Molecular methods, essentially based upon PCR, have become an indispensable tool in the diagnosis of infectious diseases. Real-time quantitative PCR (qrtPCR), as a novel technology, has revolutionized molecular diagnostics by adding reliability and speed (15, 28). However, Apfalter et al.(1) have stressed the need for caution in analyzing results from both qrtPCR and “conventional” PCR (cnPCR, herein designing any form of endpoint detection). We are also concerned by the fact that many reports tend to generalize the idea that qrtPCR in general has an improved sensitivity compared with cnPCR or enzyme-linked immunosorbent assay (ELISA)-PCR. It is absolutely undeniable that qrtPCR has greatly improved our molecular diagnostic practices and, as such, represents a considerable progress in molecular diagnosis. Indeed, it has many objective advantages over cnPCR, particularly speed, broad dynamic range of target DNA quantitation, and reduction of contamination. But it should be stressed that qrtPCR is not necessarily more sensitive than cnPCR.(Sensitivity in this paper is taken in its broader sense, whether “analytical,”“absolute,” or “diagnostic,” and may be assimilated to the lower limit of detection of the assay. Obviously, when compared examples are given, we considered studies using the same type of sensitivity [essentially “diagnostic”].) This relates to a more general consideration: in many reports of comparative studies between a given qrtPCR assay and a cnPCR assay, the conclusions drawn by the authors seem to apply to qrtPCR in general, as if their assay represented “the” qrtPCR (per se) and the other represented cnPCR per se. Multiple examples of this can be found in the literature, particularly in abstracts or introduction sections (eg, references 12, 13, 18, 27, 30–33, 37, and 38). The same phenomenon occurred when microbiological diagnosis by cnPCR was becoming more widely used in diagnostics in the 1990s and when “PCR” was considered as a single highly sensitive method, regardless of the assay parameters and the primers and probes used and whatever the performance characteristics. It is our intention to remind readers that in any such study the findings hold true only for the particular (PCR) assay used but certainly not for all PCR assays of the same type (whether nested PCR, cnPCR, or qrtPCR, etc.). What we emphasize here essentially concerns “in-house” or laboratory-developed PCR assays, which still constitute the vast majority of PCR diagnostic assays in microbiology (1). Our examples will focus the discussion on parasitology, mycology, and bacteriology, as these are our fields of competence and because commercial tests are less frequently available for the detection of these microorganisms than for virus detection; however, this commentary also concerns virology.

Considerable interlaboratory variations can easily be shown by examining reports from different groups for exactly the same diagnosis (1, 2) or from comparative multilaboratory studies or external quality assessment reports (5, 10, 20, 26, 34). What is the cause of these variations? Although this issue has already been extensively reviewed (1), we believe it is extremely important for the understanding of the biologist and clinician, and hence for patient care, to underline the following:(i) the PCR is not just one technique but a method encompassing a number of techniques, each with a variable outcome depending on a variety of factors; and (ii) qrtPCR is essentially a sophisticated technological advance of PCR, but it has many of the same limitations with respect to interlaboratory variations. A simple comparison can be made here with serology …