Table 1.
PCR-ribotypes represented in the study and identification performance of the testing laboratories at subsequent validation stages of the CE-ribotyping protocol.
Table 2.
Comparison of internal protocols from the four participating laboratories and the proposed consensus protocol.
Fig 1.
Process for multi-centre consensus method validation.
STAGE 1: 70 well characterised ribotypes disseminated from Netherlands to each laboratory for ribotyping (data (i) held locally for future comparsion/ribotype assignment, (ii) sent to UK laboratory); STAGE 2: 50 anonymised isolates disseminated from Netherlands to each laboratory for ribotype identification (assignments sent to UK laboratory for analysis); STAGE 3: 60 anonymised data files disseminated from UK to each laboratory for ribotype identification (assignments sent to UK for analysis).
Fig 2.
PCR-ribotypes with very similar profiles: (a) ribotypes 027 and 081 and (b) ribotypes 015 and 046.
PCR-ribotypes 027 and 081 differ from one another by only a ~20bp difference at position d. Similarly PCR-ribotypes 015 and 046 differ by only a ~20bp difference at position b. Discriminating features between these very similar profiles are indicated (arrows) and associated fragment sizes are highlighted in bold. Relative fragment size was the only parameter used to discriminate between ribotype profiles. Relative peak heights (relative fluorescent units, y-axis) within profiles lacked reproducibility for some ribotypes and therefore this parameter was not used.
Table 3.
PCR-ribotypes associated with minimum and maximum differences in DNA fragment sizes reported across participating centres.
Table 4.
Laboratory performance for PCR-ribotype assignment in validation stages two and three.
Table 5.
Analysis of five discrepant results generated in validation stage two (isolate challenge set).