Evaluation of the enhanced amplified Mycobacterium tuberculosis direct test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

OBJECTIVE: To evaluate the performance of the enhanced Mycobacterium Tuberculosis Direct Test (E-MTD), for the direct detection of M tuberculosis complex (MTBC) in respiratory specimens. DESIGN: Two hundred seventy-four respiratory specimens from 151 patients in respiratory isolation were tested with the E-MTD, and the results were compared with the results of mycobacterial smear, culture, and the earlier form of the test, MTD-1. RESULTS: Forty-one specimens were culture positive for mycobacteria (20 MTBC and 21 nontuberculous mycobacteria), 23 of which were smear positive (16 MTBC, 7 nontuberculous mycobacteria). Twenty-four specimens were positive by E-MTD, and 21 were positive by MTD-1. Of the 20 MTBC culture-positive specimens, 19 were positive by the E-MTD and 19 were positive by the MTD-1. The remaining specimens were MTBC negative by all methods. After resolution of discrepancies, the sensitivity, specificity, and positive and negative predictive values were 95.2%, 100%, 100%, 99.6% for the MTD-1 and 95.2%, 98.8%, 87.0%, and 99.6%, for the E-MTD. For the E-MTD smear-positive and smear-negative specimens, these same values were 93.8%, 100%, 100%, and 87.5% and 100%, 98.8%, 62.5%, and 100%, respectively. CONCLUSION: The results suggest that the E-MTD is a reliable method for the direct detection of MTBC in smear-positive respiratory specimens.     

Clinical evaluation of the Roche AMPLICOR PCR Mycobacterium tuberculosis test for detection of M. tuberculosis in respiratory specimens.

The reliability of the Roche AMPLICOR Mycobacterium tuberculosis test (AMPLICOR MTB) for the diagnosis of pulmonary tuberculosis was evaluated by testing 956 respiratory specimens from 502 patients and comparing results with results by culture and medical history. Of those 135 specimens that were culture positive for mycobacteria, 61 specimens from 31 patients grew M. tuberculosis. Fifty-two specimens were smear positive for acid-fast bacteria (AFB); M. tuberculosis was isolated from 41 of these specimens. On initial testing, the sensitivity and specificity of the AMPLICOR MTB assay, compared with culture, were 78.7 and 99.3%, respectively. After resolution of discrepancies (by review of medical history), the sensitivity, specificity, and positive and negative predictive values of the AMPLICOR MTB assay were 79.4, 99.6, 92.6, and 98.6%, respectively. Two specimens from two patients with no clinical evidence of tuberculosis were AMPLICOR MTB positive and culture positive for Mycobacterium avium complex. For AFB smear-positive specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 97.6, 100, 100, and 90.9%, respectively. For AFB smear-negative specimens, the sensitivity, specificity, and positive and negative predictive values of AMPLICOR MTB were 40.0, 99.5, 69.2, and 98.7%, respectively. Our results support the use of AMPLICOR MTB for rapid diagnosis of tuberculosis in patients whose respiratory specimens are AFB smear positive. Further studies are needed to determine the most clinically relevant and cost-effective use of this assay with AFB smear-negative specimens.     

Evaluation of Two Line Probe Assays for Rapid Detection of Mycobacterium tuberculosis,Tuberculosis (TB) Drug Resistance,and Non-TB Mycobacteria in HIV-Infected Individuals with Suspected TB

Limited performance data from line probe assays (LPAs), nucleic acid tests used for the rapid diagnosis of tuberculosis (TB), nontuberculosis mycobacteria (NTM), and Mycobacterium tuberculosis drug resistance are available for HIV-infected individuals, in whom paucibacillary TB is common. In this study, the strategy of testing sputum with GenoType MTBDRplus (MTBDR-Plus) and GenoType Direct LPA (Direct LPA) was compared to a gold standard of one mycobacterial growth indicator tube (MGIT) liquid culture. HIV-positive (HIV⁺) individuals with suspected TB from southern Africa and South America with <7 days of TB treatment had 1 sputum specimen tested with Direct LPA, MTBDR-Plus LPA, smear microscopy, MGIT, biochemical identification of mycobacterial species, and culture-based drug-susceptibility testing (DST). Of 639 participants, 59.3% were MGIT M. tuberculosis culture positive, of which 276 (72.8%) were acid-fast bacillus (AFB) smear positive. MTBDR-Plus had a sensitivity of 81.0% and a specificity of 100%, with sensitivities of 44.1% in AFB smear-negative versus 94.6% in AFB smear-positive specimens. For specimens that were positive for M. tuberculosis by MTBDR-Plus, the sensitivity and specificity for rifampin resistance were 91.7% and 96.6%, respectively, and for isoniazid (INH) they were 70.6% and 99.1%. The Direct LPA had a sensitivity of 88.4% and a specificity of 94.6% for M. tuberculosis detection, with a sensitivity of 72.5% in smear-negative specimens. Ten of 639 MGIT cultures grew Mycobacterium avium complex or Mycobacterium kansasii, half of which were detected by Direct LPA. Both LPA assays performed well in specimens from HIV-infected individuals, including in AFB smear-negative specimens, with 72.5% sensitivity for M. tuberculosis identification with the Direct LPA and 44.1% sensitivity with MTBDR-Plus. LPAs have a continued role for use in settings where rapid identification of INH resistance and clinically relevant NTM are priorities.     

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.     

Clinical evaluation of the BDProbeTec ET system for rapid detection of Mycobacterium tuberculosis

The performance of the BDProbeTec ET system (BD Biosciences, Sparks, Md.) for direct detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture performed with the BACTEC 460 TB system and Middlebrook 7H11 biplates. Patients known to have been on antituberculous therapy were excluded from the analysis. Of 600 evaluable specimens (4 specimens were excluded from the analysis due to failure of the internal amplification control [IAC]) from 332 patients, 57 grew mycobacteria; 16 were MTBC (from 12 patients), and 41 were nontuberculous mycobacteria. Of the 16 MTBC culture-positive specimens, 12 were smear positive and 4 were smear negative. BDProbeTec ET detected 14 of the 16 MTBC culture-positive specimens, resulting in initial overall sensitivity, specificity, and positive and negative predictive values of 87.5, 99.0, 70.0, and 99.7%, respectively. After resolution of discrepancies by review of medical records and retesting of samples yielding discordant MTBC culture and BDProbeTec ET results, the revised overall sensitivity, specificity, and positive and negative predictive values of the BDProbeTec ET were respectively 93.8, 99.8, 93.8, and 99.8% by specimen and 91.7, 99.7, 91.7, and 99.7% by patient. The BDProbeTec ET System offers the distinct advantage of including an IAC in the specimen well. These data suggest that the test performance is very good, especially for smear-positive samples. However, the number of patients with tuberculosis in our study, especially those with smear-negative disease, was small; therefore, additional studies are needed.     

First evaluation of an improved assay for molecular genetic detection of tuberculosis as well as rifampin and isoniazid resistances

The commercially available line probe assay MTBDRplus 2.0 (Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect Mycobacterium tuberculosis complex (MTBC) and mutations conferring resistance to rifampin (RMP) and isoniazid (INH) directly in smear-negative and smear-positive pulmonary clinical specimens under routine laboratory conditions. A total of 348 samples originating from Moldova, a high-incidence country for tuberculosis (TB), were investigated. Two hundred fifty-seven (73.9%) were smear negative, 12 samples were excluded, and 81 (23.3%) were smear positive. Two DNA extraction methods were applied. Compared to culture and clinical data as the reference standard (adapted from Vadwai V et al., J. Clin. Microbiol. 49:2540-2545, 2011), overall sensitivity and specificity were 87.6 and 99.2%, respectively. One hundred four of the 257 smear-negative samples turned out to be culture positive, and 20 were MTBC culture negative but were positive based on clinical symptoms. The combined sensitivity and specificity in the subgroup of smear-negative samples were calculated to be 79.8 and 99.2%, respectively. MTBDRplus 2.0 detected RMP and INH resistance with sensitivity and specificity of 94.3 and 96.0%, respectively. In conclusion, the MTBDRplus 2.0 assay is a rapid and highly sensitive test for the detection of M. tuberculosis strains from smear-positive and -negative clinical specimens and provides additional information on RMP and INH resistance status, which can easily be included in routine laboratory work flow.     

Rapid detection of Mycobacterium tuberculosis in contaminated BACTEC 12B broth cultures by testing with Amplified Mycobacterium Tuberculosis Direct Test

Contamination of broth cultures of acid-fast bacilli (AFB) by bacterial species other than Mycobacterium species frequently occurs. Many of these contaminated cultures require redecontamination and reincubation before the appropriate tests can be performed for identification, significantly affecting the turnaround time for reporting culture results. In this study, the Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe) was performed to detect the Mycobacterium tuberculosis complex (MTBC) in 125 BACTEC 12B broth cultures with positive growth indices. Among these, 41 grew non-AFB bacteria only, and all 41 were negative by the MTD. The remaining 84 bottles contained contaminated cultures that grew both AFB and other bacteria or yeasts. Repeat decontamination and reincubation of these specimens required a mean time of 13 days (range, 3 to 40 days). The MTD results were positive for 10 samples, 9 of which were MTBC culture positive and 1 of which grew Myobacterium celatum, a species known to cross-react in the MTD. All cultures growing other mycobacterial species were negative by the MTD. The results of this study demonstrate that the MTD is both sensitive and specific in detecting MTBC in contaminated broth cultures and that, when used selectively, the MTD can potentially rule in or out a diagnosis of MTBC as much as 12 days earlier than using nonamplified DNA probe testing alone can.     

Multicenter Evaluation of the Abbott LCx Mycobacterium tuberculosis Ligase Chain Reaction Assay

Four Australian hospital laboratories evaluated the performance of the Abbott LCx Mycobacterium tuberculosis assay with 2,347 specimens (2,083 respiratory and 264 nonrespiratory specimens) obtained from 1, 411 patients. A total of 152 specimens (6.5%) were culture positive for Mycobacterium tuberculosis complex (MTBC); of these, 79 (52%) were smear positive. After resolution of discrepant data, the overall sensitivity, specificity, and positive and negative predictive values for the LCx assay were 69.7, 99.9, 99.1, and 97.7% respectively. For smear-positive respiratory specimens that were culture positive for MTBC, the values were 98.5, 100, 100, and 98.4%, respectively, while the values for smear-negative respiratory specimens were 41.5, 99.9, 96.4, and 98%, respectively. Relative operating characteristic curves were constructed to demonstrate the relationship between sensitivity and specificity for a range of possible cutoff values in the LCx assay. These graphs suggested that the assay sensitivity for respiratory samples could be increased from 70.2 to 78.6%, while the specificity would be reduced from 99.9 to 99.4% by inclusion of a grey zone (i.e., LCx assay values of between 0.2 and 0.99). An algorithm is presented for the handling of specimens with LCx assay values within this grey zone.     

Comparison of Direct and Concentrated Acid-Fast Smears To Identify Specimens Culture Positive for Mycobacterium spp.

Microscopic examination of respiratory specimens for acid-fast bacilli (AFB) plays a key role in the initial diagnosis of tuberculosis, monitoring of treatment, and determination of eligibility for release from isolation. The objective of this study was to compare the sensitivity obtained with smears for detection of AFB (AFB smears) made directly from respiratory specimens (direct AFB smears) to that obtained with parallel smears made from concentrates of the specimens (concentrated AFB smears). A total of 2,693 specimens were evaluated; 1,806 were from the University of California Irvine Medical Center Medical Microbiology Laboratory (UCIMC), which serves a tertiary-care hospital with outpatient clinics, and 887 were from the Microbial Disease Laboratory at the California Department of Public Health (MDL), which receives specimens from outpatient facilities and clinics on Pacific islands. Of the 353 AFB culture-positive specimens at UCIMC, there was a statistically significant difference in the sensitivity of the direct AFB smear (34%) and that of the smear made from the concentrated specimen (58%) (P < 0.05). This was also true for the 208 specimens positive for Mycobacterium tuberculosis, for which the sensitivity of the direct smear was 42% (87 of 208) and that for the smear made from the concentrated specimen was 74% (154 of 208). At MDL, where all but 1 of the 45 culture-positive specimens grew M. tuberculosis, the sensitivity of the smear made from the concentrated specimen was 93% (42 of 45) and was not significantly higher than the sensitivity of the direct smear, which was 82% (37 of 45). By combining the results from both laboratories, 42 patients from whom at least three specimens were received were culture positive for M. tuberculosis. The cumulative results for the initial three specimens from these patients showed that the direct smear detected M. tuberculosis in 81% of these patients, whereas the smear made from the concentrate detected M. tuberculosis in 91% of these patients. In summary, when all culture-positive specimens are considered, the sensitivity of the direct smear compared to that of a smear made from the concentrated specimen was significantly different overall in the two different laboratory settings. However, this difference was reduced only if the cumulative results for the initial three specimens received from patients who were culture positive for M. tuberculosis were evaluated.     

Amplification of rRNA for assessment of treatment response of pulmonary tuberculosis patients during antimicrobial therapy.

The time course of persistence of Mycobacterium tuberculosis as measured by detection of rRNA, acid-fast bacillus (AFB) smear, and culture was determined for pulmonary tuberculosis patients during antimicrobial therapy. Twenty-three patients who were initially AFB smear positive and who subsequently completed a course of antimicrobial therapy were selected for the study. Sequential specimens were tested by AFB smear, culture, and rRNA amplification (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test [MTD]). The initial diagnostic specimens of all patients were positive by culture; those of 22 patients (96%) also were positive by MTD. Overall, MTD results remained positive longer than both smear and culture results. The median times to the last positive test result were 9 days for AFB smear, 26 days for culture, and 30 days for MTD. The last positive test result was the AFB smear result in 4% of cases, the culture result in 22%, and the MTD result in 52%. Fifty-six percent of patients had a period of shedding of noncultivable M. tuberculosis which was detected by MTD after culture results had converted to negative. This noncultivable period lasted 7 to 245 days. All three tests became reproducibly negative before the end of therapy and remained negative during follow-up for up to 1 year. These results indicate that during successful antimicrobial therapy, M. tuberculosis is eliminated in sputum samples as measured by amplification of rRNA, as well as by AFB smear and culture. No long-term rRNA carrier state was detected. While the time course of clearance of M. tuberculosis measured by rRNA overall was longer than with the two traditional tests, the rRNA test results allow sensitive and precise measurement of the clearance of noncultivable M. tuberculosis from respiratory specimens. This attribute may allow rRNA testing to be useful in clarifying patient response to antimicrobial therapy.     

Molecular techniques in mycobacterial detection

OBJECTIVE: To assess the clinical utility of the commercial nucleic acid amplification (NAA) tests (ie, Amplified Mycobacterium Tuberculosis Direct Test, Gen-Probe, Inc and AMPLICOR Mycobacterium tuberculosis Test, Roche Molecular Systems, Inc) for direct detection of Mycobacterium tuberculosis complex. DATA SOURCES: Review of the English-language literature. CONCLUSIONS: The performance of both NAA tests is excellent (sensitivity, > or = 95%; specificity, 100%) when testing respiratory specimens that are smear-positive for acid-fast bacilli (AFB). Only the Gen-Probe assay is approved for testing respiratory specimens regardless of the AFB smear result. Data from 3 studies showed that the sensitivity of the Mycobacterium Tuberculosis Direct Test in smear-negative patients ranged from 83% to 85%, and that the specificity was 99%. Both NAA tests have been used to test nonrespiratory specimens; in some studies, the performance was comparable to the performance obtained for respiratory specimens, whereas in others, it was lower. The NAA tests also appear to be reliable tools for rapid detection 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 result of the AFB smear. In smear-positive patients, public health and hospital infection-control resources are predominantly affected. The potential for influencing patient outcome is much greater when the AFB smear is negative. In smear-negative patients, the NAA test could provide more rapid diagnosis of tuberculosis and subsequent initiation of therapy; eliminate the need for invasive diagnostic procedures, which are both costly and pose an added risk to the patient; and allow earlier discharge of hospitalized patients. Prospective studies concerning the cost-effectiveness of the NAA tests are needed.     

Concentration of sputum by cytocentrifugation for preparation of smears for detection of acid-fast bacilli does not increase sensitivity of the fluorochrome stain.

To evaluate the usefulness of cytocentrifugation for detection of acid-fast bacilli (AFB) in sputum specimens, we compared this method to a traditional concentration method for the preparation of smears. A total of 844 sputum specimens (from 579 patients) of adequate volume that were submitted for detection of mycobacteria were evaluated. A portion of each specimen was used for cytocentrifugation; the remainder was processed by our decontamination-concentration protocol (2% sodium hydroxide-N-acetyl-L-cysteine; centrifugation at 3,600 x g for 15 min) for preparation of smears and culture. All smears were stained with auramine O. Ninety-four cultures from 46 patients gave positive results, and AFB were seen in one or both smears from 53 specimens; 3 of the latter specimens (positive by both smear methods) were culture negative. Of the 50 AFB smear-positive and culture-positive specimens, 46 were smear positive by traditional concentration, and 47 were positive by the cytocentrifugation smear (P, not significant). Cultures of all specimens that were smear positive by only one method grew nontuberculous mycobacteria. The routine use of cytocentrifugation for concentrating sputum specimens increases the cost of smear preparation but does not increase detection of AFB by auramine O staining; however, it would be useful in handling emergent requests for AFB smears.     

Comparison of the Septi-Chek AFB and BACTEC systems and conventional culture for recovery of mycobacteria.

The performance of the Septi-Chek AFB system was compared with that of the BACTEC radiometric system and that of Lowenstein-Jensen agar slants (LJ) for detection of mycobacteria in clinical specimens. A total of 642 specimens were cultured; 61 (9.5%) yielded mycobacteria. Mycobacterium tuberculosis (34 isolates) and Mycobacterium avium complex (25 isolates) were the predominant species isolated. Of the 61 culture-positive specimens, 30 were smear positive and 31 were smear negative. Overall, 95% of the positive specimens were detected by Septi-Chek and BACTEC (100% of M. tuberculosis isolates) and 75% by LJ (82% of M. tuberculosis isolates). The mean times to detection were 15 days for BACTEC, 23 days for Septi-Chek, and 27 days for LJ. Of the 30 smear-positive specimens, 100% were recovered by Septi-Chek and BACTEC and 90% were recovered by LJ. Of the 31 smear-negative specimens, 90% were detected by Septi-Chek and BACTEC and 61% were detected by LJ. The Septi-Chek and BACTEC systems are superior to the conventional (LJ) mycobacterial culture method. Although Septi-Chek requires more time for the detection of mycobacteria than BACTEC, it is comparable in terms of overall recovery.     

Evaluation of the Cobas TaqMan MTB test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens

The Cobas TaqMan MTB test, based on real-time PCR technology, was evaluated for direct detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens. A total of 1,093 samples from 446 patients, including 118 acid-fast smear-positive and 975 acid-fast smear-negative specimens, were investigated. Diagnostic cultures performed with 7H11 agar, Löwenstein-Jensen medium, and the Bactec MGIT 960 system were considered the reference methods. When discrepant results between the Cobas TaqMan MTB test and culture occurred, additional results from the BD MGIT TBc identification test and the GenoType Mycobacterium CM test performed on growth-positive and acid-fast-stain-positive MGIT tubes and review of the patient''s medical history were used for discrepancy analysis. The overall sensitivity, specificity, positive predictive value, and negative predictive value for the Cobas TaqMan MTB test were 91.5%, 98.7%, 91.5%, and 98.7%, respectively. In general, the performance of the new Cobas TaqMan MTB test was comparable to that of the replaced Cobas Amplicor MTB system. The most prominent feature of the new system was its extraordinarily high sensitivity (79.5%) for detecting MTBC in smear-negative specimens; out of 44 smear-negative but culture-positive specimens, 35 were positive by the new system. The Cobas TaqMan MTB assay, including DNA extraction, can be completed within 3 h.     

Comparison of the Xpert MTB/RIF test with an IS6110-TaqMan real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens

The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.     

Use of Transrenal DNA for the Diagnosis of Extrapulmonary Tuberculosis in Children: a Case of Tubercular Otitis Media

The diagnosis of tuberculosis (TB) is difficult in children, especially for smear-negative pulmonary and extrapulmonary TB, which are common at this age. We report an 11-year-old girl with TB otitis media with negative smear microscopy and Xpert MTB/RIF but positive Mycobacterium tuberculosis-specific transrenal DNA (Tr-MTB-DNA) test results and culture for M. tuberculosis.     

Performance assessment of the DR. MTBC Screen assay and the BD ProbeTec ET system for direct detection of Mycobacterium tuberculosis in respiratory specimens

The performance of the DR. MTBC PCR-based assay and the BD ProbeTec ET Mycobacterium tuberculosis Complex Direct Detection (DTB) assay for the direct detection of Mycobacterium tuberculosis was evaluated using 1,066 consecutive clinical respiratory samples collected from 494 patients who did not have old cases of pulmonary tuberculosis and were not receiving antituberculosis treatment at National Taiwan University Hospital from January to February 2005. The results of both assays were compared to the "gold standard" of combined culture results and clinical diagnosis. The overall sensitivity and specificity of the DR. MTBC Screen assay were 56.6% and 98.9%, respectively, and of the DTB assay were 63.2% and 98.4%, respectively. The positive and negative predictive values for the DR. MTBC Screen assay were 84.5% and 95.4%, respectively, and for the DTB assay were 81.7% and 96.0%, respectively. The DR. MTBC Screen assay produced 11 false-positive results for 11 patients, including three samples yielding non-M. tuberculosis mycobacteria (one each for M. abscessus, a mixture of M. abscessus and M. chelonae, and unidentified non-tuberculosis mycobacteria). The DTB assay produced 15 false-positive results for 13 patients, including five samples from four patients yielding non-tuberculosis mycobacteria (two for M. abscessus, one for a mixture of M. abscessus and M. chelonae, and two for unidentified non-tuberculosis mycobacteria). This study demonstrated that the DR. MTBC Screen assay has a similar diagnostic value but fewer false-positive results than the DTB assay for respiratory specimens.     

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.     

Performance of Xpert MTB/RIF RUO assay and IS6110 real-time PCR for Mycobacterium tuberculosis detection in clinical samples

The Cepheid Xpert MTB/RIF research-use-only (RUO) assay and a laboratory-developed test (LDT) targeting IS6110 were evaluated and compared to mycobacterial culture as the gold standard. The performance characteristics of both molecular assays were determined by using 112 specimens from 90 patients, including 89 pulmonary specimens and 23 extrapulmonary specimens. Of the specimens tested, 37 (33%) were culture positive for the Mycobacterium tuberculosis complex; 29 were pulmonary, and 8 were extrapulmonary. Of these culture-positive specimens, 83% of the pulmonary specimens and 50% of the extrapulmonary specimens were smear positive. There was complete concordance between the smear-positive culture-positive specimens, independent of the anatomical site (100% sensitivity). The sensitivity of the MTB/RIF RUO assay for smear-negative specimens was 60% for pulmonary and 75% for extrapulmonary specimens, while the IS6110 LDT sensitivities were 40% and 0%, respectively. There was also complete concordance among the culture-negative specimens tested. Both assays showed 95% specificity, with four culture-negative specimens testing as positive. A review of patient records indicated that there was a high likelihood of the presence of M. tuberculosis complex DNA in the false-positive specimens. Biosafety analysis was performed and showed an acceptable reduction in organism viability using the processing methods described above. Both molecular assays are suitable for the detection of M. tuberculosis isolates in smear-positive pulmonary and extrapulmonary specimens, while the sensitivity of the detection of M. tuberculosis isolates in smear-negative specimens was variable.     

Enhanced Amplified Mycobacterium Tuberculosis Direct Test for Detection of Mycobacterium tuberculosis Complex in Positive BACTEC 12B Broth Cultures of Respiratory Specimens

The reliability of the Gen-Probe enhanced Amplified Mycobacterium Tuberculosis Direct Test (MTD) for identification of Mycobacterium tuberculosis complex (MTBC) in BACTEC 12B broth cultures of respiratory specimens was evaluated by testing aliquots from 268 bottles with a growth index of >/=50. MTD results were compared to those obtained by usual laboratory protocol, whereby MTBC was identified by DNA probe (Gen-Probe, Inc.) testing sediment from broth samples or colonies on a solid medium. For the first 134 cultures, from which 68 mycobacterial isolates (including 27 MTBC isolates) were recovered, both fresh and frozen aliquots were tested. MTD results for the frozen aliquots agreed with the identification by usual protocol in all cases, whereas there was one false-negative MTD result with fresh aliquots. For the remaining 134 cultures, only frozen aliquots were tested. Of the total 268 broth cultures (from 210 patients) evaluated, 137 (51.1%) grew mycobacteria, including 60 MTBC isolates. All 60 isolates were MTD positive, as was one additional culture that grew Mycobacterium gordonae. The latter culture was from a patient who was diagnosed with tuberculosis a few months earlier and was on therapy; therefore, the MTD result was considered a true positive. Sensitivity, specificity, and positive and negative predictive values of MTD were 100%. The mean times from specimen receipt to identification of MTBC were 15 (+/-1) days (range, 4 to 27 days) for BACTEC plus MTD and 19 (+/-1) days (range, 6 to 36 days) for the usual protocol (P < 0.001). These data indicate that the MTD is a rapid, reliable method for identification of MTBC in fresh or frozen aliquots of broth from positive BACTEC 12B cultures of respiratory specimens.