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Journal of Clinical Microbiology, April 2006, p. 1614-1615, Vol. 44, No. 4
0095-1137/06/$08.00+0 doi:10.1128/JCM.44.4.1614-1615.2006
Mycobacterium tuberculosis Beijing Genotype and Mycobacterial Interspersed Repetitive Unit Typing

LETTER
Rao et al. (
3) described a method to identify
Mycobacterium tuberculosis strains of the Beijing genotype. The authors suggested
using simple PCR of mycobacterial interspersed repetitive unit-variable-number
tandem repeat (MIRU-VNTR) locus 26 to identify these strains.
This suggestion is based on the authors' assumption that MIRU
locus 26 has a specific seven-copy signature in these strains.
Rao et al. (
3) based their conclusions on 10 available Beijing
strains (DNA samples) for which they indeed found seven-copy
signatures. The authors justly mentioned that their test should
be validated and confirmed with a large number of known Beijing
strains. Prior to further experiments, it would be advisable,
however, to compare their results against an already large number
of available articles on MIRU typing of
M. tuberculosis strains,
published between 2001 and 2004, also containing information
about Beijing strains (
1-
2,
4-
6), including our article specially
focused on the MIRU typing of the Beijing genotype (
2). These
articles clearly demonstrated for a large number of representative
samples of strains from Russia (
n = 44), South Africa (
n = 38),
Singapore (
n = 160), Bangladesh (
n = 15), and other locations
that MIRU locus 26 is not monomorphic but instead moderately
polymorphic in the Beijing genotype and may consist of two,
three, four, five, six, seven, eight, or nine copies [
1,
2,
4,
5,
6]). Furthermore, a brief check of the mentioned articles
(
1,
4-
6) reveals that seven copies at MIRU locus 26 may occur
not only in some Beijing strains but also in strains of other
genotypes of
M. tuberculosis. I am afraid that for these reasons,
formally speaking, the proposed method dramatically lacks both
sensitivity and specificity and cannot be used to identify
M. tuberculosis Beijing genotype strains in any setting.

REFERENCES
1 - Banu, S., S. V. Gordon, S. Palmer, R. Islam, S. Ahmed, K. M. Alam, S. T. Cole, and R. Brosch. 2004. Genotypic analysis of Mycobacterium tuberculosis in Bangladesh and prevalence of the Beijing strain. J. Clin. Microbiol. 42:674-682.[Abstract/Free Full Text]
2 - Mokrousov, I., O. Narvskaya, E. Limeschenko, A. Vyazovaya, T. Otten, and B. Vyshnevskiy. 2004. Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations. J. Clin. Microbiol. 42:2438-2444.[Abstract/Free Full Text]
3 - Rao, K. R., N. Ahmed, S. Srinivas, L. A. Sechi, and S. Hasnain. 2006. Rapid identification of Mycobacterium tuberculosis Beijing genotypes on the basis of the mycobacterial interspersed repetitive unit locus 26 signature. J. Clin. Microbiol. 44:274-277.[Abstract/Free Full Text]
4 - Sun, Y.-J., R. Bellamy, A. S. G. Lee, S. T. Ng, S. Ravindran, S.-Y. Wong, C. Locht, P. Supply, and N. I. Paton. 2004. Use of mycobacterial interspersed repetitive unit-variable-number tandem repeat typing to examine genetic diversity of Mycobacterium tuberculosis in Singapore. J. Clin. Microbiol. 42:1986-1993.[Abstract/Free Full Text]
5 - Supply, P., R. M. Warren, A.-L. Banuls, S. Lesjean, G. D. van der Spuy, L.-A. Lewis, M. Tibayrenc, P. D. van Helden, and C. Locht. 2003. Linkage disequilibrium between minisatellite loci supports clonal evolution of Mycobacterium tuberculosis in a high tuberculosis incidence area. Mol. Microbiol. 47:529-538.[CrossRef][Medline]
6 - Supply, P., S. Lesjean, E. Savine, K. Kremer, D. van Soolingen, and C. Locht. 2001. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J. Clin. Microbiol. 39:3563-3571.[Abstract/Free Full Text]
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Igor Mokrousov*
Laboratory of Molecular Microbiology St. Petersburg Pasteur Institute 14 Mira Street St. Petersburg 197101, Russia,1
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* Phone: 7 812 233 21 49, Fax: 7 812 232 92 17, E-mail: igormokrousov{at}yahoo.com |
Authors' Reply

LETTER
We accept the arguments of Igor Mokrousov, that variants of
the Beijing genotype with or without seven allele copies at
locus 26 have been documented, and we regret the omission of
those references. The argument is based mainly on their study
of Russian and South African Beijing genotypes using MIRU-VNTR
typing (
2). MIRU locus 26 has been promoted largely as a "Beijing-discriminating"
locus for some time. However, it has been lately realized that
members of the
M. tuberculosis Beijing family are quite diverse
as regards MIRU-VNTR typing results but homogeneous with respect
to spoligotyping results. Mokrousov and colleagues (
2) have
also documented the occurrence of seven allele copies at MIRU
locus 26 for all the South African isolates that they compared
and a fraction of the Russian Beijing population called MIRU
type 11 (M11) but not in the majority of Russian isolates. This
means that locus 26 is not really a "Beijing-discriminating"
locus for the majority of Russian isolates but could still be
preliminarily used for a large number of native Beijing isolates
circulating in other countries, such as Singapore (
4).
In our study (3), we stressed that the seven allele copies at MIRU locus 26 and a spoligotype signature (hybridization corresponding to spacers 35 to 43) should together constitute a definite identification of the Beijing genotype. However, since many clinical laboratories, especially in resource-poor countries, do not have the capabilities to perform spoligotyping, we attempted to test whether a simplified MIRU locus 26-specific PCR can be used as a stand-alone test. Our findings revealed that locus 26 PCR could still be broadly applicable for most of the Beijing isolates circulating in different countries, although we now agree that it may not be a stand-alone locus for the discrimination of Beijing strains in all settings, especially the Russian setting. Given the fact that this locus has earlier been used successfully, although with a technically complicated format of allele typing on automated DNA sequencers (5), we independently attempted to simplify its use for diagnosis after validating it with a blinded collection of samples. This exercise revealed to us a good concordance of occurrence of seven alleles at locus 26 with spoligotyping when we used the genomic DNA of the strains from Libya and the Kremer collection (1). This collection is largely representative of the strains circulating throughout the world and has been tested for MIRU-VNTR typing previously (5). Indeed, the greatest shortcoming of our paper is the very small number of Beijing samples analyzed by us. We believe, and we have mentioned explicitly in our conclusion, that a large number of Beijing strains should be tested as regards this locus alone before field level testing. We also suggested that this locus might be used in rapid diagnosis preliminarily to initiate precautionary treatments until spoligotyping data are available.

REFERENCES
1 - Kremer, K., D. van Soolingen, R. Frothingham, W. H. Haas, P. W. M. Hermans, C. Martin, P. Palittapongarnpim, B. B. Plikaytis, L. W. Riley, M. A. Yakrus, J. M. Musser, and J. D. A. van Embden. 1999. Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J. Clin. Microbiol. 37:2607-2618.[Abstract/Free Full Text]
2 - Mokrousov, I., O. Narvskaya, E. Limeschenko, A. Vyazovaya, T. Otten, and B. Vyshnevskiy. 2004. Analysis of the allelic diversity of the mycobacterial interspersed repetitive units in Mycobacterium tuberculosis strains of the Beijing family: practical implications and evolutionary considerations. J. Clin. Microbiol. 42:2438-2444.[Abstract/Free Full Text]
3 - Rao, K. R., N. Ahmed, S. Srinivas, L. A. Sechi, and S. E. Hasnain. 2006. Rapid identification of Mycobacterium tuberculosis Beijing genotypes on the basis of the mycobacterial interspersed repetitive unit locus 26 signature. J. Clin. Microbiol. 44:274-277.[Abstract/Free Full Text]
4 - Sun, Y.-J., R. Bellamy, A. S. G. Lee, S. T. Ng, S. Ravindran, S.-Y. Wong, C. Locht, P. Supply, and N. I. Paton. 2004. Use of mycobacterial interspersed repetitive unit-variable-number tandem repeat typing to examine genetic diversity of Mycobacterium tuberculosis in Singapore. J. Clin. Microbiol. 42:1986-1993.[Abstract/Free Full Text]
5 - Supply, P., S. Lesjean, E. Savine, K. Kremer, D. van Soolingen, and C. Locht. 2001. Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J. Clin. Microbiol. 39:3563-3571.[Abstract/Free Full Text]
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K. Rajender Rao
Niyaz Ahmed
Sriramula Srinivas
Laboratory of Molecular and Cell Biology Centre for DNA Fingerprinting and Diagnostics Nacharam, Hyderabad, India,2
Leonardo A. Sechi
Department of Biomedical Sciences University of Sassari Sassari, Italy,3
Seyed E. Hasnain*
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* E-mail: seh{at}uohyd.ernet.in |
Journal of Clinical Microbiology, April 2006, p. 1614-1615, Vol. 44, No. 4
0095-1137/06/$08.00+0 doi:10.1128/JCM.44.4.1614-1615.2006