the physician, had GeneXpert MTB/RIF assay performed as well as traditional M. tuberculosis culture and susceptibilities using Mycobacteria Growth Indicator Tube (MGIT) 960 and Löwenstein-Jensen (LJ) slants. Surveillance data for January 2009-June 2013 was analysed to characterise sputum smear-negative but culture-positive cases. Seventy-one TB cases were reported from June 2012- June 2013. GeneXpert MTB/RIF assay identified all culture-positive cases as well as three cases that were negative on culture. Also, this rapid assay identified all six smear-negative but M. tuberculosis culture-positive cases; two of these cases are described as case reports. GeneXpert MTB/RIF assay has made rapid TB diagnosis possible, with tremendous potential in determining isolation of TB suspects on one hand, and quickly ruling out TB whenever suspected.

The predictive value of Gen-Probe's amplified Mycobacterium tuberculosis direct test compared with culturing in paraffin-embedded lymph node tissue exhibiting granulomatous inflammation and negative acid fast stain

The diagnosis of granulomatous inflammation with possible tuberculosis (TB) infection in histopathology is often difficult. There is a need for a rapid and reliable diagnostic test. Thus, we evaluated the performance of the Mycobacterium tuberculosis direct (MTD) test in specimens with granulomatous lymphadenitis and negative acid fast stains. The M. tuberculosis direct (MTD) test by Gen-Probe was performed on 45 formalin-fixed paraffin-embedded tissue samples including 34 lymph nodes. We measured the predictive values of the MTD test in specimens with granulomatous lymphadenitis and negative acid fast stains. The overall test sensitivity was 73.9%, and specificity was 95.4%. The MTD test sensitivity and specificity for lymph node tissue were 72.7% and 91.67%, respectively. In the presence of granulomatous inflammation, the MTD test sensitivity and specificity were higher than those for all tissue samples, at 75% and 100%, respectively. Based on this study, the MTD test should be used as a supportive test in addition to conventional histochemical or immunological staining methods when evaluating lymph node tissue with a granulomatous inflammation to deliver stronger evidence to support clinical decisions at a much earlier time than a culture would allow.

Comparison of Performances of MTD Gene-Probe® Test, BACTEC 960™ System and Löwenstein-Jensen Culture Methods in the Diagnosis of Smear-Negative Tuberculosis Cases

The aim of this study was to compare the results of nucleic acid amplification-based MTD (Mycobacterium tuberculosis direct test) Gene-Probe® method in samples obtained from acid-fast bacilli (ARB) smear-negative patients with suspected tuberculosis (TB), with the culture results obtained from automated BACTEC 960™ (MGIT) system and Löwenstein-Jensen (LJ) medium. In addition, the contribution of molecular methods in early diagnosis of pulmonary TB and the effect of radiological prevalence of the disease associated with or without cavity to the molecular diagnosis and/or growth time in culture media have been evaluated. A total of 107 patients (86 male, 21 female; mean age: 49.89 ± 17.1 years, age range: 18-81 years) who were clinically and radiologically suspected of having pulmonary TB and/or TB pleurisy, were included in the study. Of the samples 65 (60.7%) were sputum, 32 (29.9%) were bronchial aspiration, 5 (4.7%) were pleural fluid, and 5 (4.7%) were transthoracic fine needle aspiration biopsy materials. Patient samples were cultured in solid LJ media and liquid-based BACTEC 960 system (Becton Dickinson Co., USA) in the same working day. Meanwhile, MTD Gen-Probe test (Gen-Probe Inc., USA) was studied in two separate working days of the week as specified by the laboratory. The samples were incubated until positivity was determined in BACTEC 960 system and/or growth was detected in LJ medium. Negative cultures were incubated for 42 days and were finalized. When mycobacterial growth was determined in the culture, identification of M.tuberculosis complex (MTBC) and differentiation from nontuberculous mycobacteria were performed by conventional methods and BACTEC 460 NAP test. Forty five (42%) patients were diagnosed as pulmonary paranchimal TB (40 were active pulmonary TB, 1 was miliary TB and 4 were culture-negative pulmonary TB), while 4 (3.7%) patients diagnosed as extrapulmonary TB and 58 (57.9%) patients were diagnosed as other pulmonary diseases unrelated with TB. LJ cultures yielded positive results in 32 of 45 (71%) pulmonary TB patients, and BACTEC 960 were found positive in 84.4% (38/45) of those patients. On the other hand the positivity rate of MTD Gen-Probe test was detected as 37.4% (40/107). The sensitivity, specificity, positive and negative predictive values for MTD Gen-Probe test were estimated as 89%, 100%, 100% and 93%, respectively. Those values for BACTEC 960 system were found as 82%, 98%, 97% and 88%, and for LJ culture method as 71%, 100%, 100% and 83%, respectively. Average periods to make a decision for diagnosis of TB by MTD Gen-Probe, BACTEC 960 (MGIT) and LJ culture methods were calculated as 2.36 days, 20.11 days and 32.49 days, respectively. In comparison of the methods in terms of turnaround times, MTD Gen-Probe test was found superior to LJ culture method, however the turnaround times for BACTEC 960 and LJ culture methods were similar. When the clinical data were evaluated, no effect of radiological density of lesion was identified on the diagnosis time of molecular test and time of growth in liquid based automated BACTEC system and/or LJ culture method. However, LJ culture demonstrated earlier reactivity in patients with cavitary lesions. As a result, MTD Gene-Probe test was observed as a reliable and rapid method for the early diagnosis of pulmonary TB patients, early initiation of therapy, prevention of disease progression and transmission.

Comparison of Two Nucleic Acid Amplification Assays, the Xpert MTB/RIF Assay and the Amplified Mycobacterium Tuberculosis Direct Assay, for Detection of Mycobacterium tuberculosis in Respiratory and Nonrespiratory Specimens

We compared the performance of the Xpert MTB/RIF assay, a new real-time tuberculosis (TB) PCR test, with that of the Amplified Mycobacterium Tuberculosis Direct (MTD) assay using 162 respiratory and nonrespiratory specimens. Based on culture as the gold standard, the overall sensitivity and specificity for all sample types for the Xpert MTB/RIF assay were 90.9 and 89%, respectively, while for the MTD assay, the overall sensitivity and specificity were 97.3 and 87.1%, respectively. A higher proportion of total equivocal results were obtained for the MTD assay, at 10.5% (17/162), while the Xpert MTB/RIF assay generated 5.5% (9/162) of invalid reads.

Nucleic Acid Amplification Tests for Diagnosis of Smear-Negative TB in a High HIV-Prevalence Setting: A Prospective Cohort Study

BackgroundNucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV.MethodsWe prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD) test (Gen-Probe Inc, San Diego, CA). We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid), and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment.ResultsOf 211 patients enrolled, 170 (81%) were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203). Among HIV-seropositive patients, 94 (55%) reported taking co-trimoxazole prophylaxis and 29 (17%) reported taking antiretroviral therapy. Seventy-five patients (36%) had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28–51) and that of secA1 was 24% (95% CI 15–35). Both tests had specificities of 95% (95% CI 90–98). The MTD test correctly identified 18 (24%) TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate.ConclusionsThe secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of Mycobacterium tuberculosis in sputum, even currently available tests can provide substantial clinical impact in smear-negative populations.

頸部結核性リンパ節炎の確定診断・治療とその問題点

Unlike other advanced nations, secondary spread of tuberculosis still occurs in Japan. Cervical tuberculous lymphadenitis is still an important disease of the neck, and between 2001 to 2005, we treated 6 patients with cervical tuberculous lymphadenitis. All 6 patients were females, and their ages ranged from 28 to 77 years old (average: 62 years). One patient had received antitubercular chemotherapy for pulmonary tuberculosis 40 years earlier. Two patients had a family history of pulmonary tuberculosis. One patient was an immigrant from Thailand. Three patients underwent open biopsy of the cervical lymph node, and were diagnosed with tuberculosis histologically. The remaining three patients had an abscess, and fine-needle aspiration (FNA) biopsy was performed. The diagnosis of tuberculosis was made by detection of acid-fast bacilli, MTD (Mycobacterium tuberculosis direct test), PCR (polymerase chain reaction), and culture. All six patients were treated with antitubercular chemotherapy for 6-9 months and recovered. MTD and PCR of the FNA sample seemed to enable early treatment. Attention needs to be paid to countries around Japan where tuberculosis is spreading. We suggest that treatment should be performed while at the same time making an effort to grasp the trend of spread in other countries as well as Japan.

Specimen Dilution Increases the Diagnostic Utility of the Gen-Probe Mycobacterium Tuberculosis Direct Test

The Mycobacterium Tuberculosis Direct (MTD) Test is widely used for detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens. Based on high specificity, it is accepted that a positive MTD result, in proper clinical context, “rules in” a diagnosis of tuberculosis. Test utility for “ruling out” tuberculosis has been limited by lower sensitivity and negative predictive value (NPV), based in part on poorly defined reaction inhibitors in clinical specimens. After discovering that specimen dilution could decrease concentrations of reaction inhibitors without reaching the MTBC detection limit, the Massachusetts State Laboratory Institute in 2003 began routinely testing paired undiluted and diluted samples in parallel. We retrospectively analyzed results from 105 respiratory specimens (87 smear-positive, 18 smear-negative) tested consecutively over 23 months using this dilution protocol. MTD results for each sample pair were merged into a sum result (positive or negative) and compared with culture outcome as the “gold standard.” We found that the MTD sum result always matched the culture outcome, even for smear-negative specimens. Accordingly, the MTD using the dilution protocol had sensitivity, specificity, positive predictive value, and NPV of 100% vs culture.

Specimen Dilution Increases the Diagnostic Utility of the Gen-Probe Mycobacterium Tuberculosis Direct Test

The Mycobacterium Tuberculosis Direct (MTD) Test is widely used for detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens. Based on high specificity, it is accepted that a positive MTD result, in proper clinical context, "rules in" a diagnosis of tuberculosis. Test utility for "ruling out" tuberculosis has been limited by lower sensitivity and negative predictive value (NPV), based in part on poorly defined reaction inhibitors in clinical specimens. After discovering that specimen dilution could decrease concentrations of reaction inhibitors without reaching the MTBC detection limit, the Massachusetts State Laboratory Institute in 2003 began routinely testing paired undiluted and diluted samples in parallel. We retrospectively analyzed results from 105 respiratory specimens (87 smear-positive, 18 smear-negative) tested consecutively over 23 months using this dilution protocol. MTD results for each sample pair were merged into a sum result (positive or negative) and compared with culture outcome as the "gold standard." We found that the MTD sum result always matched the culture outcome, even for smear-negative specimens. Accordingly, the MTD using the dilution protocol had sensitivity, specificity, positive predictive value, and NPV of 100% vs culture.

Molecular methods in the detection and identification of mycobacterial infections.

Nucleic acid amplification (NAA) tests for direct detection of Mycobacterium tuberculosis complex in respiratory specimens have the potential to provide a more rapid diagnosis of pulmonary tuberculosis (TB) than is currently possible by conventional stain, culture, and identification tests. Currently, 2 NAA tests-enhanced Amplified Mycobacterium Tuberculosis Direct (MTD) Test (Gen-Probe, Inc) and Amplicor Mycobacterium tuberculosis Test (Roche Molecular Systems, Inc)-have been approved by the Food and Drug Administration for testing respiratory specimens that are smear positive for acid-fast bacilli (AFB). This restriction to AFB smear-positive specimens was based on data from the initial clinical trials conducted to evaluate these products that showed low sensitivity (ie, 48%-53%) and less-than-optimal specificity (ie, 96%-99%) in AFB smear-negative specimens. Data from the clinical trial for the enhanced MTD test and from 2 subsequent studies, however, suggest that this version of the MTD test is a reliable tool for rapid diagnosis of pulmonary TB, regardless of the AFB smear result. Both NAA tests have been evaluated for diagnosis of extrapulmonary TB, and results were comparable to the results of tests performed with respiratory specimens. The NAA tests also appear to be reliable for rapid identification of M tuberculosis complex in positive broth cultures of all specimen types except blood. The impact of the NAA tests on patient outcome varies based on the AFB smear result. With smear-positive results, public health and hospital infection control resources are predominantly affected. With smear-negative results, however, the potential for affecting patient outcome is much greater. In patients with smear-negative results, the NAA test can result in earlier diagnosis of TB and subsequent initiation of therapy. Use of these tests also may eliminate the need for invasive diagnostic procedures, which are costly and pose an added risk to the patient, and they may allow earlier discharge of hospitalized patients.

False-positive Gen-Probe direct Mycobacterium tuberculosis amplification test results for patients with pulmonary M. kansasii and M. avium infections.

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test has been approved for use in the United States for the rapid diagnosis of pulmonary tuberculosis in patients with acid-fast smear-positive sputum samples since 1996. Four patients infected with human immunodeficiency virus and one chronic pulmonary-disease patient seen in our institutions with abnormal chest radiographs and fluorochrome stain-positive sputa were evaluated for tuberculosis, including performance of the MTD test on expectorated sputum samples. Three of these five patients' sputa were highly smear-positive (i.e. , more than 100 bacilli per high-power field), while two patient's sputa contained 1 to 10 bacilli per field. MTD results on sputum specimens from these patients ranged from 43,498 to 193,858 relative light units (RLU). Gen-Probe has defined values of at least 30,000 RLU as indicative of a positive test, i.e., the presence of Mycobacterium tuberculosis RNA. Four of the patients' sputum cultures yielded growth of M. kansasii within 6 to 12 days, and the fifth produced growth of M. avium only. One patient's culture contained both M. kansasii and M. avium, but none of the initial or follow-up cultures from these five patients revealed M. tuberculosis. However, subsequent cultures from three of the patients again revealed M. kansasii. During the period of this study, in which MTD tests were performed on smear-positive sputum specimens from 82 patients, four of seven patients with culture-proven M. kansasii pulmonary infections yielded one or more false-positive MTD tests. The MTD sensitivity observed in this study was 93.8%, and the specificity was 85.3%. Five cultures of M. kansasii (including three of these patients' isolates and M. kansasii ATCC 12478), and cultures of several other species were examined at densities of 10(5) to 10(7) viable CFU/ml by the MTD test. All five isolates of M. kansasii and three of three isolates of M. simiae yielded false-positive test results, with readings of 75,191 to 335,591 RLU. These findings indicate that low-level false-positive MTD results can occur due to the presence of M. kansasii, M. avium, and possibly other Mycobacterium species other than M. tuberculosis in sputum. Low-level positive MTD results of 30,000 to 500,000 RLU should be interpreted in light of these findings. It remains to be determined if the enhanced MTD test (MTD 2) recently released by Gen-Probe will provide greater specificity than that observed in this report with its first-generation test.

Evaluation of the Gen-Probe amplified Mycobacterium tuberculosis direct detection test.

Mycobacterium tuberculosis direct (MTD) testing was performed on 423 specimens (from 293 patients) submitted for mycobacterial culture, including 121 from 47 patients with current or recent cultures positive for tuberculosis. Resolved sensitivity and specificity were 91.4% and 99.3%, respectively. The assay detected 12 of 14 patients with one specimen and 31 of 32 with > or = 2 specimens. The 10 missed specimens included 2 resembling blood clots and 6 smear negative or 1+ specimens; however, a 2+ and a 4+ specimen were missed, which implies inhibitors. Some smear positive/culture negative and smear negative/culture negative specimens from treated patients gave positive results. The number of smear negative, culture positive specimens from untreated patients was too low to evaluate rigorously. Consideration of these observations and of costs suggests that the assay is appropriate only for untreated patients with smear-positive, nonbloody specimens. They should not be reported as negative for tuberculosis until three smear positive specimens have given negative MTD results. Factors involved in the generation of false positive results, including amplicon contamination, are evaluated.

Performance of the Gen-Probe amplified Mycobacterium tuberculosis direct test in a laboratory that infrequently isolates Mycobacterium tuberculosis

The performance of the Gen-Probe Mycobacterium tuberculosis direct (MTD) test was assessed in a laboratory whose specimens were derived from a population with a low prevalence (1.3%) of tuberculosis. A total of 339 specimens from 113 patients were included in the study. Nine of 10 MTD positive samples were culture positive (smear positive n = 7; smear negative, n = 1; smear not ordered, n = 2). The 10th MTD-positive sample, which was smear and culture negative, was from a patient whose two other study specimens were smear and culture positive and who had a clinical history consistent with tuberculosis. Prior to and following resolution of discrepant results, the sensitivities and specificities of the MTD test relative to culture were 100 and 99.7% and 100 and 100%, respectively.