Many PCR assays reported in the literature amplify mitochondrial DNA sequences.2 Hence, they amplify multicopy Pneumocystis target genes, significantly heightening detection sensitivity. In addition, these assays often rely on nested PCR approaches where the initial amplification products are subsequently reamplified, yielding even greater sensitivity.3 Indeed, such approaches may be overly sensitive for many routine clinical applications. Accordingly, previously reported PCR assay approaches are known to detect Pneumocystis colonization in addition to invasive infections.4 However, the Pneumocystis PCR assay used in our clinical microbiology laboratory has unique features that circumvent many of these issues.5 To address the issues of colonization, we use a diagnostic Pneumocystis PCR assay that uses a single-copy target gene from Pneumocystis jirovecii, namely pjcdc2.5 We also perform quantitative amplification with real-time PCR and do not reamplify the reaction products with nested PCR.5 With this PCR assay, we have observed an increase (approximately 7%) in total diagnostic sensitivity over smeared stains alone. In addition, in now > 200 consecutive cases without clinical evidence of infection, we have not detected a PCR signal. Hence, the assay used in this study was specifically designed to detect PcP rather than colonization.5 On this basis, our laboratory now routinely uses this PCR assay rather than microscopic examination for the diagnosis of Pneumocystis infection. Moreover, it must be noted that the patients in the current report had active lung infiltration as well as signs and symptoms of infection. In each patient, there was no evidence of any other significant alternative organism, despite routine comprehensive culture, antigen detection, and molecular diagnostic assays performed to rigorously detect typical and atypical bacteria, viruses, and other fungi. Thus, in all three patients, the clinical evidence pointed to active PcP rather than to colonization.