Clinical validation of a new multi-Aspergillus species reverse transcriptase quantitative PCR: a diagnostic case-control study

Objectives: Invasive aspergillosis is a difficult-to-diagnose fungal disease; the diagnosis of which relies on many criteria including mycological criteria.Recently, the EORTC/MSGERC promoted Aspergillus PCR as a specific marker, given the significant progress made in optimizing and standardising Aspergillus real-time quantitative PCR (qPCR) assays.However, there is no unique and optimal assay in the literature, particularly for optimally detecting aspergillosis due to the main Aspergillus species responsible for invasive aspergillosis.Methods: We therefore designed, optimised, and validated using a diagnostic case-control study approach a new type of real-time PCR assay.This assay is based on reverse-transcriptase qPCR methodology and multi-Aspergillus section-specific primers (MAS-RTqPCR).This assay detects whole nucleic acids from species belonging to six Aspergillus sections (Fumigati, Flavi, Nigri, Terrei, Nidulantes, Usti).Results: The limit of detection of this assay was five copies per reaction, and the analytical specificity was 100% based on the screening of over 90 fungal species.We then used three groups of patients to validate the assay, testing 120 patients and 223 plasma samples.The clinical sensitivity was 0.91 [95% CI 0.71-0.98],clinical specificity was 1.00 [95% CI 0.93-1.00].Follow-up of positive patients revealed superiority as compared to the reference assay.In group C patient, our MAS-RTqPCR assay detected three patients with non-A.fumigatus infections.Conclusions: Overall, this study demonstrates that our MAS-RTqPCR assay is a promising diagnostic for invasive aspergillosis and monitoring PCR-positive patients.