Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens

Abstract

bronchoalveolar lavage (BAL) specimens from 150 patients (19 HIV-infected and 131 non-HIV-infected patients) were evaluated for identification of Pneumocystis jiroveci (formerly known as Pneumocystis carinii f. sp. hominis) using staining techniques, conventional PCR (mtLSUrRNA gene) and real-time PCR (MSG gene). Test results were compared to Pneumocystis pneumonia (PCP) confirmed by typical clinical findings and response to treatment. Sensitivity and specificity of the techniques were 60 and 100 % for staining (where either one or both techniques were positive), 100 and 87 . 0 % for conventional PCR and 100 and 84 . 9 % for real-time PCR, respectively. The use of a concentration of 10 3 copies of DNA per capillary of BAL as a cut-off (determined by real-time PCR) increased specificity from 84 . 9 to 98 . 6 % without reducing the sensitivity of the technique. This technique is rapid (,3 h) and therefore of major interest in differentiating between asymptomatic carriage and PCP. A BAL specimen with ,10 3 copies per capillary of Pneumocystis-specific DNA is more likely to indicate a chronic carrier state, but in such cases follow-up is required to ensure that the patient is not in the early stage of an active PCP.

3-Nitropropionic acid modifies neurotrophin mRNA expression in the mouse striatum: 18S-rRNA is a reliable control gene for studies of the striatum

“…The difference between semi-Q RT-PCR and quantitative RT-PCR is well recognized [25] ; however, as much of the information related to neurotrophin mRNA expression in the striatum was obtained with semi-Q RT-PCR or in situ hybridization, we used this as a reference for comparison with previous reports in the same region [3][4][5] . We are aware that the primers used in the present study were designed for semi-Q RT-PCR [20,21] .…”

“…this does not always correspond to the optimal efficiency of the reaction and therefore is less precise [25] .…”

Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay

“…However, lack of standardization and clinical validation of the laboratory-developed assays is the primary reason why the EORTC/MSG group decided to omit all fungal PCR from the definitions of invasive fungal diseases (7). In addition, several papers attest to the fact that PCR diagnosis of PCP is more sensitive than microscopy (4,10,11,13,22,24). The generally higher sensitivity of PCR assays than of microscopy may be disproportionately important for non-AIDS patients and may result in fewer missed clinical diagnoses.…”

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

“…In particular, results might differ between HIV-positive and -negative cases. Such a difference has been found in previous studies, which have indicated the difficulty of confirming a PCP diagnosis in non-HIV-infected cases due to the lack of sensitivity of PCR methods (20-25%), even in BAL specimens [5,23,25,32]. The PCR positive rate for PCP in the HIV positive patients was higher than that in the HIV negative patients (6/8:75%>2/8:25%) [7].…”

“…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.…”