Interlaboratory comparison of polymerase chain reaction for the detection ofToxoplasma gondii DNA added to samples of amniotic fluid

Abstract

To investigate the accuracy of the polymerase chain reaction (PCR) method for the detection of Toxoplasma gondii in clinical specimens, aliquots of amniotic fluid to which known amounts of Toxoplasma gondii DNA had been added were tested by five European Centres. Four laboratories were able to detect DNA at levels equivalent to ten tachyzoites or less, including two that detected DNA equivalent to a single parasite. Two laboratories erroneously found one of eight negative control samples to be positive. These findings confirm that the high level of sensitivity associated with the PCR method can be readily achieved under routine laboratory conditions, but they also underscore the potential for both false-positive and false-negative findings to occur. Furthermore, the results confirm the urgent need for an external quality assurance scheme to support laboratories employing PCR in a clinical context for the detection of Toxoplasma gondii.

Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens

“…In addition, most Toxoplasma-PCR assays used for this application are "in-house" or "laboratory-developed" methods, set up independently in each laboratory, which leads to important variations in the PCR protocols between laboratories (regarding DNA extraction, DNA target, PCR primers, amplification conditions, and amplicon detection) (32). This situation has well-known drawbacks, particularly a lack of standardization and variations in efficiency (12,17,21,25). In addition to this diversity, external quality assessments or interlaboratory comparative studies for the molecular detection of Toxoplasma gondii are scarce: five of these have been carried out in the last 10 years, all in Europe and all demonstrating wide divergences in the performances of PCR methods (2,12,17,21,25).…”

“…This situation has well-known drawbacks, particularly a lack of standardization and variations in efficiency (12,17,21,25). In addition to this diversity, external quality assessments or interlaboratory comparative studies for the molecular detection of Toxoplasma gondii are scarce: five of these have been carried out in the last 10 years, all in Europe and all demonstrating wide divergences in the performances of PCR methods (2,12,17,21,25). In France, a network has been set up for the improvement and standardization of this molecular diagnosis within the framework of the recently created National Reference Centre for Toxoplasmosis (http://www.chu-reims.fr /professionnels/cnr-toxoplasmose-1/).…”

Comparison of Two Widely Used PCR Primer Systems for Detection of Toxoplasma in Amniotic Fluid, Blood, and Tissues

“…Unfortunately, the PCR for identifying Toxoplasma remains unsatisfactory for the following reasons. (i) Only in-house PCR assays are available, and they are associated with lack of standardization and variations in efficiency (8,12,20). (ii) For most Toxoplasma infections, the diagnostic sensitivity of this molecular method remains low, e.g., 50 to 80% for prenatal diagnoses (reviewed in reference 1).…”

“…In contrast to this diversity of assays, comparative studies are scarce (reviewed in reference 1). Three European collaborative multicenter studies have been implemented in the last few years (8,12,20). However, these interlaboratory comparisons cannot distinguish between the many factors influencing the reaction outcome.…”

Preliminary experience in external quality control of RT-PCR PML-RARα detection in promyelocytic leukemia

“…Despite the widespread use of PCR in molecular laboratories, programs of external quality assurance by which individual laboratories can assess and directly compare the performance of their respective RT-PCR methods are not available. To our knowledge there are a few external quality assurance schemes for PCR methods, 1 mostly in the field of microbiology tests, [2][3][4][5] to our knowledge none to control the molecular detection of onco-hematological gene rearrangements.…”