Aspergillus PCR: one step closer to standardization

Abstract

PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as "compliant" or "noncompliant," according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 microl showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (>or=3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 microl.

FIG. 1.

Summary of the EAPCRI Aspergillus PCR standardization process.

FIG. 2.

The probability of a positive Aspergillus PCR signal when the 2008 WB panel is tested. The vertical dashed reference lines indicate (log-transformed) conidial loads of 10, 25, 50, 75, 100, 500, and 1,000. They intersect the various platform-specific logistic curves at the indicated probability levels. Twenty-one centers performed 59 tests on each specimen. Ten centers used the Roche LightCycler apparatus to perform a total of 30 replicates for each specimen, five centers used the Applied Biosystems TaqMan apparatus to perform a total of 13 replicates, two centers used the Corbett Rotorgene apparatus to perform a total of 6 replicates, and four centers used other platforms to perform a total of 10 replicates.

FIG. 3.

Forest plot of sensitivity (a) and DOR (b) for each center participating in testing of the 2008 EAPCRI WB panel. Individual centers are indicated by numbers. The centers are grouped according to their compliance with the extraction protocol. The effect size sensitivity (%) and DOR. The closed rhomboids indicate the average values of sensitivity/DOR at the group level.

FIG. 4.

Recommended protocol for extracting Aspergillus DNA from whole blood. Boldface text is expert opinion. The threshold is the proportion of centers reaching the threshold when the step is used. ppt, precipitation; a, the lysis buffer described previously (6); b, involves 5 min of processing time and 10 min of incubation time; when frozen specimens are processed, incubation is not necessary.