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Journal of Clinical Microbiology, May 2003, p. 2269-2270, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2269-2270.2003

LETTER TO THE EDITOR

Criteria for Identification of Cross-Contamination of Cultures of Mycobacterium tuberculosis in Routine Microbiology Laboratories

Top Letter References Letter  References  Letter   References

 
Recent years have seen a dramatic rise in the number of cases of tuberculosis worldwide, and as a result, there has been an upsurge in the demand for the isolation and characterization of Mycobacterium tuberculosis isolates. In their recent articles, Ruddy et al. (4) and de Boer et al. (2) described the incidence of false-positive cultures of M. tuberculosis in routine microbiology laboratories in London and The Netherlands, respectively. While we agree with their findings in principle, we feel that their experience does not translate well to the situation that prevails in laboratories serving areas with a high incidence of tuberculosis. While their rates of cross-contamination were low (0.54 and 2.4%), so was the average number of positive isolates handled by each laboratory per week, which in itself limits the potential for cross-contamination. We particularly feel that one of their criteria for the definition of a cross-contamination event, i.e., an isolate with a fingerprint that is identical to that of another isolate processed within 7 days, is unrealistic. In a recent paper (1), we reported on the implementation of a set of measures that resulted in the reduction of the cross-contamination rate for the processing of sputum for the culture of M. tuberculosis from 7.3 to 2.1%. In contrast to the situation in London and The Netherlands, our laboratory has a culture positivity rate of 55%, which translates into approximately 24 positive cultures per week. In addition, the incidence of tuberculosis in the area is very high (3), with a high degree of molecular clustering. In such a scenario, the exclusion, as a cross-contamination event, of isolates with identical DNA fingerprints that were cultured within 7 days of each other cannot always be correct. We found that the majority of cross-contamination events were associated with processing of culture-negative specimens in the same batch as smear-positive samples obtained within the first 3 days of treatment. The modifications we made to laboratory procedures were designed to limit the opportunity for the transfer of bacilli from positive to negative samples without increasing the workload. As we could not limit the number of smear-positive samples processed per day, we ensured that the order of sputum processing went from negative to positive specimens. We also designated a specific safety cabinet for dealing exclusively with sputum samples, and all positive cultures, whether in liquid or on solid media, were dealt with in another cabinet.

It is necessary to point out that these options are accessible to all laboratories, regardless of their level of expertise and financial support. The procedural modifications we implemented were effective at reducing the rate of cross-contamination but did not have an impact on our output, and they can be effectively applied by any laboratory, whether in a routine or clinical-trial situation, without extensive economic outlay.

Top Letter References Letter  References  Letter   References

 

1
1. Carroll, N. M., M. Richardson, E. Engelke, M. de Kock, C. Lombard, and P. D. van Helden. 2002. Reduction in the rate of false positive cultures of Mycobacterium tuberculosis in a laboratory with a high culture positivity rate. Clin. Chem. Lab. Med. 40:888-892.[CrossRef][Medline]
2
2. de Boer, A. S., B. Blommerde, P. E. De Haas, M. M. Sebek, K. S. B. Lambregts-van Weezenbeek, M. Dessens, and D. Van Soolingen. 2002. False-positive Mycobacterium tuberculosis cultures in 44 laboratories in The Netherlands (1993 to 2000): incidence, risk factors, and consequences. J. Clin. Microbiol. 40:4004-4009.[Abstract/Free Full Text]
3
3. Richardson, M., N. M. Carroll, E. Engelke, G. D. van der Spuy, F. Salker, Z. Munch, R. P. Gie, R. M. Warren, N. Beyers, and P. D. van Helden. 2002. Multiple Mycobacterium tuberculosis strains in early cultures from patients in a high-incidence community setting. J. Clin. Microbiol. 40:2750-2754.[Abstract/Free Full Text]
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4. Ruddy, M., T. D. McHugh, J. W. Dale, D. Banerjee, H. Maguire, P. Wilson, F. Drobniewski, P. Butcher, and S. H. Gillespie. 2002. Estimation of the rate of unrecognized cross-contamination with Mycobacterium tuberculosis in London microbiology laboratories. J. Clin. Microbiol. 40:4100-4104.[Abstract/Free Full Text]

						Nora M. Carroll* Madalene Richardson Paul D. van Helden MRC Centre for Cellular and Molecular Biochemistry Department of Medical Biochemistry Faculty of Health Sciences University of Stellenbosch Tygerberg, South Africa
						* Phone: 272 1938 9069 Fax: 272 1938 9476 E-mail: nmc{at}sun.ac.za

Authors' Reply (reference 4)

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Carroll et al. correctly point out an important point about our study of cross-contamination in London microbiology laboratories (1), i.e., that its conclusions are only relevant to laboratories with similar resources and practices. Although the number of new cases in a busy London laboratory is lower than that in South Africa, it is more usual for multiple specimens to be sent and processed, with the result that the laboratory has many times the number of isolates than positive patients. In many countries in sub-Saharan Africa, financial constraints mean that only a proportion of samples are cultured.

The higher degree of molecular clustering that van Helden's group has described (2) may indeed be a reason why two isolates may have the same IS6110 pattern without being caused by laboratory cross-contamination. It is prudent to separate work with specimens from work with cultures, although few African laboratories have the luxury of two exhaust-protective cabinets. The idea that cross-contamination may be limited by separating smear-positive and smear-negative specimens is superficially attractive but may not be effective. An important lesson of our paper was that some of our patients had presumed cross-contamination although they did have tuberculosis. In other words, the isolate that the laboratory reported on was not the one that was responsible for the infection in that patient, a finding that would be important in areas where resistance is common. We believe that in a laboratory that processes specimens with a high culture positivity rate, this series of events may occur often and would not be prevented by separating smear-positive and smear-negative specimens. Ultimately, it is only by rigorous laboratory procedures to limit aerosol production and splashing when processing specimens that cross-contamination can be kept to a minimum (1).

 
S.H.G. and T.D.M. are members of the Steering Committee, Molecular Epidemiology of Tuberculosis in London.

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1
1. Ruddy, M., T. D. McHugh, J. W. Dale, D. Banerjee, H. Maguire, P. Wilson, F. Drobniewski, P. Butcher, and S. H. Gillespie. 2002. Estimation of the rate of unrecognized cross-contamination with Mycobacterium tuberculosis in London microbiology laboratories. J. Clin. Microbiol. 40:4100-4104.
2
2. 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]

						Stephen H. Gillespie* Timothy D. McHugh Department of Medical Microbiology University College London Royal Free Campus Rowland Hill St. London NW3 2PF, United Kingdom
						* Phone: 44-207-794-0500 Fax: 44-207-794-0433 E-mail: stepheng{at}rfc.ucl.ac.uk

Authors' Reply (reference 2)

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With interest we took notice of the letter to the editor of our colleagues N. M. Carroll et al. dealing with the definition of and the measures against laboratory cross-contaminations of Mycobacterium tuberculosis. Although we appreciate the point that the detection of false-positive M. tuberculosis isolates is more difficult when the background prevalence of tuberculosis is high and/or the variety of strains circulating in the community is low, we do not agree with the main criticism of Carroll et al. of our definition of cross-contamination because this comment does not justify the criteria we used to register an M. tuberculosis isolate as false positive. We do not simply register an isolate as false positive if the fingerprint is identical to that of another isolate processed within 7 days, but rather, we only suspect the isolate to be false positive if the fingerprint is identical to that of another isolate processed within 7 days in the same laboratory. An isolate is only officially registered as false positive if the respective fingerprint is identical to that of another isolate from the same laboratory processed within 7 days, if the patient had no clear tuberculosis symptoms, and if the peripheral laboratory confirms the false-positive laboratory diagnosis after verification with the clinician involved (1).

Finally, we applaud the idea of reserving one safety cabinet for primary cultures from clinical specimens and another for dealing with positive cultures. Laboratories throughout the world can add this measure to the excellent list of procedures to minimize the occurrence of false-positive cultures described by Small et al. (2).

Top Letter References Letter  References  Letter   References

 

1
1. de Boer, A. S., B. Blommerde, P. E. W. de Haas, M. M. G. G. Sebek, K. S. B. Lambregts-van Weezenbeek, M. Dessens, and D. van Soolingen. 2002. False-positive Mycobacterium tuberculosis cultures in 44 laboratories (1993 to 2000): causes and consequences. J. Clin. Microbiol. 40::4004-4009.
2
2. Small, P. M., N. B. McClenny, S. P. Singh, G. K. Schoolnik, L. S. Tompkins, P. A. Mickelsen. 1993. Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. J. Clin. Microbiol. 31::1677-1682.[Abstract/Free Full Text]

						Annette S. de Boer* Center for Infectious Diseases Epidemiology Dick van Soolingen Diagnostic Laboratory for Intectious Diseases and Perinatal Screening National Institute of Public Health and the Environment Antonie van Leeuwenhoeklaan 9 3721 MA Bilthoven, The Netherlands
						* Phone: 31 30 274 3691 Fax: 31 30 274 4409 E-mail: Annette.de.Boer{at}rivm.nl

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Journal of Clinical Microbiology, May 2003, p. 2269-2270, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2269-2270.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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