Evaluation of a Real-Time PCR Assay for the Diagnosis of Pneumocystis pneumonia

Abstract

Summary:The aim of this study was to evaluate of the quantification of Pneumocystis jiroveci using a realtime PCR assay. We tried to verify whether quantification was really effective in differentiating between carriage and Pneumocystis pneumonia (PCP) using real-time PCR with or without sample species normalization for classifying each sample species (sputum, bronchoalveolar lavage : bronchoalveolar lavage (BAL), and total samples).Twenty-two positive samples previously examined by conventional qualitative PCR were subjected to real-time PCR. Of these 22 lower respiratory tract specimens, 10 were BAL samples and 12 were (induced) sputum samples. According to our clinical diagnostic criteria, 17 were PCP and 5 were non-PCP. In the 12 sputum samples the concentrations of Pneumocystis-specific DNA detected in the non-PCP patients did not differ significantly from those in the PCP patients. The data were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene to exclude differences due to the number of human cells in collected samples. After normalization, the Pneumocystis-specific DNA/GAPDH-DNA ratio in the non-PCP patients was higher than that in the PCP patients. In the BAL samples (10 samples), the mean concentration of Pneumocystis-specific DNA detected in the PCP patients was 9.6 times higher than that in the non-PCP patients (P=0.058), and after normalization, the Pneumocystis-specific DNA/GAPDH-DNA ratio in the PCP patients did not differ significantly (P=0.19) from that in the non-PCP patients. Although the present study indicated that normalization using GAPDH might be not helpful but BAL specimens are recommended over sputum specimens for the diagnosis of Pneumocystis Pneumonia by quantification with real-time PCR.

Pneumocystis jirovecii pneumonia in non-HIV-infected patients

“…In BAL samples (n ¼ 10), the mean concentration of Pneumocystis-specific DNA detected in patients with PJP was 9.6-fold higher than that in non-PJP patients (P ¼ 0.058) [111], but after normalization, the Pneumocystis-specific DNA/ GAPDH-DNA ratio in patients with PJP did not differ significantly from non-PJP patients. In BAL samples (n ¼ 10), the mean concentration of Pneumocystis-specific DNA detected in patients with PJP was 9.6-fold higher than that in non-PJP patients (P ¼ 0.058) [111], but after normalization, the Pneumocystis-specific DNA/ GAPDH-DNA ratio in patients with PJP did not differ significantly from non-PJP patients.…”

Improved detection of Pneumocystis jirovecii in upper and lower respiratory tract specimens from children with suspected pneumocystis pneumonia using real-time PCR: a prospective study

“…Normalization may control for variations in the extraction process, patient variability and inter-sample variability by accounting for the dilution factor in the BAL fluid. However, there is controversy about the choice of the most appropriate reference gene [ 28 ]. If normalization can be reliably performed, it may allow for more accurate estimation of colonization rates and of cut-off values to distinguish disease thus improving the specificity of the assay.…”

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

“…Indeed, Pneumocystis jirovecii cannot be cultured, and its detection is based on staining methods using respiratory specimens, which suffer from low sensitivity (214). Consequently, many studies have evaluated the performance of PCR on respiratory specimens for PCP diagnosis, and the results seem promising, with sensitivity values as high as 100% (91,(215)(216)(217)(218)(219)(220)(221)(222)(223)(224). However, in the case of PCP, colonization is a significant issue, with rates as high as 22% in high-risk populations (225), and more importantly, since DNA is fairly stable, it is difficult to distinguish between active and previous infections (226).…”

“…To overcome that obstacle, a second technique was developed that relies on the use of real-time PCR assays to quantify the number of DNA copies found in a specimen. Studies have shown that the mean concentration of DNA from BAL fluid samples can be manifold higher in infected individuals than in asymptomatic carriers (219). Thus, many different real-time PCR assays have been developed and evaluated, with studies showing sensitivity values comparable to those of conventional PCR techniques and consistently higher than 80% (91,216,217,220,222,224).…”