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Journal of Clinical Microbiology, September 1999, p. 3078-3079, Vol. 37, No. 9
0095-1137/99/$04.00+0

LETTERS TO THE EDITOR

Stability of IS6110 Restriction Fragment Length Polymorphism Patterns of Multidrug-Resistant Mycobacterium tuberculosis Strains

	LETTER

Top Letter References

We read with interest the recently published study by Alito et al. (1) concerning the stability of IS6110 restriction fragment length polymorphism (RFLP) patterns of multidrug-resistant (MDR) Mycobacterium tuberculosis strains. The authors analyzed the IS6110 RFLP patterns and spoligotypes of two groups of MDR M. tuberculosis strains which they discuss to represent two tuberculosis (TB) outbreaks. Within the first group, both IS6110 fingerprint patterns and spoligotypes of the MDR strains have been found to be identical, whereas among the strains of the second group spoligotypes were identical but the IS6110 patterns showed variations. Based on this, the authors conclude that the rate of change of IS6110 RFLP patterns in particular MDR M. tuberculosis strains may be too fast for a reliable interpretation of strain typing results over a period of a few years.

In our recent paper (3), we also elucidated the stability of IS6110 patterns of drug-resistant M. tuberculosis strains by analyzing 165 serial isolates obtained from 56 patients with drug-resistant TB. We did not observe a higher level of instability of IS6110 patterns in these isolates in comparison with the rates of changes described in other studies comprising mainly drug-susceptible isolates (e.g., references 2, 4, and 6). In addition, no particular M. tuberculosis genotypes showing a higher rate of IS6110 changes have been identified. From these data we conclude that the stability of IS6110 patterns in drug-resistant M. tuberculosis strains does not seem to differ from that of drug-susceptible isolates. The IS6110 changes observed, however, occurred only in MDR isolates (37 of the 56 patients were infected with MDR strains), which is likely to be due to the longer time intervals between the times of retrieval of the serial isolates in the patient group with MDR TB (a mean of 300 days for the MDR isolates compared to a mean of 60 days for the resistant but not MDR isolates). Moreover, we have not found a higher instability of IS6110 patterns in several cases of recent transmission of MDR strains.

It is well known that the IS6110 patterns of unrelated M. tuberculosis strains generally show a high degree of variability. However, some M. tuberculosis strain families displaying similar IS6110 fingerprint patterns and identical spoligotypes have been described, e.g., the "Beijing family" (5). Hence, the differences in IS6110 patterns among the strains of the second MDR group observed by Alito et al. (1) may be due not to a recent outbreak of a MDR strain showing a higher rate of IS6110 change but to false clustering of strains of an M. tuberculosis family in one fingerprint group. This notion is further supported by the fact that in our German strain collection we found two M. tuberculosis isolates showing the same spoligotype as and an IS6110 pattern similar to those of the strains of the second group of MDR strains described by Alito et al. (1) (Fig. 1).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   IS6110 RFLP patterns and spoligotypes of two M. tuberculosis strains obtained from two patients living in Germany.

In conclusion, we believe that the data presented by Alito et al. (1) do not give evidence to confirm higher instability of IS6110 patterns among particular MDR M. tuberculosis strains. On the contrary, our results indicate that the evolutionary clock of IS6110 RFLP seems to be identical among drug-susceptible and drug-resistant M. tuberculosis isolates.

	FOOTNOTES

^* Phone: (49) 4537-188658

Fax: (49) 4537-188311

E-mail: sniemann{at}fz-borstel.de

	REFERENCES

Top Letter References

1.	Alito, A., N. Morcillo, S. Scipioni, A. Dolmann, M. I. Romano, A. Cataldi, and D. van Soolingen. 1999. The IS6110 restriction fragment length polymorphism in particular multidrug-resistant Mycobacterium tuberculosis strains may evolve too fast for reliable use in outbreak investigation. J. Clin. Microbiol. 37:788-791[Abstract/Free Full Text].
2.	Cave, M. D., K. D. Eisenach, G. Templeton, M. Salfinger, G. Mazurek, J. H. Bates, and J. T. Crawford. 1994. Stability of DNA fingerprint pattern produced with IS6110 in strains of Mycobacterium tuberculosis. J. Clin. Microbiol. 32:262-266[Abstract/Free Full Text].
3.	Niemann, S., E. Richter, and S. Rusch-Gerdes. 1999. Stability of Mycobacterium tuberculosis IS6110 restriction fragment length polymorphism patterns and spoligotypes determined by analyzing serial isolates from patients with drug-resistant tuberculosis. J. Clin. Microbiol. 37:409-412[Abstract/Free Full Text].
4.	Van Soolingen, D., P. W. M. Hermans, P. E. W. de Haas, D. R. Soll, and J. D. A. van Embden. 1991. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J. Clin. Microbiol. 29:2578-2586[Abstract/Free Full Text].
5.	Van Soolingen, D., L. Qian, P. E. de Haas, J. T. Douglas, H. Traore, F. Portaels, H. Z. Qing, D. Enkhsaikan, P. Nymadawa, and J. D. A. van Embden. 1995. Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. J. Clin. Microbiol. 33:3234-3238[Abstract].
6.	Yeh, R. W., A. Ponce de Leon, C. B. Agasino, J. A. Hahn, C. L. Daley, P. C. Hopewell, and P. M. Small. 1998. Stability of Mycobacterium tuberculosis DNA genotypes. J. Infect. Dis. 177:1107-1111[Medline].

Stefan Niemann* Elvira Richter Sabine Rüsch-Gerdes Forschungszentrum Borstel National Reference Center for Mycobacteria Parkallee 18 D-23845 Borstel, Germany

	AUTHORS' REPLY

In response to the letter by Niemann et al. commenting on our recent publication (1), we have the following remarks. Niemann et al. stated that they did not find a higher rate of change in IS6110 RFLP patterns of serial isolates from patients with drug-resistant tuberculosis in comparison with the rate of change found in isolates from patients with mainly drug-susceptible tuberculosis in other studies. They conclude that instability of the IS6110 RFLP pattern is not associated with drug resistance. At face value, this seems to be true. In an extended study of serial isolates from 546 patients in The Netherlands (2), we also found that changes in the IS6110-based RFLP pattern of M. tuberculosis occurred as often in drug-resistant as in susceptible strains. Yet, Niemann et al. state in their letter that all changes they observed in IS6110 RFLP patterns in their recent study (3) were found in MDR strains. Their explanation for this phenomenon is that the time intervals at which their serial MDR strains were isolated were longer than the ones for isolates resistant to only one drug. However, their findings may also indicate that instability of the IS6110 RFLP pattern is higher in particular (MDR) strains. In our laboratory we have recently observed an overrepresentation of changing RFLP patterns in drug-resistant M. tuberculosis strains. We hope to present these results within a few months.

The second issue Niemann et al. raise is that the minor differences in the IS6110 RFLP pattern of the second-outbreak strain may be due not to the instability of IS6110 RFLP but to the predominance of a conserved genotype family in the area of Buenos Aires. As no others, we are aware that there are predominant M. tuberculosis genotypes circulating in particular areas and that this may interfere with the interpretation of strain typing results in molecular epidemiology. However, our conclusions are not based solely on strain typing results. As stated in our paper (1), based on conventional contact tracing we are quite sure that all of the isolates from the patients involved in the second outbreak were derived from a common ancestor within a period of 4 years. Nosocomial outbreaks caused by other M. tuberculosis strains have been recorded several times in Buenos Aires in the past years (4). Therefore, it is highly unlikely that the second outbreak described in our paper is a coincidental combination of circumstances resulting in the isolation of MDR strains with identical spoligopatterns and highly similar IS6110 RFLP types from patients with overlapping hospitalization dates.

In conclusion, we agree that our study (1) provides only an indication that the instability of IS6110 RFLP may be higher in resistant strains. Yet, our statement that the IS6110 RFLP may evolve too fast in particular MDR strains is based on a solid observation. Particular M. tuberculosis genotypes may well have different molecular clocks.

	FOOTNOTES

^* Phone: 31-2742363

Fax: 31-2744418

E-mail: d.van.soolingen{at}rivm.nl

	REFERENCES

Top Letter References

1.	Alito, A., N. Morcillo, S. Scipioni, A. Dolmann, M. I. Romano, A. Cataldi, and D. van Soolingen. 1999. The IS6110 restriction fragment length polymorphism in particular multidrug-resistant Mycobacterium tuberculosis strains may evolve too fast for reliable use in outbreak investigation. J. Clin. Microbiol. 37:788-791.
2.	De Boer, A. S., M. W. Borgdorff, P. E. W. de Haas, N. Nagelkerke, J. D. A. van Embden, and D. van Soolingen. 1999. Rate of change of IS6110 genotypes of Mycobacterium tuberculosis based on serial patient isolates. J. Infect. Dis., in press.
3.	Niemann, S., E. Richter, and S. Rusch-Gerdes. 1999. Stability of Mycobacterium tuberculosis IS6110 restriction fragment length polymorphism patterns and spoligotypes determined by analyzing serial isolates from patients with drug-resistant tuberculosis. J. Clin. Microbiol. 37:409-412.
4.	Ritacco, V., M. Di Lonardo, A. Reniero, M. Ambroggi, L. Barrera, A. Dambrosi, B. Lopez, N. Isola, and I. N. de Kantor. 1997. Nosocomial spread of human immunodeficiency virus-related multidrug-resistant tuberculosis in Buenos Aires. J. Infect. Dis. 176:637-642[Medline].

					Dick van Soolingen* Annette S. de Boer National Institute of Public Health and the Environment 3720 BA Bilthoven, The Netherlands
					Alicia Alito Pathobiology Institute CICV/INTA 1708 Morón, Argentina
					Nora Morcillo Cetrángolo Hospital Vicente Lopez, Argentina

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• Barlow, R. E. L., Gascoyne-Binzi, D. M., Gillespie, S. H., Dickens, A., Qamer, S., Hawkey, P. M. (2001). Comparison of Variable Number Tandem Repeat and IS6110-Restriction Fragment Length Polymorphism Analyses for Discrimination of High- and Low-Copy-Number IS6110 Mycobacterium tuberculosis Isolates. J. Clin. Microbiol. 39: 2453-2457 [Abstract] [Full Text]  

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