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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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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.
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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.

Mentions: a. ITG: Institute for Tropical Medicine, Antwerp, Belgium; IPB: Institute Pasteur du Brabant, Brussels, Belgium; SLZ: Streeklaboratorium Zeeland, Goes, the Netherlands; VUB: Free University of Brussels, Brussels, Belgium. b. Strains designated MCRO6 have been studied by Turenne et al.[22] and Torkko et al.[33]. c. Sequences determined in this study. Roman numbering according to Portaels et al.[16], who distinguished four groups within the M. abscessus/M. chelonae complex, based on the 16S–23S rRNA spacer region. d. The GenBank sequence (AF058712) of strain ATCC 14467, submitted to GenBank as M. peregrinum, did not cluster within the M. fortuitum complex (Figure 8) and was highly similar to sequence Y12871 (M. wolinskyi). Moreover, the ARDRA profile calculated from sequence AF058712 (10-1-1-2') did not correspond with the profile we obtained for strain ATCC 14467 (4-1-6-7). The sequence obtained in this study from strain ATCC 14467 (submitted as AJ422046) was identical to the M. fortuitum sequence X52933. Sequence AF058712 is indicated as M. species in the table. e. Received as M. xenopi.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: a. ITG: Institute for Tropical Medicine, Antwerp, Belgium; IPB: Institute Pasteur du Brabant, Brussels, Belgium; SLZ: Streeklaboratorium Zeeland, Goes, the Netherlands; VUB: Free University of Brussels, Brussels, Belgium. b. Strains designated MCRO6 have been studied by Turenne et al.[22] and Torkko et al.[33]. c. Sequences determined in this study. Roman numbering according to Portaels et al.[16], who distinguished four groups within the M. abscessus/M. chelonae complex, based on the 16S–23S rRNA spacer region. d. The GenBank sequence (AF058712) of strain ATCC 14467, submitted to GenBank as M. peregrinum, did not cluster within the M. fortuitum complex (Figure 8) and was highly similar to sequence Y12871 (M. wolinskyi). Moreover, the ARDRA profile calculated from sequence AF058712 (10-1-1-2') did not correspond with the profile we obtained for strain ATCC 14467 (4-1-6-7). The sequence obtained in this study from strain ATCC 14467 (submitted as AJ422046) was identical to the M. fortuitum sequence X52933. Sequence AF058712 is indicated as M. species in the table. e. Received as M. xenopi.

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