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

Abstract

Quantitative PCR (qPCR) is more sensitive than microscopy for detecting Pneumocystis jirovecii in bronchoalveolar lavage (BAL) fluid. We therefore developed a qPCR assay and compared the results with those of a routine immunofluorescence assay (IFA) and clinical data. The assay included automated DNA extraction, amplification of the mitochondrial large-subunit rRNA gene and an internal control, and quantification of copy numbers with the help of a plasmid clone. We studied 353 consecutive BAL fluids obtained for investigation of unexplained fever and/or pneumonia in 287 immunocompromised patients. No qPCR inhibition was observed. Seventeen (5%) samples were both IFA and qPCR positive, 63 (18%) were IFA negative and qPCR positive, and 273 (77%) were both IFA and qPCR negative. The copy number was significantly higher for IFA-positive/qPCR-positive samples than for IFA-negative/qPCR-positive samples (4.2 ؎ 1.2 versus 1.1 ؎ 1.1 log 10 copies/l; P < 10 ؊4 ). With IFA as the standard, the qPCR assay sensitivity was 100% for >2.6 log 10 copies/l and the specificity was 100% for >4 log 10 copies/l. Since qPCR results were not available at the time of decision-making, these findings did not trigger cotrimoxazole therapy. Patients with systemic inflammatory diseases and IFA-negative/qPCR-positive BAL fluid had a worse 1-year survival rate than those with IFA-negative/qPCR-negative results (P < 10 ؊3 ), in contrast with solid-organ transplant recipients (P ‫؍‬ 0.88) and patients with hematological malignancy (P ‫؍‬ 0.26). Quantifying P. jirovecii DNA in BAL fluids independently of IFA positivity should be incorporated into the investigation of pneumonia in immunocompromised patients. The relevant threshold remains to be determined and may vary according to the underlying disease.

Combined Quantification of Pulmonary Pneumocystis jirovecii DNA and Serum (1→3)-β- d -Glucan for Differential Diagnosis of Pneumocystis Pneumonia and Pneumocystis Colonization

“…Using an upper cutoff value of 1,900 trophic form equivalents/ml and a lower cutoff value of 120 trophic form equivalents/ml, this qPCR assay showed specificity and sensitivity of 100% for PCP diagnosis. Botterel et al determined P. jirovecii DNA copy numbers in BAL fluid samples using a qPCR assay also targeting the mtLSU rRNA gene (12). Using an upper cutoff value of 4 log 10 copies/l and a lower cutoff value of 2.6 log 10 copies/l, this qPCR assay showed specificity and sensitivity of 100% for PCP diagnosis.…”

“…Some of those studies determined cutoff values for P. jirovecii DNA copy numbers in order to differentiate PCP from Pneumocystis colonization (5-12, 26). However, cutoff values differed from one study to another due to methodological differences, as various genomic targets, such as the major surface glycoprotein (MSG) gene (5,6,8,9,11), the heat shock protein 70 (HSP70) gene (7), the dihydropteroate synthase (DHPS) gene (26), and the mtLSU rRNA gene (10,12), were amplified. The mtLSU rRNA gene was used in the present study because (i) it is a multicopy gene in the P. jirovecii genome that allows highly sensitive detection, (ii) it has been routinely used for P. jirovecii detection with a plus/minus PCR assay in our laboratory for several years, and (iii) it is the target most commonly used for P. jirovecii detection worldwide.…”

“…Applying these cutoff values, sensitivity and specificity values for PCP diagnosis ranged from 88% to 100% and from 85% to 98.6%, respectively, depending on the methods. Other teams have suggested applying two qPCR cutoff values in order to increase both sensitivity and specificity (9)(10)(11)(12). In this context, a P. jirovecii DNA copy number below the lower cutoff value would support a diagnosis of colonization, whereas a DNA copy number above the upper cutoff value would support a diagnosis of PCP.…”

Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

“…Indeed, in addition to avoiding false-positive results, qPCR offers the possibility of quantifying the fungal burden in the considered sample, and previous studies have shown that infected patients harbor higher fungal burdens than those who are only colonized (12,13). However, the interpretation of qPCR results remains challenging, since precise cutoffs to distinguish between these patients have rarely been calculated (13)(14)(15)(16)(17). Moreover, these cutoffs are difficult to compare, since either multicopy (mtLSU or MSG) or single-copy genes (DHPS or HSP70) were targeted with different technologies (FRET or TaqMan) in those studies (13,14,16,18,19).…”

A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

“…Clinical diagnosis of PCP is based mainly on microscopic procedures performed directly on the clinical sample and usually involves immunofluorescence microscopy (IFA) or use of Grocott's methenamine silver stain (9,10). Recently, some reports have described the utility of real-time PCR (rt-PCR) protocols to increase the sensitivity and specificity of PCP diagnosis (11)(12)(13)(14)(15)(16)(17), and some of these tests have been commercialized (18). Moreover, a recent study showed the utility of rt-PCR assays for the early diagnosis and treatment of non-HIVinfected patients diagnosed with PCP, for whom a definitive diagnosis could be performed using rt-PCR techniques that detected the low fungal burden present in the patients (19).…”