Pneumocystis jirovecii (formerly known as Pneumocystis carinii sp. hominis) infection is recognized as one of the most frequent and severe opportunistic causes of pneumonia in immunocompromised patients.
Previously diagnosis of Pneumocystis within our laboratory was based on labor-intensive and subjective staining methods such as Toluidine blue-O and immunofluorescence. In 2003 this method was replaced by a rapid and semi-quantitative touch down real-time Pneumocystis PCR Light Cycler 2.0 (Roche) assay, targeting the multicopy major surface glycoprotein (MSG) gene, as described by Larsen et al 2002.
Recently we evaluated a new real time quantitative Pneumocystis PCR assay on the CFX96 Touch instrument. The primers used in this assay are as described by Larsen et al, and the probe designed in-house, using Beacon Designer 7.29 software.
Establishment of the cycle threshold (Ct) value equivalent to the limit of detection by staining methods is important, as it is postulated that low positive results may equate with subclinical Pneumocystis infection or asymptomatic carriage, and interpreting the significance of these results should be guided by clinical assessment.
In this study serial 10 fold dilutions of a specimen containing P.jirovecii were stained and the number of cysts counted for comparison with the Ct values obtained by the Light Cycler and CFX Real time PCR assays. The latter assay detected Pneumocystis jirovecii DNA at a 10-fold greater dilution than the Light Cycler assay. In summary the CFX-96 assay has proved to be more rapid and sensitive than the semi-quantitative Light Cycler assay.