Serum-based diagnosis of Pneumocystis pneumonia by detection of Pneumocystis jirovecii DNA and 1,3-β-D-glucan in HIV-infected patients: a retrospective case control study

Abstract

Background: Pneumocystis jirovecii pneumonia (PCP) is one of the most common HIV-related opportunistic infections. The diagnosis of PCP is based on analyses from respiratory tract specimens which may require the invasive procedure of a diagnostic bronchoscopy. The objective of this study was to evaluate the diagnostic potential of Pneumocystis jirovecii PCR in serum combined with the 1,3-β-D-glucan (betaglucan) test for the diagnosis of PCP in HIV-infected patients.

Methods: This was a retrospective case-control study including serum samples from 26 HIV-infected patients with PCP collected within 5 days prior to the start of PCP treatment, 21 HIV-infected control subjects matched by blood CD4⁺ cell counts, and 18 blood donors. The serum samples were analyzed for Pneumocystis jirovecii PCR and betaglucan. The reference standard for PCP was based on previously described microbiological and clinical criteria.

Results: All patients with PCP had detectabe Pneumocystis jirovecii DNA in serum yielding a sensitivity for the Pneumocystis jirovecii PCR assay in serum of 100%. All blood donors had negative Pneumocystis PCR in serum. The specificity when testing HIV-infected patients was 71%, but with a PCR Cycle threshold (Ct) value of 34 as cut-off the specificity was 90%. At a putative pretest probaility of 20%, the negative and positive predictive value for the Pneumocystis PCR assay in serum was 0.99 and 0.71, respectively. Betaglucan with cut-off level 200 pg/ml combined with a positive Pneumocystis jirovecii PCR result had sensitivity and specificity of 92 and 90%, respectively. The concentration of Pneumocystis jirovecii DNA in serum samples, expressed by the PCR Ct values, correlated inversely to the betaglucan levels in serum.

Conclusion: In this case-control study including 70% of all HIV-infected patients with PCP treated at Sahlgrenska University Hospital during a time period of 13 years, Pneumocystis PCR analysis on serum samples had a very high sensitivity and negative predictive value for the diagnosis of PCP in HIV-infected patients. A serum-based diagnostic procedure either based on Pneumocystis jirovecii PCR alone or in combination with betaglucan analysis may thus be feasible and would facilitate the care of HIV-infected patients with suspected PCP.

Keywords: 1,3-beta-d-glucan; AIDS; Diagnosis; HIV; Pneumocystis jirovecii; Pneumocystis pneumonia; Polymerase chain reaction.

Fig. 1

Correlation of Betaglucan and PCR Ct values in serum. Values of betaglucan and PCR Ct in serum from 29 patients with positive Pj PCR in serum samples. Black dots represent patients with PCP (n = 23), and red dots represent HIV-infected controls (n = 6). Three patients with 1:2 diluted serum samples out of which one also had inconclusive BG result were excluded. Correlation was assessed by Spearman’s correlation coefficient: r = − 0.5 (− 0.7 to − 0.1), p < 0.01

Fig. 2

PCR Ct values and Betaglucan levels in serum in patients with severe and mild PCP and in HIV-infected control subjects. Ct values (a) and Betaglucan levels (b) with medians and interquartile ranges in serum samples obtained from patients with severe, mild and no Pneumocystis pneumonia (PCP). Medians were compared by Mann Whitney U-test. Three PCP patients whose serum samples had been analysed after 1:2 dilution (a) and one patient whose serum sample was not analyzed for BG due to insufficient volume of serum (b) were excluded