Comparison of a new commercial real-time PCR assay (RealCycler PJIR kit, Progenie Molecular) to an in-house real-time PCR assay for the diagnosis of Pneumocystis jirovecii infections

Abstract

The atypical fungus Pneumocystis jirovecii ( P. jirovecii ) is the causative agent of a life threatening pneumonia [ Pneumocystis pneumonia (PCP)] in immunosuppressed patients. PCP diagnosis is essentially based on the detection of the fungus in pulmonary specimens using microscopic examination and polymerase chain reaction (PCR). For the past 20 years, the use of PCR assays has improved the sensitivity of P. jirovecii detection. The main objective of the present study was to compare the efficiency of the RealCycler PJIR kit (Progenie Molecular; pmPCR), to an in-house PCR assay (ihPCR), both assays targeting the mitochondrial large subunit ribosomal rRNA gene. Thirty-four patients were retrospectively enrolled in the study. Fourteen patients were initially diagnosed with PCP, 15 were considered to be pulmonary colonized by the fungus, and five were negative for P. jirovecii detection (negative control group). Archival DNA specimens (extracted from the patient's pulmonary specimens) were examined with ihPCR and pmPCR on the same day using the same real-time PCR instrument (Applied Biosystems 7500 Real-Time PCR system). The ihPCR assay was performed as described elsewhere by Meliani et al. and Totet et al. [1,2] . The pmPCR assay was performed according to the manufacturer's recommendations. Concordant results were obtained with the two PCR assays for all DNA specimens but one (33/34). Thus, the concordance rate between the two methods was evaluated at 97% (Cohen's kappa, 0.905). The discrepant result concerned one specimen from a colonized patient previously known to have a cycle threshold (Ct) value ≥ 35 using ihPCR. This specimen was confirmed to be positive with ihPCR and was negative with pmPCR. Taking into account Ct values for positive samples, the correlation factor between the two PCR methods has been evaluated at 0.97 ( R 2 = 0.9481). Taking into account the Cohen's kappa > 0.8, the RealCycler PJIR kit (Progenie Molecular) appears to be an efficient technique to detect P. jirovecii in patients with PCP and in patients colonized by P. jirovecii .