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

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.

Figure 1

HhaI (CfoI) restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)

Figure 2

MboI restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)

Figure 3

RsaI restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)

Figure 4

HhaI (CfoI) restriction patterns of mycobacterial 16S rRNA genes, theoretically calculated using RFLP (Applied Maths) and published GenBank sequences. Graphical representation and table of restriction fragment lengths for each of the possible patterns.

Figure 5

MboI restriction patterns of mycobacterial 16S rRNA genes, theoretically calculated using RFLP (Applied Maths) and published GenBank sequences. Graphical representation and table of restriction fragment lengths for each of the possible patterns.

Figure 6

RsaI restriction patterns of mycobacterial 16S rRNA genes, theoretically calculated using RFLP (Applied Maths) and published GenBank sequences. Graphical representation and table of restriction fragment lengths for each of the possible patterns.

Figure 7

BstUI restriction patterns of mycobacterial 16S rRNA genes, theoretically calculated using RFLP (Applied Maths) and published GenBank sequences. Graphical representation and table of restriction fragment lengths for each of the possible patterns.

Figure 8

Neighbour-joining similarity tree for 16S rRNA gene sequences of most mycobacterial species. Legend: N. asteroides ATCC 49872 (Genbank Z82229) was used as the outgroup. Table 1 lists the GenBank accession numbers of the sequences used to construct this tree. ARDRA patterns for HhaI, MboI, RsaI and BstUI are listed after the species name. a: GenBank AF028712. Erroneously listed in GenBank as M. peregrinum (see also legend of Table 1). b: M. gastri clusters below 100% with M. kansasii, although it is generally agreed that the 16S rRNA gene sequences for M. kansasii and M. gastri are identical. This can be explained by the fact that the only available GenBank M. gastri sequence (X52919) contained several ambiguities. c. M. lentiflavum, initially not included in the manuscript is not presented in this tree. It clusters close to the branch including M. heidelbergense, M. simiae, M. triplex and M. genavense.