Comparison of the FXG™: RESP (Asp+) real-time PCR assay with direct immunofluorescence and calcofluor white staining for the detection ofPneumocystis jiroveciiin respiratory specimens

Abstract

assay, FXG: RESP (Asp ϩ ) from Myconostica Ltd (Manchester, UK), with immunofl uorescence and calcofl uor white staining methods for the detection of P. jirovecii in respiratory specimens. The molecular diagnostic kit targeting a portion of the Aspergillus ribosomal 18S gene and the Pneumocystis mitochondrial ribosomal large sub-unit is based on molecular beacon PCR technology. Briefl y, DNA is extracted from respiratory samples by bead beating, lysis treatment, and inhibitor removal. An aliquot of the extracted DNA is added to PCR reagents which contain P. jirovecii specifi c primers used to amplify the genetic target, if present. The assay also contains an internal amplifi cation control to confi rm the functionality of the assay. The targets amplifi ed by the PCR reaction are detected by molecular beacons, hybridization probes with fl uorophores and quenchers. The level of We compared the FXG TM : RESP (Asp ϩ ) real-time PCR assay (Myconostica Ltd) with two microscopic staining methods (direct immunofl uorescence [IFA] and calcofl uor white) for the detection of Pneumocystis jirovecii in 411 respiratory specimens submitted for P. jirovecii examination. We considered the specimen to be microscopically positive if the organism could be visualized through the use of either IFA or calcofl uor white. A second, published real-time PCR assay targeting the cdc2 gene of P. jirovecii was used to adjudicate those specimens that were microscopically negative but Myconostica PCR positive. The Myconostica PCR positive samples were deemed to be true positives if they were concordant with microscopically positive results or if they were positive by the second PCR assay. As a result, the Myconostica PCR assay was found to be more sensitive than the two microscopy methods in detecting P. jirovecii (10.5% true positivity rate by PCR, 8.0% by immunofl uorescence, and 7.1% by calcofl uor white). The Myconostica PCR assay showed 93.5% sensitivity, 95.1% specifi city, 70.5% positive predictive value, and 99.1% negative predictive value. Its high negative predictive value suggests a role of the Myconostica PCR assay in ruling out Pneumocystis pneumonia.

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).…”