The Role of Cartridge-Based Nucleic Acid Amplification Test (CBNAAT), Line Probe Assay (LPA), Liquid Culture, Acid-Fast Bacilli (AFB) Smear and Histopathology in the Diagnosis of Osteoarticular Tuberculosis.

BACKGROUND: There is a paucity of data on the role of molecular methods in the diagnosis of osteoarticular tuberculosis. The present study was conducted to define the role of molecular (CBNAAT, LPA), phenotypic (AFB smear and culture) and histopathological evaluation in the diagnosis of osteoarticular TB. METHODS: Seventy-seven consecutive cases of osteoarticular tuberculosis were grouped into presumptive TB cases (group A) and presumptive drug-resistant cases (group B). Tissue samples obtained were submitted for CBNAAT, LPA, AFB smear, liquid culture and histological examinations. The diagnostic accuracy of each test was reported against histologically diagnosed cases and in all tests in tandem. RESULTS: Group A and group B had 65 and 12 cases, respectively. The diagnostic accuracy for tuberculosis was 84.62% by CBNAAT, 70.77% by LPA, 86.15% by molecular tests (combined), 47.69% by AFB smear, 50.77% by liquid culture and 87.69% by histology in group A, and 91.67% for CBNAAT, 83.33% for LPA, 91.67% for molecular tests (combined), 25% for AFB smear, 16.67% for liquid culture and 83.33% for histology in group B. The drug resistance detection rate was 4.62% on CBNAAT, 3.08% on LPA, 6.15% on molecular tests (combined) and 1.54% on DST in group A, while it was 33.33% on CBNAAT, 58.33% on LPA, 58.33% on molecular tests (combined) and 16.67% on DST among group B cases. Similar sensitivity rates for the various tests were obtained among both the groups on comparison with histology (taken as denominator). The addition of molecular methods increased the overall diagnostic accuracy (all tests in tandem) from 93.8 to 100% in group A and from 83.3 to 100% in group B cases. CONCLUSION: No single tests could diagnose tuberculosis in all cases; hence, samples should be evaluated by molecular tests (CBNAAT and LPA), AFB smear, culture and histological examinations simultaneously. The molecular tests have better demonstration of drug resistance from mycobacterial culture. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43465-020-00326-w.

Multicentric validation of indigenous molecular test Truenat™ MTB for detection of Mycobacterium tuberculosis in sputum samples from presumptive pulmonary tuberculosis patients in comparison with reference standards.

BACKGROUND & OBJECTIVES: : Early case detection is essential to interrupt transmission and to prevent further spread of tuberculosis (TB) in high endemic settings. Nucleic acid amplification tests (NAATs) with visual read-outs are ideal as point-of-care tests. Truenat™ MTB is an indigenous chip-based NAAT for detection of Mycobacterium tuberculosis, which involves extraction of DNA and real-time polymerase chain reaction (PCR) using portable, automated, battery-operated instruments. The current multicentric study was aimed to evaluate Truenat for detection of MTB in sputum samples obtained from patients with presumptive pulmonary TB with reference to culture as gold standard and Xpert as a comparator. METHODS: : The study was conducted at four sites, namely ICMR-National Institute for Research in Tuberculosis, Chennai; All India Institute of Medical Sciences, New Delhi; ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra; and National Institute of TB and Respiratory Diseases, New Delhi. Patients suspected to have TB were screened for eligibility. Two sputum samples were collected from each patient. Tests included smear, Xpert and Truenat directly from the sputum sample and culture by Lowenstein-Jensen (L-J) medium and MGIT960 from decontaminated pellets. Sample used for Truenat assay was coded. Resolution of Truenat false positives was done using an in-house PCR with TRC4 primers. RESULTS: : The study enrolled 2419 presumptive TB patients after screening 2465 patients, and 3541 sputum samples were collected from the enrolled patients. Results of 2623 samples were available for analysis. Truenat showed a positivity rate of 48.5 per cent as compared to 37.0 per cent by Xpert. The sensitivities of Truenat and Xpert were was 88.3 and 79.7 per cent, respectively in comparison with culture. INTERPRETATION & CONCLUSIONS: : Truenat MTB identified more positives among culture-confirmed samples than Xpert and had higher sensitivity. In addition, other advantageous operational features of Truenat MTB were identified which would be useful in field settings.

Validation of an indigenous assay for rapid molecular detection of rifampicin resistance in presumptive multidrug-resistant pulmonary tuberculosis patients.

BACKGROUND & OBJECTIVES: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. METHODS: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. RESULTS: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. INTERPRETATION & CONCLUSIONS: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.

Emerging trends in microbiological diagnostics in children.

The targets of the WHO's End TB Strategy and the United Nations' (UN) Sustainable Development Goals (SDGs) have been expanded to"Find. Treat. All #EndTB" with universal access to TB diagnosis, treatment and care by 2022 in an effort to end the global TB epidemic. Trends to achieve the above targets in children have led to greater emphasis on the newer diagnostics paving way to microbiological confirmation and universal drug sensitivity in children.

Early efficacy and safety of Bedaquiline and Delamanid given together in a "Salvage Regimen" for treatment of drug-resistant tuberculosis.

BACKGROUND: Drug-Resistant Tuberculosis (DR-TB) patients for whom a WHO recommended regimen along with Bedaquiline (BDQ) cannot be prescribed, Delamanid (DLM) was added along with other drugs to provide a "Salvage Regimen". The experience of the Institute in respect of early efficacy and safety of both drugs given together is presented. OBJECTIVE: To ascertain the early efficacy, safety and tolerability of Bedaquline and Delamanid given together as a part of salvage regimen. METHODS: BDQ and DLM were used together to make regimens along with other drugs where four effective anti TB drugs could not be prescribed as per WHO recommendations. Patients were followed up for sputum smear and culture conversion and adverse events during the treatment. RESULTS: In this cohort study, 53 DR-TB patients (Median age-24) were initiated on regimens containing both BDQ and DLM. Sputum smear conversion was seen in 35% and 94% patients at the end of 1st week and 3rd month respectively. 84% patients had culture conversion at the end of 4th month. 29 adverse events (AE) were reported among 17 patients and there were 11 deaths. QTc prolongation more than 500 MS was seen in only 1 patient. CONCLUSION: BDQ and DLM given together in a salvage regimen is efficacious with low rate of adverse events. The combination provides hope to DR-TB patients with limited treatment options and should be provided as a life saving option.

Initial experience of bedaquiline implementation under the National TB Programme at NITRD, Delhi, India.

BACKGROUND: Bedaquiline (BDQ) was approved for treatment of drug resistant TB (DR-TB) under Conditional Access Programme (CAP) of Revised National Tuberculosis Control Programme (RNTCP) and was also implemented in the National Institute of TB and Respiratory Diseases (NITRD). We present early efficacy and safety of BDQ containing regimens for DR-TB. OBJECTIVE: To ascertain the early efficacy and safety of Bedaquline containing regimens in treatment of DR-TB. METHODS: BDQ containing regimens along with other drugs were designed as per WHO recommendations for DR-TB patients. They were followed up for sputum smear and culture conversion, adverse events during the treatment. RESULTS: A cohort of 290 DR-TB patients (Median age-29.77) were initiated on BDQ containing regimens. Of the available Sputum results, smear conversion was seen in 51% and 91% patients at the end of 1st week and 3rd month respectively. Similarly, 93% and 98% patients had culture conversion at the end of 3rd and 6th month respectively. 201 adverse events (AE) including 47 deaths were reported among 109 patients. QTc prolongation was seen in 29% patients but only 4 required discontinuation of BDQ. Lost to follow up of treatment was about 6%. CONCLUSION: Bedaquiline along with an optimized background regimen has shown early sputum conversion in larger number of difficult to treat patients having additional resistance of second line drugs along with INH and Rifampicin. The regimen is feasible in programmatic conditions and is relatively safe.

Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay.

PURPOSE: There is scarcity of prevalence data of multi-drug-resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. MATERIALS AND METHODS: All MDR suspects in five districts of New Delhi were referred to the laboratory from 1 st October 2011 to 31 st December 2012 as per criterion defined by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. RESULTS: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred significantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. CONCLUSION: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed significantly for both RIF and INH.

Multi-drug resistant tuberculosis among category I treatment failures--a retrospective study.

BACKGROUND: Tuberculosis (TB) remains a major global health problem and ranks as the second leading cause of death worldwide. An important cause of TB epidemic is the emergence of multi drug resistant (MDR) strains of Mycobacterium tuberculosis. Despite the availability of treatment that is expected to cure most cases of TB, levels of MDR-TB remain worryingly high in India. OBJECTIVE: This study was carried out to ascertain the prevalence of MDR-TB among category I pulmonary TB treatment failure patients. METHODS: This was a retrospective study involving 750 pulmonary tuberculosis patients enrolled at six district centres of Delhi State under RNTCP who failed to respond to CAT I treatment and whose sputum samples were submitted for culture and drug sensitivity testing (DST) over a period of three years (2009-2012). MDR-TB was defined as TB caused by bacilli showing resistance to at least isoniazid and rifampicin. RESULTS: Out of the total 750 patients included in the study, 470 (62.6 %) were culture positive. Of these, 377 (80.2%) were subjected to DST and rest 93 (19.7%) were excluded. Ultimately, DST result was available for 353 (93.6 %) cases. 239 (68%) cases were detected as multi drug resistant TB. CONCLUSION: High proportion of MDR-TB (68%) among culture positive CAT I treatment failure cases highlights the need for rapid diagnostic tests which will enable the detection of MDR-TB at an early stage and will thus minimize the risk of transmission as well as the possible errors associated with the treatment.

Primary tubercular chest wall abscess in a young immunocompetent male.

Chest wall tuberculosis is a rare entity especially in an immunocompetent patient. Infection may result from direct inoculation of the organisms or hematogenous spread from some underlying pathology. Infected lymph nodes may also transfer the bacilli through lymphatic route. Chest wall tuberculosis may resemble a pyogenic abscess or tumour and entertaining the possibility of tubercular etiology remains a clinical challenge unless there are compelling reasons of suspicion. In tuberculosis endemic countries like India, all the abscesses indolent to routine treatment need investigation to rule out mycobacterial causes. We present here a case of chest wall tuberculosis where infection was localized to skin only and, in the absence of any evidence of specific site, it appears to be a case of primary involvement.

Phylogenetic associations with drug-resistant Mycobacterium tuberculosis isolates in a paediatric population.

SETTING: Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India. OBJECTIVES: As paediatric tuberculosis (TB) is a surrogate marker for actively transmitted disease in a community, we investigated drug resistance patterns of 97 Mycobacterium tuberculosis complex strains isolated from children and explored their phylogenetic associations. DESIGN: A total of 111 paediatric patients who attended the out-patient department during the study period 2009-2011 and whose sputum samples were sent to the Microbiology Department for liquid culture and drug susceptibility testing (DST) were included in this study. DST and spoligotyping were performed on cultures positive for M. tuberculosis complex. RESULTS: DST against four first-line drugs showed that 31 of 97 (32%) strains were pan-susceptible, while 66/97 (68%) were resistant to at least one drug, including 55/97 (56.7%) that were resistant to at least isoniazid and rifampicin (i.e., multidrug-resistant). The majority of the isolates (n = 81/90, 90%) belonged to the principal genetic group 1 strains, the most predominant spoligotyping clusters being spoligotyping international type (SIT)1/Beijing (n = 28), SIT26/CAS1-Delhi (n = 27) and SIT53/T1 (n = 6). CONCLUSION: The involvement of Beijing and CAS1-Delhi clades in paediatric TB patients suggests that these two lineages play a major role in ongoing active transmission.

Xpert(®) MTB/RIF assay for tuberculosis diagnosis: evaluation in an Indian setting.

The present study was conducted to evaluate the performance of the Xpert(®) MTB/RIF assay and compare Xpert results with solid and MGIT 960 liquid culture system. A total of 134 patients who had failed the Category I or II regimen were recruited for evaluation. Xpert correctly identified all Mycobacterium tuberculosis isolates. The sensitivity and specificity of the Xpert assay for the detection of rifampicin resistance was respectively 98.2% and 97.0% when compared with MGIT 960 results.

Impact of introducing the line probe assay on time to treatment initiation of MDR-TB in Delhi, India.

SETTING: National Institute of Tuberculosis and Respiratory Diseases (erstwhile Lala Ram Sarup Institute) in Delhi, India. OBJECTIVES: To evaluate before and after the introduction of the line Probe Assay (LPA) a) the overall time to MDR-TB diagnosis and treatment initiation; b) the step-by-step time lapse at each stage of patient management; and c) the lost to follow-up rates. METHODS: A retrospective cohort analysis was done using data on MDR-TB patients diagnosed during 2009-2012 under Revised National Tuberculosis Control Programme at the institute. RESULTS: Following the introduction of the LPA in 2011, the overall median time from identification of patients suspected for MDR-TB to the initiation of treatment was reduced from 157 days (IQR 127-200) to 38 days (IQR 30-79). This reduction was attributed mainly to a lower diagnosis time at the laboratory. Lost to follow-up rates were also significantly reduced after introduction of the LPA (12% versus 39% pre-PLA). CONCLUSION: Introduction of the LPA was associated with a major reduction in the delay between identification of patients suspected for MDR-TB and initiation of treatment, attributed mainly to a reduction in diagnostic time in the laboratory.

Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry.

PURPOSE: The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). MATERIALS AND METHODS: Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. RESULTS: A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0) and conventional methods established it to be non-tuberculous Mycobacterium. CONCLUSIONS: MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.

Performance of light-emitting diode fluorescence microscope for diagnosis of tuberculosis.

BACKGROUND: Fluorescence microscopy (FM) over the years has shown the potential for increasing the performance of microscopy. The present study was aimed to access the performance of the LED microscope for the detection of acid fast bacilli in a tuberculosis (TB) endemic country. METHODS: The study was conducted at a National Reference Laboratory (NRL) in New Delhi, India. Sputum samples were collected from suspected TB patients. Each sample was processed with Auramine O and ZN methods. Auramine O stained smears were evaluated using two different excitatory light sources (MVP and LED); and ZN stained smears were examined under light microscope. The mean time required to read the smears with different modalities was recorded. Bacterial cultures provided the reference standard. RESULTS: A total of 200 patients were included in this study. Sensitivity and specificity for the LED assessment, MVP assessment and light microscopy were 83.1% and 82.4%, 78.5% and 87.5% and 81.6% and 83.5%, respectively. Mean reading time was approximately three times faster than ZN microscopy. The mean time to read a negative smear was 2min with fluorescence microscopy and 5min with light microscopy with time savings of 60%. CONCLUSION: Although the use of LED-FM only marginally increased sensitivity, the considerable time saving ability combined with very good acceptance and ease of use makes it a reliable alternative to other conventional methods available.

Presumptive identification of Mycobacterium tuberculosis complex based on cord formation in BACTEC MGIT 960 medium.

We considered samples received for culture of mycobacteria using BACTEC MGIT 960 system over a period of 1 year. Tubes flagged positive by MGIT were evaluated for presence of serpentine cording. The cord formation was compared with isolates identified as Mycobacterium tuberculosis complex (MTC) based on p-nitrobenzoic acid (PNB) test. Cords were found in 591 isolates of which 584 (98.8%) were confirmed as MTC. The sensitivity and specificity of cord formation were found to be 99.7% and 89.9%, respectively.

Prevalence of XDR TB cases--a retrospective study from a tertiary care TB hospital.

BACKGROUND: The emergence of XDR -TB strains is a major roadblock in the successful implementation of TB control programmes. This further leads to high morbidity and mortality, especially in immuno-compromised patients. Identification and observation of resistance patterns of XDR-TB strains may help clinicians manage MDR-TB cases, the treatment line of which is expensive, time-taking and involves intake of toxic drugs with many side-effects. Our study is aimed to find out the prevalence of XDR-TB among the MDR-TB strains isolated in a tertiary care hospital. MATERIAL & METHODS: The study population consisted of 223 patients of tuberculosis who were culture positive and Mycobacterium tuberculosis was resistant to Rifampicin and Isoniazid during January 2007 to December 2009. Each patient had submitted two sputum samples i.e. spot and morning. The identified Mycobacterium tuberculosis complex was subjected to drug sensitivity testing by first and second line drugs by proportion and absolute concentration methods as per standard procedure. RESULTS: The results showed that 20.17% strains (45/223) were XDR-TB strains. Most of these strains showed resistance to four drug combination viz. KM, ETH, OFX & PAS (5.82%), KM & OFX (3.13%), OFX, KM and ETH (1.79%), 1.34% strains showed resistance to all the drugs i.e. pan resistance and other combinations in the remaining strains. Nearly 80% of the XDR-TB strains showed resistance to three or more drugs combination pattern. CONCLUSION: The multidrug resistant TB cases need urgent and timely sensitivity report for second line ATT drugs to help clinicians start proper drug combinations to treat MDR-TB patients.

A pilot study of same day sputum smear examination, its feasibility and usefulness in diagnosis of pulmonary TB.

INTRODUCTION: A large number of tuberculosis cases are continuously being reported from India and other developing countries leading to high morbidity and mortality. In spite of many newer tests available for diagnosing a case of tuberculosis, smear microscopy of sputum is still the preferred test under programmatic conditions. The current national and international guidelines recommend two sputum smear examinations in two days for diagnosing cases of tuberculosis, which is time-consuming, tedious, needs multiple visits, leading to high dropout of infectious cases. In the background of existing limitations of smear microscopy, we attempted to complete the diagnosis of tuberculosis on same day by serial collection of the spot sputum specimen and analyze its advantages, feasibility and viability. MATERIAL & METHODS: The study was undertaken by the Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases during May 2010 to April 2011. Sputum specimens were collected from 330 randomly selected tuberculosis suspects who attended OPD of hospital, patients submitted spot and home collected morning sputum sample in a standard method and spot and additional spot sputum (X- spot) collected one hour after the first spot sample as per the proposed front loading method. All the samples received were stained by acid fast Ziehl-Neelsen (ZN) stain and examined on the same day. The sputum sample was pooled and cultured in Lowenstein Jensen (LJ) media in duplicate set of bottles. The results of two different microscopic methods were compared with the gold standard culture test. RESULTS: Out of the total 330 TB suspects, 70.60% were males and 29.39% females. The most common complaint was of cough with sputum (88.18%), chest pain (70.21%), fever (55.15%) and loss of appetite (43.03%). Upon examining the total sputum slides, 18.48 % were positive for acid fast bacilli. The smear positivity was 61/330 (18.48%) by standard methods and in proposed new method 43/330 (13.03%). Sensitivity of the standard and proposed new method smear microscopy was 58.25% and 40.07% respectively and specificity was 99.55% in both the methods. CONCLUSION: Same day smear microscopy for diagnosing tuberculosis by a proposed new method of smear examination in the case of suspected tuberculosis seems not a promising step towards improving the quality of sputum smear examination. The results of sensitivity and specificity of the two approaches were not similar. More than eighty per cent responded in favour of same day sputum delivery system and getting result on same day. This study can be confirmed on larger scale and preference of patients can be examined in peripheral laboratory also before taking it up for consideration in the national tuberculosis programme.

Direct detection of mycobacterial species in pulmonary specimens by two rapid amplification tests, the gen-probe amplified mycobacterium tuberculosis direct test and the genotype mycobacteria direct test.

Nucleic acid amplification tests have improved tuberculosis diagnostics considerably. This study evaluates a new amplification test, the GenoType Mycobacteria Direct (GTMD) test, for detection of the Mycobacterium tuberculosis complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium malmoense directly in 61 sputum samples. Thirty (49.2%) samples were auramine smear positive, and 31 (50.8%) were smear negative. The GTMD results were compared to the Gen-Probe Amplified M. tuberculosis Direct (MTD) test results, using culturing and sequencing of the 16S rRNA gene as reference methods. The GTMD test could identify 28 of 29 samples containing the M. tuberculosis complex and was negative in a sputum sample containing M. intracellulare. The overall sensitivity and specificity results were 93.3% and 90.0% for the GTMD test, respectively, and 93.1% and 93.5% for the MTD test, respectively. The GTMD test is rapid and can be easily included in routine clinical laboratories for the direct detection of the M. tuberculosis complex in smear-positive sputum samples as an adjunct to microscopy and culture. Further studies are needed to evaluate the performance of the GTMD test for the detection of atypical mycobacteria.