Received 12 June 2002/Returned for modification 31 July 2002/Accepted 2 September 2002

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2002, p Vol. 40, No /02/$ DOI: /JCM Copyright 2002, American Society for Microbiology. All Rights Reserved. Stability of Variable-Number Tandem Repeats of Mycobacterial Interspersed Repetitive Units from 12 Loci in Serial Isolates of Mycobacterium tuberculosis Evgueni Savine, 1 Robin M. Warren, 2 Gian D. van der Spuy, 2 Nulda Beyers, 3 Paul D. van Helden, 2 Camille Locht, 1 and Philip Supply 1 * Laboratoire des Mécanismes Moléculaires de la Pathogenèse Bactérienne, INSERM U447, Institut Pasteur de Lille, F Lille Cedex, France, 1 and MRC Centre for Molecular and Cellular Biology, Department of Medical Biochemistry, 2 and Department of Pediatrics and Child Health, 3 Stellenbosch University, Tygerberg 7505, South Africa Received 12 June 2002/Returned for modification 31 July 2002/Accepted 2 September 2002 Variable number tandem repeats (VNTRs) of elements named mycobacterial interspersed repetitive units (MIRUs) have previously been identified in 12 minisatellite loci of the Mycobacterium tuberculosis genome. These markers allow reliable high-throughput genotyping of M. tuberculosis and represent a portable approach to global molecular epidemiology of M. tuberculosis. To assess their temporal stability, we genotyped 123 serial isolates, separated by up to 6 years and belonging to a variety of distinct IS6110 restriction fragment length polymorphism (RFLP) families, from 56 patients who had positive sputum cultures. All 12 MIRU VNTR loci were completely identical within the groups of serial isolates in 55 out of 56 groups (98.2%), although 11 pairs of isolates from the same patients with conserved MIRU VNTRs displayed slightly different profiles. In a single case, serial isolates with an unchanged profile showed a change in 1 out of 12 MIRU VNTR loci. These results indicate that MIRU VNTRs are stable over time and therefore are suitable for reliable follow-up of patients chronically infected with tuberculosis over long periods. Moreover, they support MIRU VNTR genotyping as a powerful first-line method followed by subtyping by to define ongoing transmission clusters. Molecular epidemiology is a powerful approach to monitoring infectious diseases. In its simplest form, it is based on the assumption that patients infected with strains of identical types are epidemiologically linked, while those infected with strains of different types are not. The validity of this assumption depends on the evolutionary properties of the genetic markers used. They should be sufficiently polymorphic to distinguish unrelated strains yet stable enough to identify isolates of the same strains. Hypervariable markers blur epidemiological links and lead to their underestimation, whereas markers evolving too slowly erroneously link distantly related isolates, leading to overestimation of transmission rates (36). This issue is particularly important in the study of chronic diseases such as tuberculosis, where patients with recurrent tuberculosis can be chronically infected with a given strain and relapse due to reactivation of that strain or, in contrast, can be reinfected by a different strain after cure (30). A correct distinction between these alternatives is essential for accurate estimation of the success rates of tuberculosis control programs (5). Restriction fragment length polymorphism (RFLP) analysis using the IS6110 insertion sequence as a probe is the current gold standard tool for study of the molecular epidemiology of Mycobacterium tuberculosis (29, 31). This analysis is based on the observation that the numbers and/or the genomic locations of IS6110 are highly variable in unrelated M. tuberculosis * Corresponding author. Mailing address: Laboratoire de Mécanismes Moléculaires de la Pathogenèse Bactérienne, INSERM U447, Institut Pasteur de Lille, 1, rue du Prof. Calmette, F Lille Cedex, France. Phone: (33) Fax: (33) Philip.Supply@pasteur-lille.fr. clinical isolates, whereas they are usually identical in epidemiologically related isolates. This method shows the highest discriminatory power among the typing methods currently available (12). However, the method is labor intensive, and the results are difficult to compare between laboratories. Furthermore, the stability of the fingerprint and its adequacy for tracking epidemiological links is still under debate (1, 3, 4, 18, 34, 36), as illustrated by studies on the rates of changes in serial patient isolates. Changes in one or more IS6110 bands are quite frequently observed in paired isolates from patients for whom exogenous reinfection is unlikely (3, 4, 18, 35, 36). In addition, it has been suggested that the degree of instability may be influenced by differences in the genetic backgrounds of the strains (1, 4, 32, 36). To compensate for this instability, it has been proposed that at least 1-band differences may be included in IS6110 genotype-defined clusters (36). However, the result is that there are no clear-cut guidelines for defining which strains should be included or excluded from such clusters. Therefore, the use of alternative, more stable markers would be desirable. Variable-number tandem repeat (VNTR) sequences have emerged as valuable markers for the genotyping of several bacterial species, especially for genetically homogenous pathogens such as Bacillus anthracis (10, 13), Yersinia pestis (11, 13), and the M. tuberculosis complex members (see below). These methods rely on PCR amplification using primers specific for the flanking regions of the VNTRs and on determination of the sizes of the amplicons, which reflect the numbers of the amplified VNTR copies. Their use provides typing data in a portable numerical format, adequate for studying the global molecular epidemiology of infectious agents (6, 11, 16). 4561

2 4562 SAVINE ET AL. J. CLIN. MICROBIOL. Initial VNTR typing systems for M. tuberculosis complex strains made use of very limited sets of loci (6, 8, 14, 15, 17), which turned out not to be sufficiently discriminatory (12). More-extensive sets of VNTR loci have been described subsequently (19, 20, 21, 28), including a system based on 12 loci (28), which has been shown to be applicable for reliable genotyping and molecular epidemiology studies of M. tuberculosis (16, 26). These loci contain VNTRs of original genetic elements named mycobacterial interspersed repetitive units (MI- RUs) that are located mainly in intergenic regions dispersed throughout the M. tuberculosis genome (27, 28). MIRUs are typically 51 to 77 bp long. A MIRU-based high-speed genotyping system has been developed; it combines analysis of four multiplex PCRs for the 12 loci on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of strains by our laboratory demonstrated that MIRU VNTR genotyping is 100% reproducible and 100% sensitive, as all M. tuberculosis complex isolates tested were fully typeable, including those with only a few IS6110 copies or without IS6110 copies (26). Moreover, full reproducibility was also observed when 28 isolates were analyzed in a blinded fashion by our laboratory, and the results were compared to those obtained by Steinlein Cowan and coworkers for the identical isolates (25) (data not shown). In two distinct collections of strains, the discriminatory power of MIRU VNTR typing was found to be close to that of IS6110 RFLP analysis (16, 26). However, the temporal stability of these MIRU VNTR loci had been examined only in a small set of uncharacterized serial isolates (five pairs), separated by at most 18 months (16). In this study, we analyzed the stability of these loci by genotyping well-characterized serial isolates, separated by up to 6 years and belonging to a variety of distinct families, from 56 patients with sputum-positive cultures. MATERIALS AND METHODS Strains and genomic DNA. DNA was extracted from 123 M. tuberculosis isolates obtained from 56 different patients with persistent disease. They were selected from a large collection of serial isolates from 346 patients at the MRC Centre for Molecular and Cellular Biology, Stellenbosch University, Cape Town, South Africa (35). This collection is part of a private database and a sample bank containing isolates collected between January 1992 and December 1998 from approximately 900 different patients attending the health care clinics within adjacent suburbs in Cape Town, South Africa (representing a recovery of approximately 70% of all culture-positive patients), an area with a very high incidence of disease ( 1,000/1,000,000) (2). The isolates were previously typed according to the internationally standardized protocol using the IS-3 probe (29, 31), as described previously (34). All the isolates were assembled into families of similar patterns (IS-3 similarity index of 65%, calculated by using the Dice coefficient and UPGMA [unweighted pair group method with arithmetic averages] clustering method with GelCompar 4.1 software), as described previously (33) and recently updated (28a). Each different pattern within these families was assigned a specific and arbitrary genotype number. To avoid biases due to exogenous reinfections in this highincidence area, patients who had serial isolates featuring more than four changes in the IS6110 banding pattern were considered to be reinfected with a genotypically unrelated strain and were treated as having two separate cases of disease. Furthermore, if any serial isolate featuring a change in pattern was found to be present in the community prior to appearance in the patient, the patient was excluded from the study, since in that case, the patient may have been reinfected from a community contact. In addition, isolates for which laboratory error, contamination, or loss of viability had been noted were excluded. The 123 isolates were selected to contain representatives of the main IS6110 RFLP pattern families present in the database, previously described (33) and recently updated (28a) and on the basis of the following strain and patient characteristics. Twenty-four groups of serial isolates from different chronically infected patients were fully drug sensitive, whereas 21 groups of serial isolates were obtained from patients with drug-resistant tuberculosis. In these groups the paired isolates showed identical banding patterns. Finally, we included 11 groups of two to four serial isolates with minor variations in their banding patterns (35). MIRU VNTR typing. All 123 isolates were genotyped by amplifying the 12 MIRU VNTR loci by using four different multiplex PCRs, as described previously (26). Briefly, the multiplex PCR mixtures were prepared by using the HotStartTaq DNA Polymerase kit (Qiagen, Hilden, Germany). The oligonucleotides used in the PCRs (26) corresponded to the flanking regions of the 12 MIRU VNTR loci identified in the M. tuberculosis genome (28). The samples were subjected to electrophoresis using a 96-well ABI 377 as described in reference 26. Sizing of the PCR fragments and assignment of the various MIRU VNTR alleles were done using the GeneScan and Genotyper software packages (Perkin-Elmer Applied Biosystems), as described in reference 26 and based on the conventions described in reference 28. The tables used for MIRU VNTR allele scoring are available at MIRU VNTR loci 4 and 31 are identical to VNTR D and VNTR E of Frothingham and Meeker-O Connell (6), while loci QUB-5 (20) or ETR 3007c (21), VNTR 0960, and VNTR 2531 (19, 21) correspond to MIRU VNTR loci 27, 10, and 23, respectively. ETR 0154, 2059, and 4348, VNTR 1644 and 2996, and DR 2687 (21) are identical to MIRU VNTR loci 2, 20, 39, 16, 26, and 24. RESULTS A total of 123 isolates originating from 56 patients were included in this study in order to cover the diversity of strain genotypes and patient characteristics encountered in a highincidence region of the Cape Town area (South Africa). They were grouped into three main categories. Twenty-four groups of isolates came from patients with chronic drug-sensitive disease. The serial isolates from different episodes, with time intervals ranging from 8 to 1,759 days, showed identical IS6110 RFLP banding patterns and also showed identical MIRU VN- TRs throughout all 12 loci analyzed (Table 1). Twenty-one groups of isolates were obtained from patients with drug-resistant tuberculosis, with time intervals between isolates ranging from 76 to 2,185 days. Again, the paired isolates showed identical banding patterns and identical MIRU VNTR profiles, except for one pair (from patient 44) (Table 2). In the latter case, the two serial isolates had 11 identical loci but differed in locus 26 by a single repeat difference. Eleven patients had two to four serial isolates obtained with time intervals of 0 to 1,143 days, showing minor variations in their banding patterns. The MIRU VNTR genotypes were identical within all these groups of serial isolates (Table 3). Interestingly, all isolates coming from distinct patients but featuring identical IS6110 fingerprints with more than five copies (compare isolates with fingerprints 209 and 213 in Tables 1, 2, and 3) also displayed identical MIRU VNTR genotypes. In contrast, some strains with identical MIRU VNTR genotypes were identified within previously defined families and had distinct but related IS6110 RFLP patterns (compare isolates in the same families in Tables 1, 2, and 3). These results are in agreement with previous observations (16, 26) suggesting that the combined evolution rate of the 12 MIRU VNTR loci is slightly lower than that of in strains with high IS6110 copy numbers. Also in agreement with these previous results,

3 VOL. 40, 2002 STABILITY OF M. TUBERCULOSIS VNTRs 4563 TABLE 1. Stability of MIRU VNTR genotypes in drug-sensitive serial isolates from 24 chronically infected patients Patient no. No. of isolates tested family no. a genotype no. b No. of IS6110 bands Time span (days) c Copy no. for MIRU locus: , , , , , , N/A a Defined by an similarity index of 65%, as previously described (33) and recently updated (28a). b pattern as defined in the MRC database. c Time span between the first and last isolates from one patient. we confirmed here that some strains from different patients displaying identical IS6110 fingerprints with five or fewer copies (e.g., fingerprints 191 and 338) could be distinguished by MIRU VNTR genotyping, suggesting that in strains with low Patient no. No. of isolates tested IS6110 copy numbers, the evolution rate of the 12 MIRU VNTR loci is higher than that of. Altogether, 23 different MIRU VNTR genotypes were identified compared to 38 different fingerprints. The discrimination TABLE 2. Stability of MIRU VNTR genotypes in drug-resistant serial isolates from 21 patients family no. a genotype no. b No. of IS6110 bands Time span (days) c Copy no. for MIRU locus: , , , , 4 d a Defined by an similarity index of 65%, as previously described (33) and recently updated (28a). b pattern as defined in the MRC database. c Time span between the first and last isolates from one patient. d Five repeats in the first isolate and four repeats in the last isolate.

4 4564 SAVINE ET AL. J. CLIN. MICROBIOL. TABLE 3. Stability of MIRU VNTR genotypes in serial isolates with changes from 11 infected patients Patient no. No. of isolates tested family no. a genotype no. b Change in IS6110 RFLP c Time span (days) d Copy no. for MIRU locus: S S1 1, a Defined by an similarity index of 65%, as previously described (33) and recently updated (28a). b pattern as defined in the MRC database. c 1or 1, one additional or one less IS6110 band, respectively; S1, shift of one IS6110 band. d Time between the first and the last isolate from one patient. level of MIRU VNTRs relative to that of was lower in this study than in previous ones (16, 26). This is likely to be due to the reduced bacterial diversity in this patient population from a 2.4-km 2 Cape Town suburb area compared to populations from France or from various countries previously studied, which renders more prominent the overall lower evolution rate of the 12 MIRU VNTRs relative to that of. DISCUSSION In this study we analyzed the temporal MIRU VNTR genotype stability in M. tuberculosis by using serial isolates from patients chronically infected over long periods (up to 6 years) within a defined high-incidence geographic area. The 12 MIRU VNTR loci were identical within groups of serial isolates in 55 out of 56 cases (98.2%). The total set of 123 isolates included both drug-sensitive and drug-resistant strains from a range of 20 distinct genotype families as previously defined by fingerprints (33). The strains contained 2 to 24 IS6110 bands. The 12 MIRU VNTR loci thus appear to be highly stable over a period as long as 6 years, within a large range of strain lineages and genetic backgrounds. Even the 11 paired isolates with slightly different IS6110 RFLPs all displayed conserved MIRU VNTR genotypes. However, in one case serial isolates with an unchanged IS6110 RFLP pattern showed a change in the MIRU VNTR genotype, which was limited to 1 out of the 12 loci. Interestingly, this locus (locus 26) is one of the most polymorphic MIRU VNTR loci in M. tuberculosis strains, suggesting that its molecular clock is faster than those of the other loci (16, 26). The change in this locus consisted of a single repeat difference. Such a change is consistent with a stepwise VNTR mutation mechanism (sequential additions or deletions of repeat units), supported by a previous analysis of MIRU VNTR changes in locus 4intheMycobacterium bovis BCG genealogy (28). It should be noted that these isolates were separated by 309 days, which is far less than the time spans between many other first and last isolates (up to 2,185 days) during which MIRU VNTR genotypes were stable. Thus, this change is best interpreted by stochastic occurrence of a VNTR mutation event concomitant to emergence of a corresponding clonal variant in the bacterial population in the second isolation. In agreement with previous studies (16, 26, 28), these results provide additional evidence that the mechanisms driving MIRU VNTR and pattern polymorphisms are essentially independent from one another and that the combined evolution rate of the 12 MIRU VNTR loci is slightly lower than that of. Therefore, the MIRU VN- TRs appear suitable for reliable follow-up of patients chronically infected with M. tuberculosis over long periods. On the basis of the stability of MIRUs, the detection of changes in MIRU VNTR genotypes will help to clarify the definition of exogenous reinfection, which is currently based on observation of changes of at least three or four bands in patterns (24, 35). This issue is particularly important for accurate estimation of success rates of tuberculosis control programs and for evaluation of clinical trials of new therapies. Extrapolation of the rates of changes within serial isolates (3, 4, 18, 35, 36) has led to proposal of the use of primarily as an inclusion tool for tracking ongoing transmission. Two strains with identical fingerprints are considered to be included in the same epidemiologically linked cluster (7, 36), while the status of strains differing by one or more bands remains undefined. In previous studies, IS6110 RFLP changes were observed in 4 to 29% of serial isolates (3, 4, 18, 35, 36) and in an estimated 18% of isolates within chains of transmission in a period covering 6.5 years (34). Exceptionally, no changes were observed in patterns obtained from isolates collected sequentially from patients in Texas, but most of these organisms were obtained within 60 days of one another, which may be a period too short for variation to be observed (22). Therefore, if one considers only absolutely identical s as the sole criterion for inclusion of isolates in the same cluster, a significant proportion of isolates would be erroneously excluded (36). The results presented here support MIRU VNTR genotyping as an efficient alternative to as an exclusion/ inclusion method for tracking ongoing transmission. Based on the stability of the 12 MIRU VNTR loci observed here, less than 2% of the isolates could be extrapolated to be erroneously

5 VOL. 40, 2002 STABILITY OF M. TUBERCULOSIS VNTRs 4565 excluded from a cluster if one considered absolute identity of all 12 MIRU loci as the sole criterion. Given the advantages of MIRU VNTR genotyping with respect to speed and ease of analysis, reproducibility, and discriminatory power (16, 26), we suggest the use of MIRU VNTR typing as a first-line analysis to identify clusters during ongoing transmission. Optionally, this first-line analysis could be complemented by spoligotyping (9). This simple PCR-based method targets a single locus and has lower discriminatory power but can nevertheless help distinguish between some MIRU VNTR or other VNTR genotypes with no apparent epidemiological links (12, 16, 23, 25). In cases where MIRU VNTR (and possibly spoligotype) profiles are identical, could be used as a second-line analysis to confirm potential epidemiological links between isolates. This two-step strategy would be expected to increase the accuracy of outbreak investigations and to considerably accelerate epidemiological studies of M. tuberculosis. ACKNOWLEDGMENTS We thank Pablo Bifani for critical reading of the manuscript, Sarah Lesjean for technical assistance, and Vincent Vatin and Philippe Boutin for providing laboratory facilities. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur de Lille, Région Nord-Pas-de-Calais, a joint grant from the Ministère de la Recherche and Ministère des Affaires Etrangères, and the South African Medical Research Council (MRC) and National Research Foundation (NRF). P.S. is a Chercheur du Centre National de Recherche Scientifique. E.S. holds a Poste Vert from INSERM. REFERENCES 1. Alito, A., N. Morcillo, S. Scipioni, A. Dolmann, M. I. Romano, A. 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