Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay

Abstract

Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations.Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, <3.5 copies/l of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.

Validation of the MycAssay Pneumocystis Kit for Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Specimens by Comparison to a Laboratory Standard of Direct Immunofluorescence Microscopy, Real-Time PCR, or Conventional PCR

“…Previous studies comparing real-time PCR assays to microscopy for the detection of P. jirovecii in respiratory specimens have also noted that substantial numbers of samples were positive by realtime PCR but negative by microscopy (2,7,9,13,15,19). Together with these studies, our results suggest that real-time PCR is more sensitive than microscopy for detecting P. jirovecii in BAL specimens.…”

“…Previous studies have suggested that in some individuals P. jirovecii may colonize lung tissue without causing infection. Consequently, positive results, especially those with high C T values, must be interpreted in the context of patient symptoms in order to be able to distinguish colonization from infection (1,5,7,8,15,19). Likewise, negative results could be the result of insufficient sampling, resulting in a less than detectable number of P. jirovecii cells in the BAL specimen.…”

“…The results show a high negative predictive value (98% to 99%), indicating the value of the test in ruling out Pneumocystis infection. However, a substantial number of real-time PCR-positive results were unconfirmed by microscopy or clinical diagnosis, resulting in relatively low positive predictive values (59% to 70%) (7,15). It is not known whether the MycAssay Pneumocystis kit returns false positives or whether these results represent true analytical positives reflecting a low fungal burden of P. jirovecii not detected by microscopic examination.…”

“…Recently, a commercial real-time PCR assay, the MycAssay Pneumocystis kit developed by Myconostica, Ltd. (Manchester, United Kingdom), was evaluated by comparison to microscopic examination of respiratory specimens and clinical diagnosis (7,15). The results show a high negative predictive value (98% to 99%), indicating the value of the test in ruling out Pneumocystis infection.…”

“…Various real-time PCR assays that show increased sensitivity over microscopic examination have been developed. These include real-time PCR assays that target the dihydrofolate reductase gene (11), dihydropteroate synthase (9), heat shock protein 70 (8), the cdc2 gene (2,19), and the mitochondrial ribosomal large-subunit gene (mtLSU) (1,7,15). Diagnosis of PCP is typically made on the basis of laboratory results, including microscopy and real-time PCR results, when available, as well as clinical information, including patient symptoms and underlying immune status (5,6).…”