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Journal of Clinical Microbiology, May 2003, p. 2272, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2272.2003

LETTER TO THE EDITOR

Contamination Management of Broad-Range or Specific PCR: Is There Any Difference?


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LETTER
 
We read with interest the exchange of correspondence between Millar et al. (5a) and Vandecasteele et al. (6) about the contamination management of broad-range ribosomal DNA PCR. The latter should certainly require more stringent measures than specific PCR, but this may prove theoretical. As noted by Millar et al., some specific PCR applications do require zero contamination. This is particularly true in some areas of protozoology, where the PCR has become an indispensable diagnostic tool. An accurate diagnosis of toxoplasmosis or visceral leishmaniasis often requires the detection of less than one organism per reaction tube, as very low levels of Toxoplasma or Leishmania may be present and swamped by a mass of human host DNA. Hence the development of optimized PCR methods aimed at achieving maximal sensitivity (1-4). In fact, one can detect 0.5 Toxoplasma or Leishmania DNA equivalent per reaction and still miss truly infected placenta or blood samples (4; our unpublished data). Furthermore, a highly sensitive PCR method, able to detect 10-4 Leishmania DNA equivalent, is necessary to reveal parasitemia in all infected dogs with clinically overt disease (5). In these scenarios, the presence of a so-called low-level contamination of "only 55 copies" quoted by Vandecasteele et al. is strictly unacceptable; indeed, it would yield >90% false positives. Cutoff values in PCRs are acceptable and even necessary in some pathological conditions when established from clinical and biological criteria; by contrast, defining those cutoffs for adapting to contaminations constitutes an undesirable drift. In any case, whichever their application field (microbiology or genetics), all PCRs are not equal; therefore, their management does not necessarily follow the same rules.

Vandecasteele et al. are right in pointing out that evidence may be lacking for the efficiency of many of the measures recommended for eliminating contamination. Studies are indeed needed to clarify these issues. Nevertheless, we entirely support the statement of Millar et al. that the ultimate aim of a molecular diagnostic laboratory is to achieve zero contamination and not low levels of contamination.


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REFERENCES
 
    1
  1. Bastien, P. 2002. Molecular diagnosis of toxoplasmosis. Trans. R. Soc. Trop. Med. Hyg. 96:S1205-S1215.
    2. 2
  2. Costa, J. M., C. Munoz, D. Kruger, R. Martino, T. K. Held, M. L. Dardé, C. Cordonnier, and S. Bretagne. 2001. Quality control for the diagnosis of Toxoplasma gondii reactivation in SCT patients using PCR assays. Bone Marrow Transplant. 28:527-528.[CrossRef][Medline]
    3. 3
  3. Lachaud, L., J. Dereure, E. Chabbert, J. Reynes, J. M. Mauboussin, E. Oziol, J. P. Dedet, and P. Bastien. 2000. Optimized PCR using patient blood samples for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients. J. Clin. Microbiol. 38:236-240.[Abstract/Free Full Text]
    4. 4
  4. Lachaud, L., S. Machergui-Hammami, E. Chabbert, J. Dereure, J. P. Dedet, and P. Bastien. 2002. Comparison of six PCR methods using peripheral blood for the detection of canine visceral leishmaniasis. J. Clin. Microbiol. 40:210-215.[Abstract/Free Full Text]
    5. 5
  5. Lachaud, L., E. Chabbert, P. Dubessay, J. Dereure, J. Lamothe, J. P. Dedet, and P. Bastien. 2002. Value of two PCR methods for the diagnosis of canine visceral leishmaniasis and the detection of asymptomatic carriers. Parasitology 125:197-207.[Medline]
    6. 5
  6. Millar, B. C., J, Xu, and J. E. Moore. 2002. Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J. Clin. Microbiol. 40:1575-1580.[Free Full Text]
    7. 6
  7. Vandecasteele, S. J., J. Frans, and M. Van Ranst., and Millar, B. C., X. Jiru, and J. E. Moore. 2002. Contamination management of broad-range ribosomal DNA PCR: where is the evidence? J. Clin. Microbiol. 40:3885-3886.[Free Full Text]
Patrick Bastien*
Elisabeth Chabbert
Laurence Lachaud
Laboratoire de Parasitologie-Mycologie
Centre Hospitalier Unversitaire de Montpellier
163 Rue A. Broussone
F-34070 Montpellier, France

* Phone: 33.499.23.26.78
Fax: 33.467.63.00.49
E-mail: p-bastien{at}chu-montpellier.fr


Authors' Reply


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LETTER 
 
We read with interest the letter of Bastien et al. in response to our original minireview on contamination management of broad-range ribosomal DNA PCR (1), as well as the previous letter of Vandecasteele et al. (2). Bastien et al. further develop our original minireview by questioning if there is any difference in contamination management practice between broad-range and specific PCRs? We agree that the ultimate aim in both specific and broad-range PCRs is to achieve zero contamination; however, the control measures that should be implemented in order to achieve this status may differ. The level of stringency should range from minimum to maximum control measures, as outlined in the previous minireview (1). As there are numerous permutations of PCR types, e.g., broad-range or specific, nested/seminested or single-round PCR, for detection or identification purposes, ultimately, as previously stated in our minireview, workers should perform a detailed risk assessment of the particular PCR application they wish to employ and subsequently establish the required contamination management stringency pro rata the risk assessment outcomes.


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REFERENCES 
 
    1
  1. Millar, B. C., J, Xu, and J. E. Moore. 2002. Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J. Clin. Microbiol. 40:1575-1580.
    2. 2
  2. Vandecasteele, S. J., J. Frans, M. Van Ranst, B. C. Millar, X. Jiru, and J. E. Moore. 2002. Contamination management of broad-range ribosomal DNA PCR: where is the evidence? J. Clin. Microbiol. 40:3885-3886.
Beverley C. Millar
Xu Jiru
John E. Moore*
Northern Ireland Public Health Laboratory
Department of Bacteriology
Belfast City Hospital
Lisburn Rd.
Belfast BT9 7AD, Northern Ireland

* Phone: 44 (28) 90263554
Fax: 44 (28) 25892887
E-mail: jemoore{at}niphl.dnet.co.uk

Journal of Clinical Microbiology, May 2003, p. 2272, Vol. 41, No. 5
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.5.2272.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




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