The Development of Three Strand Displacement Amplification Assays for Culture Confirmation of Mycobacterium tuberculosis Complex, Mycobacterium avium Complex and Mycobacterium kansasii on the BDProbeTecTM ET System

■ The clinical importance of mycobacterial infections is increasing, particularly as a result of AIDS. The diagnosis of these infections has been traditionally dependent on culture followed by biochemical analysis. These procedures are time consuming and labor intensive; diagnosis can take as long as six weeks. In combination with automated culturing systems such as the BACTEC460TB and the BACTEC MGIT 960 Systems, a nucleic acid amplification test can significantly reduce the time to diagnosis. Here we report the development of three homogeneous strand displacement amplification (SDA) assays for the detection of either Mycobacterium tuberculosis complex (Mtb), Mycobacterium avium complex (MAC) or Mycobacterium kansasii from culture. Each assay simultaneously amplifies and detects specific target DNA and an internal amplification control. Assay performance was optimized by evaluating different reaction components of the systems in statistically designed experiments. These experiments identified a common sample buffer, thereby allowing one sample to be tested in each of the three assays. Sensitivities of the assays were evaluated across multiple strains of organisms. The Mtb, MAC and M. kansasii assays demonstrated sensitivities of 100% (10/10), 100% (50/50) and 100 (137/137), respectively. An analytical sensitivity using plasmid target DNA was estimated for each assay: M. tuberculosis = 54 copies/reaction, M. avium = 771 copies/ reaction, M. intracellulare = 58 copies/reaction and M. kansasii = 155 copies/reaction. These sensitivities allow for the identification of these organisms on the BACTEC 460TB and BACTEC MGIT 960 Systems at a Growth Index of ≥ 50 and Growth Units of ≥ 75, respectively. The specificity of each assay was evaluated against 56 other mycobacterial and non-mycobacterial species at 10 genome equivalents/ml. No significant crossreactivity to any clinically relevant mycobacterial or non-mycobacterial species tested was observed in any of the three assays. The culture confirmation assays on the BDProbeTec ET System are sensitive, specific and can be used for the identification of Mtb, MAC and M. kansasii from a single culture specimen in less than four hours. INTRODUCTION Mycobacterium tuberculosis (Mtb) and nontuberculous (NTM) mycobacterial diseases pose an increasing public health challenge. Mtb and NTM infections frequently occur in patients with AIDS or other immunocompromised illnesses. Mycobacterium avium complex (MAC) and M. kansasii are the two leading causes of NTM infections in humans. The diagnosis of Mtb and NTM infections has been traditionally dependent on acid fast staining and culture of organisms followed by biochemical analysis. These methods can be either insensitive or require weeks to perform. Even with automated culturing systems such as the BACTEC 460TB and BACTEC MGIT 960 Systems (BD Biosciences, Sparks MD), time to identification usually extends to greater than two weeks. Amplification technologies have allowed for the increase in sensitivity and specificity as well as decreased time to organism identification. Here we report the development of three assays for the detection of M. tuberculosis complex, Mycobacterium avium complex or M. kansasii on the BDProbeTec ET System. The assays utilize homogeneous strand displacement amplification (SDA) to simultaneous amplify and detect a target region of the IS6110, dna J, or KATS1 region of the genome for the identification of Mtb, MAC, and M. kansasii organisms, respectively. The assays also include an internal amplification control (IAC) to validate negative results. Optimization of the assays has allowed for the testing of all three organisms from a single culture aliquot with time to results in under four hours.