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 l 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 (>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 l.

ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients

“…Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the use of bead-beating, white cell lysis buffer, a starting volume of 3 mL, and an elution volume less than 100 mL. 64 A recent reanalysis reinforces the importance of the different stages of DNA extraction. 65 New, standardized, commercially available PCRs might also contribute to improve inter-laboratory reproducibility of results; however, these expensive and technically complex assays need a validation in specific clinical settings at high risk of IFD.…”

Impact of fluconazole versus posaconazole prophylaxis on the incidence of fungal infections in patients receiving induction chemotherapy for acute myeloid leukemia

“…[39] The working group of the International Society for Human and Animal Mycology is preparing to propose a harmonization of protocols at the European level [European Aspergillus PCR initiative (EAPCRI)]. [40][41][42] Finally, the evaluation of the serum β-glucan (present in fungal membranes) could be of global interest, but no evaluation in patients receiving prophylaxis is available. [43] The absence of difference between the two groups in our study could eventually be explained by the limited sample size.…”

Biomarkers for Invasive Aspergillosis: The Challenges Continue

“…• Asserted that the efficiency of Aspergillus PCR is limited by the DNA extraction procedure and not by PCR amplification [165];…”