Comparison of PCR protocols for detecting Histoplasma capsulatum and Coccidioides spp. DNA through a multi-center study

Introduction: In-house real-time PCR (qPCR) is increasingly used for the diagnosis of endemic mycoses and diverse assays are in use in specialized laboratories. External quality control is currently lacking. Objective: To compare the performance of different molecular detection protocols for the detection of Histoplasma cap-sulatum and Coccidioides spp. in a multicenter study involving five European laboratories. Methods: Two test sample panels were sent to each laboratory which performed the analysis with their in-house assays. Recipients were blinded to sample content.The Histoplasma-panel included 14 samples representing a range of concentrations of Histoplasma DNA (n = 7), as well as a negative control and DNA from other fungi to test for specificity (Paracoccidioides lutzii n = 1; Blastomyces dermatitidis n = 1; Aspergillus fumigatus n = 1; Emergomyces spp. n = 2, and Candida albicans n = 1). The Coccidioides-panel included 10 samples representing a range of DNA concentrations of Coccidioides posadasii (n = 6), as well as a negative control and DNA from other fungi to test specificity (Uncinocarpus reesii n = 1; Trichophyton violaceum n = 1; and Candida albicans n = 1).Regarding techniques used, four laboratories used Histoplasma qPCRs, and one laboratory a conventional PCR and a broad-range PCR (brPCR) for fungal DNA.Four laboratories used different Coccidioides qPCRs and one laboratory a brPCR to detect Coccidioides DNA. Results: Concerning the Histoplasma panel, qPCR assays were the most sensitives and agreement in the lowest detected amount of Histoplasma DNA was very suitable, ranging from 1 pg to 4 pg [ < 1 genomic equivalent (mean sensitivity: 96.4%) ]. The lowest detected amount of Histoplasma DNA by cPCR (sensitivity 71.4%) and the brPCR (sensitivity 42.9%) was 0.1 and 10 pg, respectively. Overall, sensitivity ranged from 42.9-100% (mean 83.3%). Overall specificity ranged from 78.6%-100%, with falsepositive resultsoccurring withhigh DNAconcentrations (200 pg/μl) of Blastomycesspp.intwo laboratoriesthat used qPCR, Emergomycesspp. by qPCRinone laboratory andAspergillus in onelaboratory thatused cPCR.Concerningthe Coccid-ioides panel, sensitivity ranged from 33.3-100% (mean 76.6%), and agreement of the lowest detected amount of Coccidioides DNA by qPCR ranged from 1-16 pg (< 1 genomic equivalent) (mean sensitivity: 87.5%) and in the brPCR 10 pg (sensitivity 33.3%).Specificity wasbetween 87.5%-100%, with onefalse positive result occurringwith high DNAconcentrations (20 pg/μ) of Uncinocarpus in one laboratory using qPCR. Conclusion: Specific protocols based on qPCR showed better sensitivity than conventional and brPCR.These methods are useful for the rapid and sensitive detection Histoplasma and Coccidioides. Application of these tests on clinical samples may speed up diagnosis and potentially limit laboratory exposure to these fungi.Comparisons of in-house tests are essential to assess the performance and detect potential cross-reactivities and achieve a consensus.