Development and Evaluation of a Quantitative, Touch-Down, Real-Time PCR Assay for Diagnosing Pneumocystis carinii Pneumonia

Abstract

A rapid (time to completion, <4 h, including DNA extraction) and quantitative touch-down (QTD) real-time diagnostic Pneumocystis carinii PCR assay with an associated internal control was developed, using fluorescence resonance energy transfer (FRET) probes for detection. The touch-down procedure significantly increased the sensitivity of the assay compared to a non-touch-down procedure. Tenfold serial dilutions of a cloned target were used as standards for quantification. P. carinii DNA has been detected in respiratory specimens from patients with P. carinii pneumonia (PCP) and from patients without clinical evidence of PCP. The latter probably represents colonization or subclinical infection. It is logical to hypothesize that quantification might prove helpful in distinguishing between infected and colonized patients: the latter group would have lower copy numbers than PCP patients. A blinded retrospective study of 98 respiratory samples (49 lower respiratory tract specimens and 49 oral washes), from 51 patients with 24 episodes of PCP and 34 episodes of other respiratory disease, was conducted. PCR-positive samples from colonized patients contained a lower concentration of P. carinii DNA than samples from PCP patients: lower respiratory tract samples from PCP and non-PCP patients contained a median of 938 (range, 2.4 to 1,040,000) and 2.6 (range, 0.3 to 248) (P < 0.0004) copies per tube, respectively. Oral washes from PCP and non-PCP patients contained a median of 49 (range, 2.1 to 2,595) and 6.5 (range, 2.2 to 10) (P < 0.03) copies per tube, respectively. These data suggest that this QTD PCR assay can be used to determine if P. carinii is present in respiratory samples and to distinguish between colonization and infection.The opportunistic fungus Pneumocystis carinii f. sp. hominis is an important cause of morbidity and mortality, causing P. carinii pneumonia (PCP) in AIDS and other immunocompromised patients. The standard method for diagnosis of PCP is microscopic examination of stained (immunofluorescent or conventional tinctorial) invasive lower respiratory tract specimens: bronchoalveolar lavage (BAL), lung biopsy, or induced sputum specimens, the latter being the least sensitive, with reports of sensitivity varying from Ͻ50% to Ͼ90% (4-6, 9, 17, 18, 21, 23). Molecular detection systems have the potential to provide a higher degree of sensitivity than microscopic examination. PCR methods have been applied to lower respiratory tract specimens, and recently to non-invasive oral washes as well (1-5, 10, 12-14, 19-36, 38-42). However, some of these techniques are cumbersome, often requiring several steps in order to increase sensitivity and leaving them open to possible contamination.A single-round, nonnested, PCR assay, with no manipulations of amplicons required, would significantly reduce risks of contamination problems and be ideal for use in clinical diagnostic laboratories. A rapid PCR assay for detection of PCP, with a turnaround time comparable to smears, would enhance the clinical utility of molecula...

Comparison of three cost effective staining methods for detection of Pneumocystis jirovecii

“…PCP has nonspecific clinical symptoms such as dyspnea, fever and nonproductive cough that are similar to many infectious agents. Although a variety of polymerase chain reaction (PCR) methods, such as nested-PCR and real-time PCR, have been developed for the diagnosis of PCP, these assays are not conventional in most clinical microbiology laboratories and are not easily available, especially in developing countries (Bandt and Monecke 2007;Contini et al 1998;Larsen et al 2002). Therefore, the standard laboratory method for diagnosis of PCP is microscopic examination of the clinical specimen such as induced sputum, bronchoalveolar lavage (BAL) and lung biopsy after some types of staining methods (Takahashi et al 2002).…”

Pneumocystis jirovecii Pneumonia in AIDS Patients

“…Thus, a quantitative real-time PCR assay that targeted Pneumocystis Major Surface Glycoprotein (MSG) multigene family was applied to OW samples, and revealed significant differences between PcP patients and Pneumocystis colonized subjects in the number of MSG copies. The authors suggested a cutoff value of 50 MSG gene fragment copies/tube for distinguishing between the two conditions (Larsen et al, 2002). However, quantitative PCR results seemed difficult to use on the field.…”

“…In common practice, this difficulty is often solved on the basis of a careful clinical, radiological and laboratory assessment of the patient pathological condition, as it is usually done to other infectious diseases, especially when their agents are opportunistic pathogens. However, the alternative of quantifying parasite rates was also explored (Larsen et al, 2002). Thus, a quantitative real-time PCR assay that targeted Pneumocystis Major Surface Glycoprotein (MSG) multigene family was applied to OW samples, and revealed significant differences between PcP patients and Pneumocystis colonized subjects in the number of MSG copies.…”

Evaluation of a Real-Time PCR Assay for the Diagnosis of Pneumocystis pneumonia

“…It is thought that real-time PCR, as recently described [5,14], might be useful in distinguishing between colonization and clinical disease. This sugges-tion is based on the hypothesis that PCP patients should have a higher organism burden than colonized or subclinically infected patients, and that this would be reflected by a higher amount of Pneumocystis jiroveci DNA present in the extracted specimens from PCP patients.…”

“…Standards and external controls: To obtain standards for real-time PCR, P. jiroveci-positive BAL samples (by Grocott smear) were used. The extracted DNA (described above in Preparation of DNA specimens) was used for amplification of the major surface glycoprotein (MSG) gene of Pneumocystis [14]. A PCR method previously described [11] was modified for the real-time assay.…”