Diagnostic accuracy of (1→3)-β-D-glucan to predict Pneumocystis jirovecii pneumonia in non-HIV-infected patients

Pneumocystis jirovecii is a cause of Pneumocystis pneumonia (PCP) in immunocompromised patients. PCP most often occurs in human immunodeficiency virus (HIV) infected patients associated with high pathogen burdens but also in non-HIV immunocompromised patients.1, 2, 3 The diagnosis is mainly based on clinical and radiographic examinations, which are in majority of cases inconclusive, so microscopic or molecular detection of P. jirovecii in lower respiratory tract samples are necessary.2, 3 The mechanism by which Pneumocystis organisms induce lung inflammation remains incompletely understood, but studies imply that Pneumocystis spp. cell wall constituent (1→3)-β-D-glucans are released and contribute to the development of extensive lung inflammatory reaction.4, 5, 6, 7, 8, 9, 10 Various studies have reported high serum (1→3)-β-D-glucan levels in patients with PCP.7, 8, 9, 10, 11, 12, 13, 14, 15 The (1→3)-β-D-glucan has high sensitivity for PCP but, it is a not species-specific marker for P. jirovecii.2,11,13,15

Detection of P. jiroveci DNA in respiratory tract specimens without signs and symptoms of PCP is defined as colonization. Molecular diagnostic techniques like quantitative PCR (qPCR) are more sensitive than staining methods like direct immunofluorescence method (DIF). However, qPCR can give sometimes false signal that patient has PCP, although patient is just colonized with fungi.16, 17, 18, 19, 20, 21 Studies have reported prevalence of colonization in susceptible adults from 10.0% to 43.8%.17, 18, 19, 20, 21, 22, 23 Real-time qPCR assays have been shown as most accurate for discrimination of true PCP from colonization with P. jirovecii. Flori et al. compared the performance of standard staining, standard PCR, and qPCR using 173 BAL specimens from 150 patients. Real-time qPCR gave the best results.24

A systematic review, that evaluated PCR kits to confirm PCP, revealed that positive PCR results do not always confirm PCP. Low amounts of DNA can be expected in patients with P. jirovecii colonization of and no clinical signs of PCP.25

(1→3)-β-D-glucan can be helpful to distinguish PCP from P. jirovecii colonization. However, not as standalone test. (1→3)-β-D-glucan is positive in colonization of gastrointestinal tract with candida. It is positive when patients are colonized with P. jirovecii.13,16 Combining best tests is a advisable in immunocompromised cancer patients to be able to treat patients with the right therapy.

We designed a retrospective multicentric study that included patients from 3 tertiary care hospitals and 2 secondary care hospitals. The objective of our study was to distinguish true PCP from colonization with P. jirovecii with diagnostic algorithm that includes measurement of (1→3)-β-D-glucan and real-time PCR (qPCR).

Our experience with (1→3)-β-D-glucan and qPCR indicated that cut-off of > 400 pg/mL for (1→3)-β-D-glucan could be used to predict micro-biologically confirmed PCP with greatest statistical significance. Results of the analysis indicated that colonization is possible when levels of (1→3)-β-D-glucan are moderate, in our case < 400 pg/mL, and low concentrations of P. jirovecii DNA with qPCR are detected, in our case > 35 cycles.

Fifty percent of the patients in our study had (1→3)-β-D-glucan > 500 pg/mL and mean qPCR Ct 28.9 ± 5.18. The second largest group of patients (24 patients) with possible PCP were those with (1→3)-β-D-glucan < 100 pg/mL, and mean qPCR Ct 35.43 ± 3.51. Patients with microbiologically confirmed PCP had mean (1→3)-β-D-glucan of 467.5 ± 95.5 and mean qPCr Ct 27.2 ± 4.6. Even more, true PCP where patients with it, had response to therapy, can be predicted when cut-off for (1→3)-β-D-glucan is set to 496.45 pg/ml. At this cut-off, AUC to predict PCP was 0.817.

Similar results were produced in recent studies.26,27 In an older study cut-off value of 340 copies/ mL was used to discriminate probable PCP from colonization. At this qPCR cut-off, (1→3)-β-D-glucan levels were significantly higher in patients with both definite PCP and probable PCP than in colonized patients.26 It was recently shown that serum (1→3)-β-D-glucan levels of 143 pg/mL can be used to distinguish PCP from colonisation with P. jirovecii.28 To diagnose and treat PCP you have to have a lot of experience.29 Our results were not concordant with latter studies because we measured much higher (1→3)-β-D-glucan levels in the patients with true PCP. The clinicians in majority of included institutions seldomly get the chance to treat PCP and are not experienced enough.

When PCR was introduced, it remarkably increased sensitivity to diagnose PCP, resulting in an increase in the number of documented PCP cases.30, 31, 32, 33, 34 On the other hand multiple evidences indicate that P. jirovecii can colonize the mucosal epithelium of both healthy individuals and those with compromised immunity.34, 35, 36 Results of qPCR in the area where differentiation between PCP and colonization is demanding, if we look at them separately from (1→3)-β-D-glucan. It was suggested that every (1→3)-β-D-glucan values should be interpreted with qPCR and vice versa in patients with possible PCP.16,26,29

Montesinos et al. also showed with their in-house PCR test cut-off value Ct = 34 to discriminate definite PCP from unlikely PCP with 65% sensitivity and 85% specificity.37 Other studies have suggested applying two qPCR cut-off values to increase sensitivity and specificity of qPCR. There is a grey zone of unclear clinical significance between these two cut-off values where the differential diagnosis of PCP versus colonization cannot be determined.16,24,38, 39, 40 In recent literature two cut-offs were proposed to predict definite PCP. The two cut-offs for qPCR provide 100% sensitivity and 100% specificity to diagnosis definite PCP. The higher cut-off value represents the value below which the diagnosis of PCP is unlikely. The authors suggested that the range between these two cut-off values represents an indeterminate zone.16,39,41, 42

According to our study, P. jirovecii DNA copy number above the cut-off value Ct ≥35 would support a diagnosis of colonization, and a copy number below the cut-off value would support a diagnosis of PCP. However, good sensitivity of qPCR can be misleading, since qPCR detects very low fungal load that can lead to over-diagnosis of PCP due to misinterpretation of findings.33,42 Our cut-off was determined with the help of (1→3)-β-D-glucan, which is a good indicator for PCP if the values are high and we detect a lot of P. jirovecii DNA in lower respiratory tract.

Our study had some limitations. We could not gather the information that treatment with antimicrobials helped patients which would confirm that patients really had PCP for all included individuals. The influence of different samples from lower respiratory tract on qPCR was not assessed. Although our qPCR displayed high accuracy for discriminating colonization from PCP, the cut-off values used in our study should be standardized to copies/ml and compared to other qPCRs. The cutoff values for (1→3)-β-D-glucan was not always measured the same day as qPCR was done which could influence on the concentration of (1→3)-β-D-glucan. For future studies, patients that have their clinical picture and response to therapy consistent with possible PCP, and are randomized according to the analysed samples, should be included to resolve the discrepancy seen in our study.

The protocol we presented here could better support clinicians in their decisions whether patient in front of them has PCP. Our diagnostic cut- off values allow identification of true PCP versus colonization with P. jirovecii with better diagnostic accuracy. However, according to our results qPCR cannot be used alone to confirm PCP. PCP has to be confirmed with qPCR and with (1→3)-β-D-glucan.