Issues regarding the use of PCR for fungal diagnostics

FactorPotential solutionsAdvantages and/or disadvantages
Choice of sampleMany different sample types have been proposedThe best sample varies depending on the target pathogen and the site where it is preferentially accumulated. Tissue samples can be ideal to evaluate a new test, as they can differentiate between colonization and infection. However, less invasive samples, such as BAL fluid, serum, and whole blood, are favorable because they can be utilized as screening tests. Serum also allows for multiple tests to be performed with the same sample.
DNA extractionUse of larger sample volumes, lower elution volumes, and appropriate cell and fungal wall lysis methodsThe general idea is to maximize and concentrate the amount of fungal cells or free fungal DNA in the tested sample. However, even with perfect DNA extraction, some fungal species may be found in the circulation only transiently when they establish deep-seated infections.
Primer selectionrDNA versus mitochondrial DNA versus other DNAThe target amplification sequence should be found in multiple repeats and should differ from the respective host sequence. rDNA seems to be superior to mitochondrial DNA for diagnosis of aspergillosis.
Type of PCRStandard versus nested versus real-time PCRNested PCR requires additional time and might be more prone to contamination due to the additional amplification step. Real-time PCR allows for quantitation of the amplified DNA and thus could help to differentiate infection from colonization.
In vitro validation of a certain PCRUse of reference strains or DNA calibrator materialsThese methods are of paramount importance for the accurate evaluation of the sensitivity of any PCR and allow for interlaboratory comparisons of the results.
Coinfection by multiple microbial speciesBroad-range PCR with postamplification identification methodsThis technique allows for the simultaneous identification of multiple microbial pathogens from the same sample. The broadest method that has been proposed is the multiplex SeptiFast PCR. However, this has not yet been tested for use for patients at high risk for fungal infections.