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Open Access Highly Accessed Methodology article

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory

Thierry De Baere1, Ricardo de Mendonça2, Geert Claeys1, Gerda Verschraegen1, Wouter Mijs3, Rita Verhelst1, Sylvianne Rottiers2, Leen Van Simaey1, Catharine De Ganck1 and Mario Vaneechoutte1*

Author affiliations

1 Department Clinical Chemistry, Microbiology & Immunology, Ghent University Hospital, Ghent, Belgium

2 Université Libre de Bruxelles, Hôpital Erasme, Service de Microbiologie, Route de Lennik 808,1070 Bruxelles, Belgium

3 Innogenetics, Industriepark 7, Box 5, 9052 Zwijnaarde, Belgium

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Citation and License

BMC Microbiology 2002, 2:4  doi:10.1186/1471-2180-2-4

Published: 1 March 2002

Abstract

Background

The development of DNA amplification for the direct detection of M. tuberculosis from clinical samples has been a major goal of clinical microbiology during the last ten years. However, the limited sensitivity of most DNA amplification techniques restricts their use to smear positive samples. On the other hand, the development of automated liquid culture has increased the speed and sensitivity of cultivation of mycobacteria. We have opted to combine automated culture with rapid genotypic identification (ARDRA: amplified rDNA restriction analysis) for the detection resp. identification of all mycobacterial species at once, instead of attempting direct PCR based detection from clinical samples of M. tuberculosis only.

Results

During 1998–2000 a total of approx. 3500 clinical samples was screened for the presence of M. tuberculosis. Of the 151 culture positive samples, 61 were M. tuberculosis culture positive. Of the 30 smear positive samples, 26 were M. tuberculosis positive. All but three of these 151 mycobacterial isolates could be identified with ARDRA within on average 36 hours. The three isolates that could not be identified belonged to rare species not yet included in our ARDRA fingerprint library or were isolates with an aberrant pattern.

Conclusions

In our hands, automated culture in combination with ARDRA provides with accurate, practically applicable, wide range identification of mycobacterial species. The existing identification library covers most species, and can be easily updated when new species are studied or described. The drawback is that ARDRA is culture-dependent, since automated culture of M. tuberculosis takes on average 16.7 days (range 6 to 29 days). However, culture is needed after all to assess the antibiotic susceptibility of the strains.