Abstract
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.
Highlights
Pneumocystis jirovecii is an ascomycetous fungi that thrives in human alveoli and is constantly transmitted among immunocompetent individuals, who are most likely to be the reservoir ofJ
Platforms for the detection of six different dilutions master mixes were tested on four different quantitative PCR (qPCR) platforms for the detection of six different dilutions of 336 tests tests in in duplicate
This study confirmed the impact of qPCR platform and amplification kit/enzyme on P. jirovecii nucleic acid quantification
Summary
Pneumocystis jirovecii is an ascomycetous fungi that thrives in human alveoli and is constantly transmitted among immunocompetent individuals, who are most likely to be the reservoir ofJ. P. jirovecii causes Pneumocystis pneumonia (PCP), mostly in patients with AIDS, hematological malignancies, solid organ transplant, or under immunosuppressive therapy, especially corticosteroids [2,3,4]. In these different populations, the symptoms and the disease severity depend on the underlying disease, and on the fungal load, which is important to quantify [5]. P. jirovecii is responsible for numerous outbreaks in hospital settings and early detection of carriers with low fungal load and infected patients is essential [6]
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