Evaluation of Roche Amplicor PCR assay for Mycobacterium tuberculosis.

The Roche Amplicor Mycobacterium tuberculosis PCR test (RMtb-PCR) was compared with mycobacterial culture, with the BACTEC 460 system and inoculation on Lowenstein-Jensen media. Results were interpreted with an adjusted "gold standard" incorporating clinical diagnosis. A total of 1,480 clinical specimens from 1,155 patients, including tissues and fluids, as well as 141 specimens which demonstrated a positive growth index on the BACTEC 460 system were assessed. The sensitivity, specificity, and positive and negative predictive values of RMtb-PCR compared with the adjusted gold standard for clinical specimens were 79, 99, 93, and 98%, respectively. In smear-positive specimens, the sensitivity of RMtb-PCR was 98% versus 53% for smear-negative specimens. When RMtb-PCR was performed two times per week, PCR results were available an average of 21 days before the culture results. For specimens demonstrating a positive growth index on the BACTEC 460 system, RMtb-PCR had a sensitivity and specificity of 98 and 100%, respectively. This study demonstrates the value of a commercial nucleic acid amplification kit for rapid diagnosis of M. tuberculosis, particularly in smear-positive specimens or BACTEC culture-positive specimens.     

Diagnosis of tuberculosis by Amplicor Mycobacterium tuberculosis test: a multicenter study.

The Amplicor Mycobacterium tuberculosis test is a new PCR assay for the direct detection of Mycobacterium tuberculosis from clinical samples. A multicenter study that included six laboratories was done to evaluate the Amplicor test in comparison with direct microscopy and culture (solid or radiometric media), and the culture method was used as the "gold standard." A total of 2,073 specimens, i.e., 1,749 respiratory specimens and 324 other specimens, were tested. A total of 184 cultures yielded M. tuberculosis. Of these 184 cultures, 77 (42%) were smear negative and 23 (12.5%) concerned extrapulmonary specimens. The sensitivity of the Amplicor test for all of the specimens and for extrapulmonary, smear-positive, and smear-negative specimens was 86, 83, 94.5, and 74%, respectively. The sensitivity of direct microscopy in comparison with that of culture was 58%. A total of 95% of patients with culture-proven tuberculosis were diagnosed by the Amplicor test, whereas direct microscopy detected mycobacteria in only 72% of these patients. The Amplicor test exhibited a high degree of specificity (98%). The assay was very rapid and easy to perform.     

Clinical evaluation of a Mycobacterium tuberculosis PCR assay.

On the basis of previously published PCR primer sequences, we have designed a sensitive system for detecting DNA of the Mycobacterium tuberculosis complex (MTB) in patient sputum samples which employs a fast and simplified sample preparation method appropriate for routine diagnostic testing. In order to evaluate the accuracy of the PCR assay, we performed a prospective study with 103 patients, comparing PCR results with culture results of samples obtained from a parallel culture assay as well as with subsequent culture results. Using two MTB-specific PCR primer systems, we found 48 of 49 tuberculosis (Tb) patients to be PCR positive (PCR sensitivity, 0.98). Sixteen of 54 presumably non-Tb patients showed amplifiable MTB DNA (specificity, 0.7). The study demonstrates that for diagnostic applications of MTB PCR two MTB-specific primer pairs should be used. MTB infection is extremely unlikely in cases of MTB PCR-negative samples: with our method for the exclusion of active Tb, the validity of one PCR assay seems to be equivalent to those of at least three culturing procedures. Positive PCR results do not necessarily reflect active MTB infection. It remains to be shown whether positive PCR results in Tb-negative patients mean false-positivity, an early laboratory finding which predicts a subsequent reactivation of a prior Tb infection, or whether asymptomatic patients may carry PCR-amplifiable MTB DNA without any clinical relevance. It is important to point out that the validity of PCR results in clinical studies depends on the use of contamination controls parallel to all PCR steps and the simplicity of the DNA extraction method as well as on the specificity of the PCR results.     

Comparison of Amplicor, in-house PCR, and conventional culture for detection of Mycobacterium tuberculosis in clinical samples.

Five hundred four clinical specimens (337 sputum and 167 bronchial samples) from 340 patients were tested for the presence of M. tuberculosis complex by the Amplicor M. tuberculosis test and by an in-house PCR. The results were compared with those obtained by conventional culture and by direct microscopy. Thirty specimens (from 14 patients) were positive by in-house PCR, 25 (from 13 patients) were positive by the Amplicor M. tuberculosis test, and 24 (from 10 patients) were positive by culture. Cultures from 16 specimens were contaminated with other bacteria. Strong inhibition of in-house PCR was found with three samples. After discordancy analyses, with clinical data as supportive evidence for tuberculosis, 27 true-positive and 458 true-negative samples were defined. On the basis of these figures, the sensitivities of the Amplicor M. tuberculosis test, in-house PCR, culture, and microscopy were 70.4, 92.6, 88.9, and 52.4%, respectively. The specificities of all four tests were higher than 98%. The good performance of the in-house PCR for detection of M. tuberculosis makes it a very useful additional tool in M. tuberculosis diagnostics. In contrast, the Amplicor test needs to be improved. Twenty-three of the Amplicor-negative samples were further tested for inhibition of the Amplicor system by retesting the DNA extracts after the addition of M. tuberculosis DNA. In 15 of these samples, 5 true positives and 10 true negatives, inhibition of the Amplicor test was demonstrated. This might explain the lack of sensitivity of the Amplicor test. If the inhibition problem can be solved, the Amplicor M. tuberculosis test, which is already rapid, very user-friendly, and reasonably priced, may certainly become very useful in microbiological laboratories.     

Detection and identification of Mycobacterium tuberculosis directly from sputum sediments by Amplicor PCR.

Sputum specimens received for the diagnosis of tuberculosis or other mycobacterial infections were tested by a PCR-based assay and culture techniques. Results of the PCR assay (Amplicor Mycobacterium tuberculosis Test) were compared with results of standard culture techniques with cultures held for 6 weeks. One thousand nine specimens were included: 301 retrospective specimens (frozen at -70 degrees C and later tested by PCR) and 708 prospective specimens (tested within 1 day of processing). One hundred sixty-two (16%) of the specimens were culture positive for M. tuberculosis; 83 (51%) of these were also fluorochrome stain positive. The sensitivity and specificity of the Amplicor PCR compared with those of culture were 83% (134 of 162 specimens) and 97% (800 of 827 specimens), respectively. The sensitivity for fluorochrome stain-positive specimens was 99%, and that for fluorochrome stain-negative specimens was 66%. The great majority of the 28 PCR-negative, culture-positive specimens were low positives; 27 were smear negative and 19 contained < 100 CFU of M. tuberculosis per ml. The 27 PCR-positive, culture-negative specimens included 24 that were positive by repeat testing by alternate primer PCR and were from patients with tuberculosis on antimicrobial therapy. With these considered culture misses, the final sensitivities of PCR and culture were 85, and 87%, respectively, while the specificities were 99.6 and 100%, respectively. After normal laboratory processing of sputum specimens, the Amplicor PCR assay can be completed in 8 h. Thus, it is possible to have results available within 10 h of specimen submission.     

Comparison of Roche Cobas Amplicor Mycobacterium tuberculosis Assay with In-House PCR and Culture for Detection of M. tuberculosis

The new Roche Cobas Amplicor Mycobacterium tuberculosis assay, which is a semiautomated version of the manually performed Roche Amplicor M. tuberculosis test, was compared to culture and an IS6110-based in-house PCR protocol. A total of 1,681 specimens from 833 patients, including specimen types other than sputum, were tested in parallel by both the in-house PCR and the Cobas Amplicor M. tuberculosis assay. After we resolved discrepant PCR results, the sensitivity, specificity, and positive and negative predictive values for the Cobas Amplicor M. tuberculosis assay were 66.33, 99.71, 94.36, and 97.66%, respectively. The corresponding values for the in-house PCR were 91.08, 99.85, 97.87, and 99.37%, respectively. For culture- and smear-positive specimens, the sensitivity of the Cobas Amplicor M. tuberculosis test was 96.42% (in-house PCR, 100%). If only smear-negative sputum specimens were considered, the Cobas Amplicor M. tuberculosis assay exhibited a sensitivity of 45.45% (in-house PCR, 63.63%) relative to that of culture. With a modified protocol for DNA extraction (washing of samples plus ultrasonication), both PCR methods performed better with gastric aspirates than with sputum samples (sensitivity of the Cobas Amplicor M. tuberculosis assay with smear-negative gastric aspirates, 70.00%; sensitivity of in-house PCR, 90.00%). With dithiothreitol being used for liquefaction of specimens in this study, the Cobas Amplicor M. tuberculosis assay exhibited an inhibition rate of 9.16%. In our view, the new Cobas Amplicor M. tuberculosis test (i) is well suited for typing of smear-positive specimens, (ii) may also be applied to gastric aspirates and other types of specimens if DNA extraction methods are modified appropriately, and (iii) exhibits a sensitivity with smear-negative sputum specimens which makes it recommendable that a minimum of three samples from the same patient be tested.     

Detection of Mycobacterium tuberculosis in BACTEC 12B broth cultures by the Roche Amplicor PCR assay.

To evaluate the ability of the Amplicor MTB Assay to detect Mycobacterium tuberculosis complex (MTBC) organisms in BACTEC 12B broth cultures, 249 cultures with a growth index (GI) of > or = 20 from 160 patients were tested retrospectively. Specimens were processed by standard methods, and then BACTEC 12B vials and Middlebrook 7H11/7H115 plates were inoculated, incubated, and interpreted in accordance with the manufacturer's instructions and laboratory protocol. From 12B vials with a GI of > or = 20, and aliquot of broth was removed and frozen at -20 degrees C until assayed by PCR. PCR results were compared to those obtained by the usual laboratory protocol, whereby MTBC organisms were identified by a DNA probe assay performed on broth from 12B vials with a GI or > or = 300 or on colonies from solid medium. Of the 249 broth cultures evaluated, 142 contained mycobacteria, including 44 that contained MTBC organisms. Of these 44 cultures, 41 were PCR positive; the 3 that were PCR negative were blood specimens collected in an Isolator tube. All 98 cultures with nontuberculous mycobacteria and the 107 that did not contain mycobacteria were PCR negative. Thus, the sensitivity and specificity of PCR were 93 and 100%, respectively. For those culture sin which MTBC organisms were identified by both the DNA probe and PCR assays, the mean time from specimen inoculation to detection and identification of MTBC organisms was 16 (range, 4 to 26) days for the PCR and 28 (range, 13 to 43) days for the DNA probe assay (P < 0.0001). In summary, PCR is a rapid, reliable method for detection of MTBC organisms in BACTEC 12B broth cultures with a GI of > or = 20.     

Comparison of in-house PCR with conventional techniques and Cobas Amplicor M. tuberculosis kit for detection of Mycobacterium tuberculosis

PURPOSE: Polymerase chain reaction (PCR) assay, introduced as a fast and sensitive diagnostic method, is useful in detecting Mycobacterium tuberculosis. The purpose of this study was to evaluate the usefulness of in-house PCR assay in the detection of Mycobacterium tuberculosis by comparing PCR results with conventional diagnostic techniques and Cobas Amplicor M. tuberculosis kit. MATERIALS AND METHODS: We retrospectively assessed the diagnostic yield of in-house PCR method employed for the amplification IS6110 sequences in 2,973 specimens. We also compared in-house PCR with Cobas Amplicor M. tuberculosis kit in 120 specimens collected from June to July 2006. Routine acid-fast stain (AFS) and culture assay were also performed and analyzed. RESULTS: Of 2,973 cases, 2,832 cases (95.3%) showed consistent results between in house PCR, AFS and culture methods, whereas 141 (4.7%) displayed inconsistent results. The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 77.5%, 99.7%, 95.5%, and 98.0%, respectively for PCR; 49.2%, 100%, 100%, and 95.7%, respectively, for AFS method; and 80.7%, 100%, 100%, and 98.3%, respectively, for culture assay. Consistent results between PCR and Cobas Amplicor M. tuberculosis kit were shown in 109 cases (90.8%). The sensitivities, specificities, and positive and negative predictive values of each method were as follows: 81.3%, 98.9%, 96.3%, and 93.5% respectively for PCR and 71.9%, 100%, 100%, and 90.7%, respectively, for Cobas Amplicor kit. CONCLUSION: In-house PCR and Cobas Amplicor kit show high sensitivity and specificity, and are reliable tests in the diagnosis of tuberculosis.     

Quantitative culture of Mycobacterium tuberculosis from clinical sputum specimens and dilution endpoint of its detection by the Amplicor PCR assay.

The minimum number of Mycobacterium tuberculosis CFU detectable in clinical sputum specimens by the Amplicor PCR test was estimated by performing the test on duplicate samples of quantitatively cultured serial dilutions of sputum. Positive PCR test results were obtained for all samples that contained 42 CFU of M. tuberculosis. The detection limits of the PCR assay for decontaminated (N-acetyl-L-cysteine [NALC]-NaOH) and nondecontaminated (NALC only) specimens were equivalent, even though the number of CFU cultured from decontaminated samples was only 11 to 20% of the number cultured from nondecontaminated samples. Thus, the 42 CFU that could be detected in nondecontaminated specimens by the Amplicor PCR test correspond to the approximately 8 CFU (0.20 x 42) that could be recovered in culture after decontamination with NALC-NaOH.     

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.     

Removal of PCR inhibitors by silica membranes: evaluating the Amplicor Mycobacterium tuberculosis kit

The effectiveness of PCR inhibitor removal by silica membranes in combination with the Amplicor Mycobacterium tuberculosis kit was analyzed for 655 respiratory and nonrespiratory specimens. The overall inhibition rate was reduced from 12.5%, when applying the Amplicor kit alone, to 1.1% with the addition of silica membrane DNA purification.     

Large-scale human immunodeficiency virus rapid test evaluation in a low-prevalence ugandan blood bank population

The use of rapid tests for human immunodeficiency virus (HIV) has become standard in HIV testing algorithms employed in resource-limited settings. We report an extensive HIV rapid test validation study conducted among Ugandan blood bank donors at low risk for HIV infection. The operational characteristics of four readily available commercial HIV rapid test kits were first determined with 940 donor samples and were used to select a serial testing algorithm. Uni-Gold Recombigen HIV was used as the screening test, followed by HIV-1/2 STAT-PAK for reactive samples. OraQuick HIV-1 testing was performed if the first two test results were discordant. This algorithm was then tested with 5,252 blood donor samples, and the results were compared to those of enzyme immunoassays (EIAs) and Western blotting. The unadjusted algorithm sensitivity and specificity were 98.6 and 99.9%, respectively. The adjusted sensitivity and specificity were 100 and 99.96%, respectively. This HIV testing algorithm is a suitable alternative to EIAs and Western blotting for Ugandan blood donors.     

Rapid diagnosis of pulmonary tuberculosis by using Roche AMPLICOR Mycobacterium tuberculosis PCR test.

A rapid PCR-based test for the diagnosis of pulmonary tuberculosis, the Roche AMPLICOR Mycobacterium tuberculosis test (AMPLICOR MTB), was evaluated. Results from AMPLICOR MTB were compared with culture results and the final clinical diagnosis for each patient. A total of 985 specimens from 372 patients were tested. When AMPLICOR MTB results were compared with resolved results, i.e., a specimen grew M. tuberculosis or was obtained from a patient with a clinical diagnosis of tuberculosis, the sensitivity, specificity, positive predictive value, and negative predictive value for the AMPLICOR MTB test were 66.7, 99.6, 91.7, and 97.7%, respectively. These results were comparable to those obtained from culture. Test results were available approximately 6.5 h after specimen receipt in the laboratory. Our data demonstrate that AMPLICOR MTB will provide rapid, valuable information for the diagnosis and control of tuberculosis.     

Evaluation of Amplicor MTB test as adjunct to smears and culture for direct detection of Mycobacterium tuberculosis in the French Caribbean.

A total of 784 specimens collected from 370 individuals between January and August 1995 were analyzed by using the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic System, Basel, Switzerland), a PCR-based test for the direct detection of organisms of the M. tuberculosis complex. The PCR results were compared with standard bacteriological data, including those obtained by acid-fast microscopy, culture, and biochemical identification as well as final clinical diagnosis for each patient. Several parallel controls were used: the kit DNA positive control, 10(3) CFU of M. tuberculosis, and three negative controls for each independent assay. No false-positive PCR results were obtained, and overall, M. tuberculosis was detected in 20 of 370 individuals screened. Five additional patients during the same time were found to be infected with mycobacteria other than tubercle bacilli; their specimens gave positive smear and/or culture test results, but Amplicor tests were always negative. The sensitivity, specificity, positive predictive value, and negative predictive value for the Amplicor MTB test compared with culture per specimen were 76.7, 97.7, 66.0, and 98.6%, respectively. For resolved cases, these values were, respectively, 69.4, 100, 100, and 96.8%; however, the sensitivity and negative predictive value increased to 90.9 and 99.2%, respectively, if PCR-negative nonrespiratory specimens (gastric washings) were not considered. When only specimens from proven tuberculosis patients were considered (n = 114) and the sum of PCR-positive and/or culture-positive samples from proven tuberculosis patients was considered the total number of positive samples, PCR had a sensitivity of 83.3% compared with 71.6% for culture. Results per patient (about three samples each) yielded 100% sensitivity and 100% specificity. We conclude that the Amplicor MTB test is highly specific and rapid for routine use in a clinical laboratory. However, in order to obtain a higher degree of sensitivity, it should be run as an adjunct to smears and culture with at least three samples for each patient, and a single-sample PCR-negative results must be considered carefully because of potential false-negatives.     

A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples.

Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.     

Comparison of the real-time PCR method and the Gen-Probe amplified Mycobacterium tuberculosis direct test for detection of Mycobacterium tuberculosis in pulmonary and nonpulmonary specimens

Real-time PCR was compared to Amplified Mycobacterium tuberculosis Direct Test (AMTDII) for 100 clinical specimens. The overall sensitivities of the real-time PCR method and AMTDII were similar for respiratory and nonrespiratory specimens. However, real-time PCR seemed to be less susceptible to amplification inhibitors than AMTDII.     

Prospective evaluation of a whole-blood test using Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 for diagnosis of active tuberculosis

A new immunodiagnostic test based on the Mycobacterium tuberculosis-specific antigens CFP-10/ESAT-6(QFT-RD1) has been launched as an aid in the diagnosis of latent tuberculosis (TB) infection (LTBI). The aim of this study was to evaluate this test for the diagnosis of active TB. Eighty-two patients with suspicion of TB and 39 healthy BCG-vaccinated persons were enrolled. Forty-eight had active TB, 25 did not, and 9 were excluded. Sensitivity and specificity of the test for active TB were evaluated in a prospective blinded manner in patients suspected of TB. The sensitivity of the QFT-RD1 was 85% (40/48; confidence interval [CI], 75 to 96), and it was higher than the sensitivity of microscopy, 42% (20/48; CI, 27 to 56; P = 0.001), and culture, 59% (27/46; CI, 44 to 73; P = 0.009). Of patients with extrapulmonary TB, 92% (12/13) were QFT-RD1 positive, whereas only 31% (4/13) were positive by microscopy and 42% (5/12) by culture (P < 0.05), and 87% (13/15) of those who were negative by both microscopy and culture were QFT-RD1 positive. By combining microscopy and culture with the QFT-RD1 test, sensitivity increased to 96% (CI, 90 to 102). Ten of 25 (40%) non-TB patients were QFT-RD1 positive, resulting in a specificity of 60%. However, 80% (8/10) of these had risk-factors for TB, indicating latent infection in this group. In healthy controls, only 3% (1/39) were QFT-RD1 positive. In conclusion, the QFT-RD1 test is sensitive for diagnosis of TB, especially in patients with negative microscopy and culture. The accuracy of the QFT-RD1 test will vary with the prevalence of LTBI. We suggest that the QFT-RD1 test could be a very useful supplementary tool for the diagnosis of TB.     

Evaluation of a semi-automated Seegene PCR workflow with MTB,MDR, and NTM detection for rapid screening of tuberculosis in a low-prevalence setting

In areas of low tuberculosis (TB) prevalence, laboratory diagnosis of TB may essentially cover non-tuberculous mycobacteria (NTM) in addition to Mycobacterium tuberculosis (MTB). In this study, a semi-automated PCR workflow distinguishing MTB and NTM (Anyplex™ MTB/NTMe, Seegene) and subsequently detecting MTB isoniazid/rifampicin resistance (Allplex™ MTB/MDRe, Seegene) was evaluated for replacing smear microscopy of acid-fast bacilli as the rapid screening method for TB. With 279 clinical samples, 47 cultures positive for MTB and 76 for NTM, the Anyplex™ MTB/NTMe assay and smear microscopy showed equal sensitivities (49.6% vs 50.8%, respectively) but Anyplex™ MTB/NTMe was more sensitive for MTB (63.8% vs 25.6%) than for NTM (40.8% vs 64.5%). Allplex™ MTB/MDRe showed a slightly higher sensitivity of 68.1% for MTB (32/47 positive, n = 222). Antibiotic resistance profiles were correctly identified for all MTB isolates (one MDR isolate). Specificity was 100% for both assays. Anyplex™ MTB/NTMe detected all the 18 NTM species present in the study. The analytical performance of the evaluated high-throughput workflow was relatively weak compared to culture but potentially adequate as a rapid screening method analogous to smear microscopy with additional differentiation between TB, MDR-TB, and NTM.