Use of a cutoff range in identifying mycobacteria by the Gen-Probe Rapid Diagnostic System

Abstract

Commercial DNA probes (Gen-Probe Corp., San Diego, Calif.) for Mycobacterium tuberculosis complex Mycobacterium avium, and Mycobacterium intracellulare were compared with conventional methods for accuracy, applicability, and speed for the identification of putative isolates of the M. tuberculosis and M. avium complexes. Results are expressed as percent hybridization. Values of >15% were considered positive, and values of <5% were negative. Cultures having hybridization values within an indeterminate range of 5 to 15% were repeated. Mycobacterial isolates resembling M. tuberculosis and M. avium complex from cultures of 589 specimens, representing 432 patients, were entered into this study; 294 cultures were tested with the M. tuberculosis complex probe, and 326 cultures were tested with the M. avium probe. In ail cases, probe results agreed with our biochemical identification of the isolates. The M. intracellulare probe was used with 117 isolates morphologically resembling M. avium complex, and one false-negative result was observed. Seventy-two cultures gave initial hybridization results that fell within the indeterminate range and were repeated. If the manufacturer's recommended 10% cutoff value had been used, the original hybridization values would have resulted in 27 misidentified cultures, 16 false-negatives and Il false-positives. In this report we describe the diagnostic evaluation of the Gen-Probe Rapid Diagnostic Systems (Gen-Probe Corp., San Diego, Calif.) assay for Mycobacterium tuberculosis complex and Mycobacterium avium complex in culture. The M. tuberculosis complex system detects M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, and M. microti; the M. avium complex system distinguishes between M. avium and M. intracellulare. These assays are based on the ability of single complementary nucleic acid strands to hybridize and form stable double strands under appropriate conditions. The assays employ DNA probes complementary to the corresponding rRNA. The objectives were (i) to compare the results of culture confirmation by nucleic acid hybridization probe with the results of our classical biochemical methods of identification (specificity), (ii) to evaluate the sensitivity of probes compared with biochemical methods, and (iii) to determine how much sooner the final results would be available with DNA technology. MATERIALS AND METHODS DNA probe test. All materials, lysing reagent, probe solutions, hydroxyapatite separation suspension, and wash solution, were obtained in kit form. The Gen-Probe Rapid Diagnostic Systems kit for M. tuberculosis complex contained only the M. tuberculosis complex probe solution; the M. avium complex kit contained probe solutions for both M. avium and M. intracellulare. Control organisms. Positive controls included with the appropriate probes were M. tuberculosis H37Rv and ATCC 25618, M. avium serotype 4 (laboratory isolate), and M. intracellulare (laboratory isolate). These same organisms also were run as negative controls, i.e., M. avium was used with the M. tuberculo...

Assessment of the Accuprobe Listeria monocytogenes culture identification reagent kit for rapid colony confirmation and its application in various enrichment broths

“…In PALCAMY and UVM, 109 to 1010 microorganisms were necessary to detect a positive result. The probes developed by Gen-Probe are specifically optimized for rapid colony confirmation of identification of various bacteria, including mycobacteria (4,12). Our study confirms that the Accuprobe kit can also identify isolates of L. monocytogenes with a very high level of sensitivity and specificity.…”

The Mycobacterium avium complex.

“…The identification of clinically significant mycobacteria can be achieved within a few hours, once sufficient growth is available, using nonradioactively labelled DNA probes (134,178,304,383,425). Positive broth cultures can be concentrated by centrifugation and directly tested with the probes; some investigators believe the results should be confirmed by testing organisms isolated on solid media (142,380).…”

Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction

“…An oligonucleotide probe, based on the 16S rRNA gene sequence of M. tuberculosis, is commercially available (Gen-Probe, San Diego, Calif.). This probe permits the identification of the species M. tuberculosis, and other similar species-specific probes have been developed for Mycobacterium avium and Mycobacterium intracellulare (7,30,35). The sequence of part of the 16S rRNA gene of Mycobacterium leprae was recently published (6), permitting the development of similar DNA probes for the diagnosis of leprosy.…”