Performance of a Real Time PCR for Pneumocystis jirovecii Identification in Induced Sputum of AIDS Patients: Differentiation between Pneumonia and Colonization

Abstract

Pneumocystis jirovecii pneumonia (PcP) remains an important cause of morbimortality worldwide and a diagnostic challenge. Conventional methods have low accuracy, hardly discriminating colonization from infection, while some new high-cost or broncho-alveolar lavage-based methods have limited usefulness in developing countries. Quantitative PCR (qPCR) tests may overcome these limitations due to their high accuracy, possibility of automation, and decreasing cost. We evaluated an in-house qPCR targeting the fungus mtSSU gene using induced sputum. Sensitivity of the assay (ten target gene copies/assay) was determined using recombinant plasmids. We prospectively studied 86 AIDS patients with subacute respiratory symptoms in whom PcP was suspected. qPCR results were determined as quantification cycles (Cq) and compared with a qualitative PCR performed in the same IS, serum 1,3-β-D-Glucan assay, and a clinical/laboratory/radiology index for PcP. The qPCR clustered the patients in three groups: 32 with Cq ≤ 31 (qPCR+), 45 with Cq ≥ 33 (qPCR-), and nine with Cq between 31-33 (intermediary), which, combined with the other three analyses, enabled us to classify the groups as having PcP, not P. jirovecii-infected, and P. jirovecii-colonized, respectively. This molecular assay may contribute to improve PcP management, avoiding unnecessary treatments, and our knowledge of the natural history of this infection.

Keywords: 1,3-β-D-glucan; HIV/AIDS; Pneumocystis jirovecii; molecular diagnosis; real time PCR.

Figure 1

Distribution of the Cq results of the qPCR performed with IS of patients with AIDS and suspected Pneumocystis pneumonia (PcP) according to the low (green) or high (red) clinical index for PcP. Each symbol represents one patient. PcP (n = 32): positive qPCR with low Cq values indicating high fungal load. Intermediary group (n = 9): positive qPCR with high Cq values indicating low fungal load. qPCR-: n = 45.

Figure 2

Comparison of the Cq values of the positive qPCR (n = 41) with the results of the cPCR performed in IS of patients with AIDS and suspected PcP. Each symbol represents one patient.

Figure 3

Comparison of the Cq values of the positive qPCR (n = 41) performed in IS with the serum 1,3-beta-d-glucan assay of patients with AIDS and suspected PcP. Each symbol represents one patient.