Journal of Clinical Microbiology, July 2004, p. 3379-3380, Vol. 42, No. 7
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.7.3379-3380.2004
| LETTER TO THE EDITOR |
Internal Amplification Control for PCR Should Not Be Mandatory in the Clinical Medical Environment
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Hoorfar et al. propose making internal amplification control (IAC) mandatory for diagnostic PCR (J. Hoorfar, N. Cook, B. Malorny, et al., Letter, J. Clin. Microbiol. 41:5835, 2003). This proposal appears to be specific to foodstuffs, but it might also be applied to clinical diagnostics. It is interesting to consider IAC for PCR in light of other methods in clinical microbiology, where it is uncommon to test for the presence of inhibitors despite numerous false negatives. For example, 51% of urine specimens were shown to contain antibiotics, with a 78% loss of expected positive cultures (unpublished data).
With foodstuffs, "a false negative turns a risk into a threat for the population" (Hoorfar et al., letter), as it might allow contaminated foods to reach the market. In clinical medicine a test result is merely one piece of evidence and should always be interpreted in view of the clinical assessment. We do not withdraw treatment from patients whose tests are negative. We may repeat the test, try another method of confirming the clinical suspicion, pursue an alternative diagnosis, or do nothing! I don't deny that false negatives are undesirable. They are damaging. They prevent us from focusing on a specific disease, bringing the extra costs and complications of continuing with unnecessary drugs and investigations while searching for a diagnosis. This damage extends from all test types, not just PCR. Don't doubt that we are working to improve the collection of urine samples!
Insisting that internal controls are included in articles on PCR is less attractive than encouraging reports on whether a test is "robust" enough for use in the intended environment, with data produced by diagnostic staff working in real-life situations on unselected clinical samples. This would be much more helpful to clinical diagnostic laboratories assessing the literature. Knowing the reason for a false negative is helpful, as it may provide an opportunity to correct the problem, but it is not an essential piece of information when considering implementing a test in medicine. It is the overall use and clinical performance of the test in field conditions that interests us.
PCR is relatively new but is not so special that it deserves an IAC in preference to other tests. If the standards organizations were to require IAC for PCR in clinical work, then surely, for the sake of standardization, they should also require similar controls for culture methods; perhaps we have accepted undetected false negatives for too long. However, this added cost might prevent the introduction of a test that otherwise outperforms alternative methods. In medicine the benefit of extra diagnoses with a sensitive system such as PCR may outweigh losses due to some false negatives caused by inhibition, which is only one of the causes of a false negative.
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TopLetterReferencesLetter |
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- Abu Al-Soud, W., and P. Rådström. 2001. Purification and characterization of PCR-inhibitory components of blood cells. J. Clin. Microbiol. 39:485-493.
[Abstract/Free Full Text] - Hoorfar, J., A. Abdulmawjood, N. Cook, B. Malorny, M. Wagner, and P. Fach. 2004. Practical considerations in design of internal amplification controls for diagnostic PCR assays. J. Clin. Microbiol. 42:1863-1868.
[Free Full Text] - Rossen, L., P. Nørskov, K. Holmstrøm, and O. F. Rasmussen. 1992. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int. J. Food Microbiol. 17:37-45.[CrossRef][Medline]
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Timothy Barkham
Tan Tock Seng Hospital 11 Jalan Tan Tock Seng Singapore 308433 Republic of Singapore
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* Phone: 65-63578957 Fax: 65-62536507 E-mail: timothy_barkham{at}ttsh.com.sg |
Authors' Reply
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TopLetterReferencesLetter |
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Including an internal amplification control (IAC) in PCR tests offered by medical laboratories was not sufficiently addressed in our Letter to Editor (J. Hoorfar, N. Cook, B. Malorny, M. Wagner, D. De Medici, and P. Fach, Letter, J. Clin. Microbiol. 41:5835, 2003). It is therefore much appreciated that Dr. Barkham has raised the issue in the context of clinical diagnostics. It is correct that the presence of an IAC in a PCR response is "merely one piece of evidence and should be always interpreted in view of the clinical assessment." It is also justified that publication reports on PCR testing should include "data produced by diagnostic staff working in real-life situations on unselected clinical samples."
However, the letter has mentioned two reasons for not making IACs mandatory: first, the test is only part of the diagnosis and subsequent treatment strategy; second, mandatory IACs would risk increasing the cost of PCR testing.
As for the use of PCR testing in differential diagnosis and disease treatment, we have all to win and nothing to lose by including an IAC. PCR is a completely enzymatic reaction; i.e., inhibition of the DNA polymerase by the biological constituents of a sample matrix can be damaging (1, 3). This is not the case for culture methods. Pathogens will grow, perhaps at a slower speed, even in the presence of sample-inhibiting constituents. It would be desirable to include an internal control for culture methods; however, this is technically not possible, to our knowledge.
With regard to the additional cost of including an IAC to a diagnostic PCR, this is minimal (approximately 25 cents per test) compared to the cost of other reagents, thermalcyclers, and personnel. Construction of IACs can be made in many different ways (2), e.g., a simple synthesis of an entire chimeric IAC through commercial manufacturers of DNA oligonucleotides. However, careful consideration must be given to the design of an IAC, depending on the purpose of the test and its significance in the overall clinical diagnosis. Here, the issues of good laboratory practice and overall validation on real-life samples should be taken into consideration, as correctly pointed out in the aforementioned letter.
In conclusion, we are convinced that the inclusion of an IAC would be essential to PCR and would substantially improve the quality of scientific reports describing new diagnostic (not investigative) PCRs.
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Jeffrey Hoorfar
Danish Institute for Food and Veterinary Research 27 Bülowsvej DK-1790 Copenhagen V, Denmark
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* Phone: 45-72346251, Fax: 45-72346001, E-mail: jho@dfvf.dk |
Journal of Clinical Microbiology, July 2004, p. 3379-3380, Vol. 42, No. 7
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.7.3379-3380.2004
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