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.Worldwide, tuberculosis (TB) is a major cause of illness and death. WHO estimates that in 2006, 9.2 million new cases and 1.7 million deaths occurred from TB globally (25), and the incidence is increasing. The emergence of multidrug-resistant TB, and recently also extensively drug-resistant TB, and the human immunodeficiency virus-TB coinfection are further worsening the situation, and effort to accelerate progress in global TB control is needed. Important factors for TB control are increased case detection and treatment success rates (25). The slow growth of most pathogenic mycobacteria results in diagnosis and treatment delay and has stimulated the development of nucleic acid amplification (NAA) tests for identification of mycobacteria directly in clinical specimens. NAA tests provide test results within 1 day. In general, the specificity result for NAA tests ranges from 95% to 100% (1, 12, 16, 23), but the sensitivity result, especially for acid-fast bacillus (AFB) smear-negative samples, varies greatly, from 33 to 96% (1, 12, 16, 23). For AFB smear-positive respiratory specimens, the sensitivity level is approximately 95%.Two direct systems approved by the United States Food and Drug Administration (FDA) for detection of pulmonary TB are commercially available, as follows: the Amplicor Mycobacterium tuberculosis test (Roche Diagnostic Systems, Indianapolis, IN) and the Gen-Probe Amplified M. tuberculosis Direct test (MTD test; Gen-Probe, San Diego, CA). Both tests use the 16S rRNA gene as the target amplification gene. The 16S rRNA gene represents a stable property of microorganisms and is widely used as the target for identifying mycobacterium species. Several studies have confirmed an excellent test proficiency (sensitivity and specificity levels of more than 95%) in AFB smear-positive sputum samples but a reduced sensitivity level (82 to 85%) when applied on AFB smear-negative samples (1, 16, 23, 24). Thus, their use was limited to respiratory smear-positive samples from untreated patients. An enhanced version of the MTD test was later approved for use in both smear-positive and smear-negative specimens (5). A novel, commercially available NAA test for diagnosis of TB directly in patient specimens which has not yet been FDA approved is the BD ProbeTec ET test (Becton Dickinson Diagnostic Systems, Sparks, MD). The test is based on strand-displacement amplification of target sequences in IS6110 and the 16S rRNA gene and has a sensitivity level of 90 to 100% and a specificity level of 92% in smear-positive sputum samples (16). To make the NAA tests more rapid, robust, and applicable in laboratories without substantial technical infrastructure, the following novel NAA tests have been developed: the loop-mediated isothermal amplification (LAMP) test (Eiken Chemical Co., Ltd., Tokyo, Japan) (2, 3), the GeneXpert system (Cepheid, Sunnyvale, CA) (9), and the gold nanoparticle probes assay (21). Simple sample processing, amplification, and detection steps make these NAA tests more applicable in low-income countries with high incidence of TB. However, data on test proficiencies are limited so far. Ongoing studies will show if these rapid molecular tests can be alternatives to the conventional TB diagnostic tests.Recently, a new DNA strip test for detection of mycobacteria directly in smear-positive and smear-negative respiratory samples has been developed. The GenoType Mycobacteria Direct (GTMD) test (Hain Lifescience GmbH, Nehren, Germany) is based on nucleic acid sequence-based amplification and amplifies single-stranded nucleic acids from the 23S rRNA gene in an isothermal reaction. The biotinylated amplified DNA product is hybridized to specific oligonucleotide probes immobilized on the strip. The GTMD test detects members of the M. tuberculosis complex (MTC), Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium malmoense directly from decontaminated respiratory specimens, and the result is available within 1 day. Few studies have previously evaluated the GTMD test (7, 15, 20).The aim of this study was to evaluate the performance of the GTMD test and compare that test to the MTD test. Thus, the GTMD and MTD tests were evaluated for sensitivity and specificity using 61 respiratory specimens from patients suspected to suffer from pulmonary TB. Amplification and sequencing of the 16S rRNA gene of strains isolated from specimen culture (solid and automated liquid media) were used as reference methods.