Development of a Rapid Real-Time PCR Assay for Quantitation ofPneumocystis cariniif. sp.carinii

Abstract

A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r ‫؍‬ 0.99) over 6 log values for standards containing >5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r ‫؍‬ 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.

Clinical Significance of Quantifying Pneumocystis jirovecii DNA by Using Real-Time PCR in Bronchoalveolar Lavage Fluid from Immunocompromised Patients

“…However, there are marked differences among studies, which prevents direct comparison. For instance, the target chosen for amplification was either a multicopy gene [major surface glycoprotein (MSG) gene (10,11,17,18) or ribosomal RNA gene (1,3,12,14,22)] or a single-copy gene (3,5,15,18,26). The use of UNG for preventing cross-contamination has been reported only in the present study.…”

“…Additionally, cross-study comparison is difficult when the type of clinical specimen sampled is different. Most studies have used BAL fluids only (2,3,5,10,11,15,18,22,26), but some have included mixed sputa and BAL fluid (1,14) or sputa and/or oral washes (12,17). Even though oral washes or sputum samples can be used for P. jirovecii detection, we used the BAL fluids only to keep the quantification consistent.…”

“…The use of UNG for preventing cross-contamination has been reported only in the present study. An internal control has been effective (1,5,11,14,17), omitted (2,3,12,15,18,22,26), or inadequately chosen (10). Results have been expressed in copy numbers using a plasmid clone (2,3,5,12,15,17,22) or as Cq values (1,10,11,14,18,26).…”

Comparison of Quantitative Reverse Transcription-PCR to Viral Culture for Assessment of Respiratory Syncytial Virus Shedding

“…Quantitative RT-PCR has been used successfully to monitor disease progression and assess response to therapy for malignant diseases and other viral infections that are difficult to cultivate, such as human immunodeficiency virus and hepatitis C virus (2,12,15,20,29). However, the relationship of RNA copy to viable organisms and clinical illness is less well defined for respiratory pathogens, including RSV (9,16,17,19,22). Therefore, we developed a quantitative real-time RT-PCR and compared it to viral culture to assess viral load in adult volunteers challenged with the RSV A2 strain.…”

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

“…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).…”