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Reply to “Evaluation of Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping as Performed in Laboratories in Canada, France, and the United States”

With great interest, we have taken notice of the letter of Cowan et al. (1). Indeed, the very high degree of worldwide standardization in typing of Mycobacterium tuberculosis is unique compared to the variety of techniques used for typing of other bacteria. However, the quality of this variable-number tandemrepeat (VNTR) typing has hardly been studied in the international context. Thanks to the enthusiastic participation of 37 laboratories worldwide, the first external quality assessment has been conducted within the framework of an European Centre for Disease Prevention and Control (ECDC) project on the molecular surveillance of multidrug-resistant/extensively drug-resistant tuberculosis (MDR/XDR-TB) in the European Union (2). The main purpose of the initiation of this proficiency study was to identify the flaws in VNTR typing in international laboratories worldwide, and indeed, multiple structural aberrations were observed. This provided laboratories the opportunity to identify important problems in the use of the locally adapted typing technique. In the letter of Cowan et al., our study was described as “a beneficial learning experience which will likely improve the typeability.” We would like to add to these positive comments that this first proficiency study indeed revealed that global standardization of a molecular typing technique cannot even be done without proper quality control. The 24-locus VNTR typing of M. tuberculosis complex strains was proposed as the new gold standard in 2006 (4). The initial multicenter proficiency study in 2009, however, demonstrated that the reproducibility was limited. With an average interlaboratory reproducibility of 60% (range, 0 to 100%) and intralaboratory reproducibility of 72% (range, 0 to 100%), we realized that not all details of the technique had been worked out sufficiently and the interpretation of the protocols was not unambiguous. We do agree that the outcome of an external quality assessment is highly dependent on the selection of strains. Indeed, the inclusion of one Mycobacterium canettii isolate in the first panel may be questioned, since these bacteria are rarely encountered outside East Africa (3, 5). However, if only strains without difficulties in typing are included, all laboratories will score 100%, but we do not learn anything. In contrast, if strains with a higher degree of difficulty are included, the true performance of individual laboratories becomes visible. Nevertheless, for the already-performed and analyzed second round of the quality control panel, we selected samples in order to focus on specific problems in VNTR typing, e.g., double alleles and a high number of repeats. Another issue, not yet addressed in the quality control is the sensitivity of VNTR to the quality of DNA of the Mycobacterium samples. The DNA samples so far used in the proficiency studies were of a high purity. To challenge the participating laboratories and reflect the daily routine more accurately, future panels should also contain samples with a lower quantity and quality of DNA. The participating laboratories perform VNTR typing with significant variation in methodologies; some use a commercial test, while others explore in-house approaches. The determination of PCR fragment lengths is done by gel electrophoresis or sophisticated capillary electrophoresis (CE)-based DNA analyzers. In addition, the laboratories have different degrees of experience with typing techniques. In order to produce reliable typing data in all parts of the world that allow meaningful interpretation by local and international health authorities, quality control within networks on molecular epidemiology remains highly important. Thanks to the active role of European networks since the early 1990s, molecular typing techniques were already standardized from their early development. Within the framework of an ECDC project, the VNTR typing (4) has now become the new standard. To consolidate this unique degree of standardization and ensure the quality in the molecular epidemiology of one of the most important infectious diseases, an active network with central coordination needs to be preserved.