Advances and Prospects for Molecular Diagnostics of Fungal Infections

Abstract

The polymerase chain reaction (PCR) methods published for the diagnosis of invasive fungal infections are still not included in the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group definitions of IA. This could be achieved with consensual PCR procedures. A checklist of items has been proposed to improve the reliability of the results and clinicians' confidence in them, with emphasis on limiting false-positive results from contamination with either previously amplified products or environmental commensals. Internal amplification controls are mandatory to expose false-negative results. However, our ignorance of the origin and the kinetics of fungal DNA during an infection hamper the choice of the best specimen and DNA extraction protocol. Evidence is increasing that serum could be a good compromise between sensitivity and ease of DNA extraction. Once a technical consensus is achieved, clinical studies should be initiated to integrate quantitative PCR in the diagnostic armamentarium.

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

“…The current trend is to use qPCR because this format decreases the risk of contamination with previously amplified products, and can control the yield of amplification to give reliable quantitative results. 59 A recent meta-analysis of studies in hematological patients with IA using different home-made PCRs in blood reported a sensitivity of 75% (95% CI: 54-88) and a specificity of 87% (95% CI: 78-93) for two PCR-positive results, and a sensitivity of 88% (95% CI: 75-95) and a specificity of 75% (95% CI: 63-84) for a single positive result. 60 When used as a screening test, authors favor the negative predictive value of the results.…”

Clinical Significance of Quantifying Pneumocystis jirovecii DNA by Using Real-Time PCR in Bronchoalveolar Lavage Fluid from Immunocompromised Patients

“…This format is not intended to give quantitative results, and the test is prone to contamination with previously amplified products, leading to false-positive results. In contrast, realtime quantitative PCR (qPCR) dramatically reduces the risk of false-positive results because of the closed-tube nature of the amplification process (6), and the resulting data are quantitative if guidelines for interpretation of qPCR results are followed (7).…”

Difficulties with molecular diagnostic tests for mould and yeast infections: where do we stand?

“…To prevent false‐positives due to amplicon contamination, uracil‐N‐glycosylase treatment has been proposed for more than 20 years , but enzymatic methods have been slowly implemented in PCR assays. Currently, the best means to prevent amplicon contamination are the closed format provided by real‐time quantitative PCR (qPCR) .…”

“…With “pan‐fungal” primers, the specificity of the amplicon cannot be assessed unless specific probes are used . The analysis of melting curves using SYBR Green dye is only indicative as this method lacks the required specificity for clinical diagnostic assays .…”