CLINICAL-APPLICATION OF MICROPLATE DNA-DNA HYBRIDIZATION PROCEDURE FOR RAPID DIAGNOSIS OF MYCOBACTERIAL INFECTIONS

Setting: As an alternative to biochemical analysis, microplate DNA-DNA hybridization was applied for rapid diagnosis of mycobacterial infection. Objective: To assess how rapidly and correctly the microplate hybridization method can progress from clinical sample to final species identification of mycobacteria. Design: Clinical samples (pooled sputa or bronchial lavage fluid) were obtained from patients. Depending on the estimated bacterial amounts, genetic identification was performed either directly or following primary culture. Extracted DNA labeled by photobiotin was hybridized in microdilution wells with type-strain DNAs from 4 species (Mycohacterium tuberculosis, M. avium, M. intracellulare, and M. kansasii), then identified on the basis of genetic relatedness, which was quantitated by colorimetric detection. Results: With samples containing more than 10(8) colony-forming units [CFU] (5 cases), species identification was successfully performed on the day of sample preparation. With samples of not more than 10(7) CFU (14 cases). although 4-21 days' primary culture were necessary, species were also correctly identified by the microplate method. Furthermore, M. avium and M. intracellulare were distinctly identified. All the results precisely corresponded to those of biochemical analysis, which took 4-12 weeks to complete identification. Conclusion: We consider that microplate DNA-DNA hybridization is a dependable technique for rapid diagnosis of mycobacterial infection.