First worldwide proficiency study on variable-number tandem-repeat typing of Mycobacterium tuberculosis complex strains

Abstract

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.

Fig 1

Interlaboratory reproducibility of VNTR typing of 30 M. tuberculosis complex DNA samples scored by 36 participating laboratories and the reference laboratory. The black bars represent the reproducibility on the basis of VNTR patterns that were completely identical to those obtained by the reference laboratory (laboratory 37). The gray bars represent the cumulative reproducibility when VNTR patterns that contained missing results were included but which were identical to those obtained by the reference laboratory when not considering the loci with missing results. Most laboratories used 24-locus VNTR typing, and three laboratories used either 14 loci (#) or 15 loci (*) for typing.

Fig 2

Intralaboratory reproducibility of VNTR typing of 10 duplicated DNA samples of M. tuberculosis scored by 36 participating laboratories and the reference laboratory (laboratory 37). The laboratory numbers correspond to those shown in Fig. 1. Most laboratories used 24-locus VNTR typing, and three laboratories used either 14 loci (#) or 15 loci (*) for typing.

Fig 3

Distribution of detected errors by locus in the 24-locus VNTR panel calculated on the basis of the results of typing of 30 M. tuberculosis complex DNA samples by 35 laboratories (excluding laboratories 2 and 5, the systematic errors, and the two incidents of sample exchange described in detail in the text). Among the errors, wrong results and missing results were distinguished.