Diagnosis of Pneumocystis jirovecii Pneumonia in Immunocompromised Patients by Real-Time PCR: a 4-Year Prospective Study

Abstract

Pneumocystis jirovecii pneumonia (PCP) is a life-threatening infection in immunocompromised patients. Quantitative real-time PCR (qPCR) is more sensitive than microscopic examination for the detection of P. jirovecii but also detects colonized patients. Hence, its positive predictive value (PPV) needs evaluation. In this 4-year prospective observational study, all immunocompromised patients with acute respiratory symptoms who were investigated for PCP were included, totaling 659 patients (814 bronchoalveolar lavage fluid samples). Patients with negative microscopy but positive qPCR were classified through medical chart review as having retained PCP, possible PCP, or colonization, and their clinical outcomes were compared to those of patients with microscopically proven PCP. Overall, 119 patients were included for analysis, of whom 35, 41, and 43 were classified as having retained PCP, possible PCP, and colonization, respectively. The 35 patients with retained PCP had clinical findings similar to those with microscopically proven PCP but lower fungal loads (P < 0.001) and were mainly non-HIV-infected patients (P < 0.05). Although the mean amplification threshold was higher in colonized patients, it was not possible to determine a discriminant qPCR cutoff. The PPV of qPCR in patients with negative microscopy were 29.4% and 63.8% when considering retained PCP and retained plus possible PCP, respectively. Patients with possible PCP had a higher mortality rate than patients with retained PCP or colonization (63% versus 3% and 16%, respectively); patients who died had not received co-trimoxazole. In conclusion, qPCR is a useful tool to diagnose PCP in non-HIV patients, and treatment might be better targeted through a multicomponent algorithm including both clinical/radiological parameters and qPCR results.

Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients

“…In our study, the C T values were defined to obtain a sensitivity of 80% and a specificity of 80%, allowing the C T values to provide physicians with useful insight. Some studies determined various PCR cutoff values that may be used to discriminate PCP from colonization, but very few of them gave different values according to HIV infection status (62,63). The C T values given in our study could be used by other laboratories, provided that extraction and qPCR are performed as described in our Materials and Methods.…”

Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia

“…Several groups have proposed to classify patients according to the probability of having PCP (5,(16)(17)(18). Criteria analyzed included the presence of risk factors, clinical and radiological signs, outcomes of anti-P. jirovecii treatment, and the results of DE of respiratory samples.…”

“…To facilitate the interpretation of PCR results and to differentiate PCP from colonization, cutoff values of P. jirovecii DNA copy number or C T values were suggested (6,11,16,17,(21)(22)(23)(24). However, as the fungal load during PCP can vary according to the population of the patients tested (e.g., HIV infected versus non-HIV infected) and is often higher in HIV-infected patients (5,17,18), cutoffs need to be adapted to the population. Nevertheless, the lack of standardization of pulmonary samples (volume, number of cells, etc.)…”

Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

“…However, the interpretation of qPCR results remains challenging, since precise cutoffs to distinguish between these patients have rarely been calculated (13)(14)(15)(16)(17). Moreover, these cutoffs are difficult to compare, since either multicopy (mtLSU or MSG) or single-copy genes (DHPS or HSP70) were targeted with different technologies (FRET or TaqMan) in those studies (13,14,16,18,19).…”

“…This is probably due to differences in the pathophysiology of the disease, and particularly in the host immune response. Moreover, some studies have suggested that the fungal burden may be lower in HIV-negative patients (14,18,19). However, the determination of qPCR cutoffs to distinguish colonization from infection in each of these populations of patients has never been performed, to the best of our knowledge.…”