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1Lariboisière-Saint Louis hospital, APHP, Parasitology-Mycology laboratory, Paris, France; Université Paris Diderot, Sorbonne Paris Cité, France; Molecular Mycology Unit, CNRS URA 3012, Institut Pasteur, Paris, France.
Abstract:
PCR assays have not reached the same level of acceptance for the detection of human fungal pathogens as for other micro-organisms, mainly because the low number of micro-organisms challenges the detection limits of PCR. Therefore, whereas meta-analyses focusing on clinical validation suggest interest in adding PCR results to the diagnostic workup for invasive fungal disease (IFD) along with clinical evaluation, CT scans, classical mycology and antigen detection, no consensual PCR method has emerged. Compared with the end-point format of the 1990s, real-time quantitative PCR is a major breakthrough. This format prevents contamination with previously amplified products, provides the yield of amplification, allows for developing consensus procedures and should therefore be the only format used. An internal control is now mandatory to avoid false-negative results. Primer design strongly impacts on the objectives: pan-fungal primers can provide false-positive results due to environmental fungal DNA contamination; conversely, species-specific primers miss infections caused by untargeted fungi. Unresolved issues include the best specimens to be used; serum is currently preferred to blood because of the ease of the DNA extraction step. Work is in progress to establish standards at least for Aspergillus PCR, and the implementation of quality controls should help centres to improve assays. Eventually, the classical analysis of biomarker performance does not consider the evolving risk factors and changing treatments during IFD, which can lead to variable conclusions. New statistical methods such as event history analysis should be considered.
Insights
Real-time quantitative PCR offers improved detection of fungal pathogens, overcoming limitations of older methods. Standardization and quality controls are crucial for reliable results in diagnosing invasive fungal disease (IFD).
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Polymerase Chain Reaction (PCR) assays face challenges in detecting low fungal pathogen loads, limiting acceptance in clinical diagnostics.
- Despite meta-analyses suggesting PCR's value in diagnosing invasive fungal disease (IFD), a standardized method remains elusive.
- Traditional PCR formats are susceptible to contamination and lack quantitative data, hindering widespread adoption.
Purpose of the Study:
- To review the current status and challenges of PCR-based detection of human fungal pathogens.
- To highlight the advantages of real-time quantitative PCR (qPCR) over end-point PCR for fungal diagnostics.
- To discuss critical factors for developing reliable and standardized PCR methods for IFD.
Main Methods:
- Review of PCR assay performance for fungal pathogen detection.
- Comparison of end-point PCR versus real-time quantitative PCR (qPCR).
- Analysis of primer design strategies (pan-fungal vs. species-specific) and specimen choice (serum vs. blood).
Main Results:
- Real-time qPCR is a significant advancement, preventing contamination and providing amplification yield.
- Internal controls are essential for accurate qPCR results, avoiding false negatives.
- Primer selection and specimen type (serum preferred) impact assay reliability; standardization efforts are underway.
Conclusions:
- Real-time qPCR should be the standard format for fungal PCR detection due to its enhanced reliability.
- Standardization, quality controls, and appropriate primer/specimen selection are vital for improving PCR accuracy in IFD diagnosis.
- Advanced statistical methods like event history analysis may be needed to interpret biomarker performance in the context of evolving IFD treatments and risk factors.
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