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Open AccessArticle

Quantification of Pneumocystis jirovecii: Cross-Platform Comparison of One qPCR Assay with Leading Platforms and Six Master Mixes

1
Laboratoire de Parasitologie-Mycologie, Groupe Hospitalier Saint-Louis-Lariboisière-Fernand-Widal, Assistance Publique-Hôpitaux de Paris (AP-HP), Université de Paris, 75475 Paris, France
2
Molecular Mycology Unit, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR2000), Institut Pasteur, CEDEX 15, 75724 Paris, France
3
National Reference Center for Invasive Mycoses and Antifungals (NRCMA), Institut Pasteur, CEDEX 15, 75724 Paris, France
4
Public Health Wales, Microbiology Cardiff, Heath Park, University Hospital of Wales (UHW), Cardiff CF14 4XW, UK
5
Department of Molecular Medicine, University of Padua, 35122 Padua, Italy
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
J. Fungi 2020, 6(1), 9; https://doi.org/10.3390/jof6010009
Received: 11 December 2019 / Revised: 23 December 2019 / Accepted: 23 December 2019 / Published: 26 December 2019
(This article belongs to the Special Issue Molecular Diagnostics of Fungal Infections)
Diagnosis of Pneumocystis jirovecii pneumonia relies on nucleic acid quantification in respiratory samples. Lack of standardization among molecular assays results in significant differences among assays/centers. To further promote standardization, we compared four thermocyclers and six master mixes for the detection of P. jirovecii. Whole nucleic acid (WNA) was extracted from broncho-alveolar lavages. Positive and negative sample extracts were pooled to get enough homogeneous materials. Three master mixes were tested to detect DNA by qPCR (D1, D2, and D3), and three to detect WNA by reverse transcriptase qPCR (W1, W2, and W3) manufactured by Roche, Eurogentec, Applied Biosystem, Invitrogen and Thermofischer Scientific. Experiments were performed on four thermocyclers (Roche LightCycler 480, Qiagen Rotor-Gene Q, Applied Biosystem ABI7500, and QuantStudio). Comparison of quantitative cycle (Cq) values between the methods targeting WNA versus DNA showed lower Cq values for WNA, independently of thermocycler and master mix. For high and low fungal loads, ∆Cq values between DNA and WNA amplification were 6.97 (±2.95) and 5.81 (±3.30), respectively (p < 0.0001). Regarding DNA detection, lower Cqs were obtained with D1 compared to D2 and D3, with median ∆Cq values of 2.6 (p = 0.015) and 2.9 (p = 0.039) respectively. Regarding WNA detection, no mix was superior to the others. PCR efficiency was not significantly different according to the qPCR platform (p = 0.14). This study confirmed the superiority of WNA over DNA detection. A calibration method (e.g., an international standard) for accurate comparative assessment of fungal load seems necessary. View Full-Text
Keywords: pneumocystis; qPCR; diagnosis; standardization; efficiency; threshold; DNA; whole nucleic acid; quantification cycle pneumocystis; qPCR; diagnosis; standardization; efficiency; threshold; DNA; whole nucleic acid; quantification cycle
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Dellière, S.; Gits-Muselli, M.; White, P.L.; Mengoli, C.; Bretagne, S.; Alanio, A. Quantification of Pneumocystis jirovecii: Cross-Platform Comparison of One qPCR Assay with Leading Platforms and Six Master Mixes. J. Fungi 2020, 6, 9.

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