A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis

Abstract

Background: In-house real-time PCR (qPCR) is increasingly used to diagnose the socalled endemic mycoses as commercial assays are not widely available.Objectives: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories.Methods: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing.Results: qPCR assays were the most sensitive for the detection of H. capsulatum DNA.The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA.

The Current and Future States of Diagnostic Tests for Histoplasmosis with a Focus on People with HIV and Disseminated Histoplasmosis

“…PCR has great potential, but implementation has been inconsistent. Sensitivity can be high in PWH and disseminated disease, but the lack of protocol and target standardization has limited utility and its role in diagnosing histoplasmosis is not clear [ 25 , 26 ]. Further, even in people without HIV, PCR can be utilized in targeted specimen types where infection is suspected, such as respiratory specimens [ 27 ].…”

“…PCR protocols are not yet standardized, which is a big limitation. Yet, a multicenter external quality assessment has been recently published, and although samples were few, this was really seen as a potential first step towards standardization [ 26 ].…”