Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1->3)-β-D-glucan for differential diagnosis of pneumocystis pneumonia and Pneumocystis colonization

Abstract

This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-β-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/μl, P < 0.05). A lower cutoff value (1.6 × 10(3) copies/μl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 10(4) copies/μl) 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/μl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

Fig 1

Pneumocystis jirovecii DNA copies per μl of extracted DNA in patients with Pneumocystis pneumonia and patients with Pneumocystis colonization. Gray boxes, 50% of the sample data; black horizontal bars, median values. The median value for patients with Pneumocystis pneumonia was 4.2 × 10⁵ copies/μl (interquartile range, 3.3 × 10⁴ to 1.5 × 10⁷ copies/μl). The median value for patients with Pneumocystis colonization was 4.4 × 10² copies/μl (interquartile range, 1.6 × 10² to 3.7 × 10³ copies/μl). Pneumocystis jirovecii DNA copy numbers were determined by a qPCR assay targeting the mitochondrial large subunit rRNA gene.

Fig 2

Receiver operating characteristic curve for Pneumocystis jirovecii DNA copy numbers for the diagnosis of Pneumocystis pneumonia. Arrows, cutoff values for the qPCR assay used for quantification of Pneumocystis jirovecii DNA copy numbers in bronchoalveolar lavage fluid samples from patients infected with Pneumocystis.

Fig 3

Results of Pneumocystis jirovecii DNA copies per μl of extracted DNA in bronchoalveolar lavage fluid samples combined with (1→3)-β-d-glucan levels in serum samples for patients with Pneumocystis pneumonia and patients with Pneumocystis colonization. ▲, patients initially assigned to the Pneumocystis colonization group; ■, patients initially assigned to the Pneumocystis pneumonia group with negative microscopic detection of Pneumocystis jirovecii in bronchoalveolar lavage fluid samples; □, patients initially assigned to the Pneumocystis pneumonia group with positive microscopic detection of Pneumocystis jirovecii in bronchoalveolar lavage fluid samples.

Fig 4

Characterization of patients infected with Pneumocystis using pulmonary quantification of Pneumocystis jirovecii DNA copy numbers combined with serum (1→3)-β-d-glucan levels. BAL, bronchoalveolar lavage fluid.

Fig 5

Flow diagram for the diagnosis of Pneumocystis infections in patients with clinically suspected Pneumocystis pneumonia. Microscopic examination using methanol-Giemsa staining and an immunofluorescence assay (Bio-Rad, Marnes la Coquette, France) and a qPCR assay targeting the mitochondrial large subunit rRNA gene of P. jirovecii were used. BAL, bronchoalveolar lavage fluid; BG, (1→3)-β-d-glucan; PCP, Pneumocystis pneumonia; ∗, results expressed in DNA copies/μl; #, without factors that interfere with (1→3)-β-d-glucan level determinations, particularly concurrent invasive fungal infections.