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

De Baere T, de Mendonça R, Claeys G, Verschraegen G, Mijs W, Verhelst R, Rottiers S, Van Simaey L, De Ganck C, Vaneechoutte M - BMC Microbiol. (2002)

Bottom Line: 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.The existing identification library covers most species, and can be easily updated when new species are studied or described.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department Clinical Chemistry, Microbiology & Immunology, Ghent University Hospital, Ghent, Belgium. thierry.debaere@rug.ac.be

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.

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RsaI restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)
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Figure 3: RsaI restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)

Mentions: The restriction patterns obtained with the enzymes HhaI (isoschizomer of CfoI), MboI and RsaI for the different species are numbered arbitrarily and are presented in Figures 1, 2 and 3 respectively. Figures 4,5,6 represent the restriction patterns obtained when digital restriction is carried out with the same enzymes on published GenBank sequences. The combination of these patterns is designed as ARDRA profiles and these are listed in Table 1. For example, strains of the M. tuberculosis complex can be recognized by an ARDRA profile 1-1-1, while M. grodonae strains have ARDRA profile 8-4-2. For some species the ARDRA pattern obtained with enzyme is already characteristic, e.g. HhaI 1 is observed only for species of the M. tuberculosis complex.


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

De Baere T, de Mendonça R, Claeys G, Verschraegen G, Mijs W, Verhelst R, Rottiers S, Van Simaey L, De Ganck C, Vaneechoutte M - BMC Microbiol. (2002)

RsaI restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC101405&req=5

Figure 3: RsaI restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)
Mentions: The restriction patterns obtained with the enzymes HhaI (isoschizomer of CfoI), MboI and RsaI for the different species are numbered arbitrarily and are presented in Figures 1, 2 and 3 respectively. Figures 4,5,6 represent the restriction patterns obtained when digital restriction is carried out with the same enzymes on published GenBank sequences. The combination of these patterns is designed as ARDRA profiles and these are listed in Table 1. For example, strains of the M. tuberculosis complex can be recognized by an ARDRA profile 1-1-1, while M. grodonae strains have ARDRA profile 8-4-2. For some species the ARDRA pattern obtained with enzyme is already characteristic, e.g. HhaI 1 is observed only for species of the M. tuberculosis complex.

Bottom Line: 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.The existing identification library covers most species, and can be easily updated when new species are studied or described.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department Clinical Chemistry, Microbiology & Immunology, Ghent University Hospital, Ghent, Belgium. thierry.debaere@rug.ac.be

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.

Show MeSH
Related in: MedlinePlus