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Journal of Clinical Microbiology, April 2006, p. 1612-1613, Vol. 44, No. 4
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.4.1612-1613.2006

LETTER TO THE EDITOR

Use of Mycobacterial Interspersed Repetitive Unit Locus 26 for Rapid Identification of Beijing Genotype Mycobacterium tuberculosis Strains


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LETTER
 
In a recent paper by Rao et al. (3), the easy and rapid mycobacterial interspersed repetitive unit (MIRU) locus 26 PCR assay was found to be 100% specific and sensitive for the discrimination of the Beijing genotype Mycobacterium tuberculosis strains from the non-Beijing strains based on the assumption that all Beijing strains have seven copies and non-Beijing strains have fewer or more than seven MIRU copies at locus 26.

Unfortunately, however, it is known that not all Beijing isolates carry seven MIRU copies at locus 26 (2, 4). Particularly, in some geographic regions, such as the countries of the former USSR, Beijing genotype strains with fewer than seven MIRU copies at locus 26 are in striking predominance over strains with seven copies (2). Recently, we showed that Beijing genotype strains recovered in Istanbul were carried over from the countries of the former USSR, and of the 46 Beijing genotype strains, 37 strains were found to have fewer than seven MIRU copies at locus 26 (1).

After having performed spoligotyping and MIRU typing for more than 2,000 M. tuberculosis strains in Istanbul, we also encountered some non-Beijing strains which had seven repeats at MIRU locus 26. These non-Beijing strains with seven repeats at locus 26 were of the T1, LAM-9, Haarlem 1, CAS 1, 703740004000760, 703777740003371, and 777777774760771 spoligotypes.

In conclusion, the sensitivity and specificity of the MIRU locus 26 PCR assay may not be 100% as was found in this study (3), and this assay seems to be far from reliable as a screening tool to identify and discriminate Beijing genotype strains from non-Beijing strains, since there are Beijing genotype strains with fewer or more than seven repeats at locus 26 and, conversely, non-Beijing strains with seven repeats.


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REFERENCES
 
    1
  1. Köksalan, O. K., Z. Kiliçaslan, G. Zanlier, R. Güzel, and E. Seber. 2006. Prevalence of the Beijing genotype Mycobacterium tuberculosis strains in Istanbul. Int. J. Tuberc. Lung Dis. 10:469-472.
    2. 2
  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. 3
  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. 4
  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]
Orhan Kaya Koksalan
Molecular TB Epidemiology Laboratory
Institute for Medical Experimental Research
Istanbul University
Capa 34270, Istanbul, Turkey,1


Authors' Reply


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LETTER 
 
We thank O. K. Koksalan for his interest in our paper, and we agree that variants of the Beijing genotype with or without seven allele copies at locus 26 have been documented (1, 2) and regret the omission of those reports. MIRU locus 26 has been promoted largely as a "Beijing-discriminating" locus for some time. However, there is evidence that members of the M. tuberculosis Beijing family are quite diverse as regards the MIRU-variable-number tandem repeat typing results but homogeneous with respect to spoligotyping results. We agree that locus 26 is not really a "Beijing-discriminating" locus for the majority of Russian isolates as described by Mokrousov and colleagues (1) but believe that it could still be preliminarily used for a large number of native Beijing isolates circulating in other countries, such as Singapore (2), where a large number of Beijing isolates display seven alleles at MIRU locus 26. We are not yet aware of the applicability of this locus to isolates in Turkey, as O. K. Koksalan pointed out based on his findings, which are yet to be published.

We understand 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. Given the fact that this locus has earlier been used successfully, although with a technically complicated format of allele typing on automated DNA sequencers (3), we independently attempted to simplify its use for diagnosis after validating it with a blinded collection of samples. Indeed, the greatest shortcoming of our paper is the very small number of Beijing samples analyzed by us. Regarding the field applicability of this method, we already 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 any precautionary treatments until spoligotyping data are available.


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REFERENCES 
 
    1
  1. Mokrousov, I., O. Narvskaya, E. Limeschenko, A. Vyazovaya, T. Otten, and B. Vyshnevskyi. 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]
    2. 2
  2. Sun, Y. J., R. Bellamy, A. S. 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]
    3. 3
  3. 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]
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*

* E-mail: seh{at}uohyd.ernet.in

Journal of Clinical Microbiology, April 2006, p. 1612-1613, Vol. 44, No. 4
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.4.1612-1613.2006





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