This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1¡3)-␤-D-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 ؋ 10 7 versus 3.4 ؋ 10 3 copies/l, P < 0.05). A lower cutoff value (1.6 ؋ 10 3 copies/l) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 ؋ 10 4 copies/ l) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of >100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 ؋ 10 3 and 2 ؋ 10 4 copies/l, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

The results provide to our knowledge the first data on the role of colonized patients as potential sources of P. jirovecii in a context of nosocomial acquisition of the fungus.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77–100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

This article describes positive (1→3)-β- d -glucan levels in serum from infants with primary Pneumocystis infection and from immunosuppressed patients with Pneumocystis pneumonia (PCP) and negative levels in serum from patients colonized by Pneumocystis jirovecii . Glucan detection is a complementary tool for the diagnosis of the diverse clinical presentations of P. jirovecii infection.

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We retrospectively investigated 76 patients with cystic fibrosis for the presence of Pneumocystis jirovecii, by performing real-time PCR and nested-PCR assays on 146 archival sputum specimens. P. jirovecii was detected in only 1 patient (1.3%) showing that in our region (Brest, France), the fungus is rarely involved in pulmonary colonization in patients with cystic fibrosis.

The burden of nosocomial Pneumocystis infections in transplantation units in France was evaluated through a retrospective survey. Over 12 years, 16 outbreaks occurred, including 13 among renal transplant recipients (RTRs). We performed Pneumocystis jirovecii genotyping in 5 outbreaks, which suggested that specific strains may have been selected by RTRs.