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Pneumocystis jirovecii (carinii) pneumonia (PJP) is a major cause of mortality in immunocompromised individuals. Therefore, early diagnosis and therapy is vital.
Currently, the laboratory reference standard for the detection of P. jirovecii is staining of the organism with monoclonal antibodies, followed by immunofluorescence microscopy in bronchoalveolar lavage (BAL) fluid. Together with a compatible clinical presentation and confirmation by computed tomography, this allows the diagnosis to be made. However, diagnosis of PJP is hampered by the fact that the performance of BAL is not always possible, owing to the limited respiratory function of some patients. Furthermore, detection of P. jirovecii is not necessarily equivalent to infection, because of the existence of clinically insignificant P. jirovecii colonization [1,2]. Until recently, no reliable serological parameters for the diagnosis of PJP were available.
(13)-β-d-Glucan (BG) is a cell wall component of P. jirovecii and of various other fungi. During the course of an invasive infection, BG is released into the serum. Data collected over recent years have shown that serum BG measurement might be a powerful new tool for the diagnosis of PJP (Table 1). However, in these studies various test systems with different characteristics have been used, and direct comparison of the reported observations is not possible. The Fungitell assay is a Limulus amebocyte lysate-based test for the measurement of BG. So far, there have been only two retrospective studies with sizeable numbers of patients (20 and 28, respectively) and a relevant control group that have used the Fungitell assay for the diagnosis of PJP [3,4]. Furthermore, information on the prognostic benefit of BG measurement is limited, and results are inconclusive [5–8].
Table 1. Published studies investigating (13)-β-d-glucan (BG) levels of patients with Pneumocystis jirovecii pneumonia
|Author, year [Reference]||Number of PJP patients||Immunosuppression of the PJP group||Median BG level (pg/mL)||Number of control patients||Immunosuppression of the control group||Sensitivity||Specificity||Reference method||Test system||Cut-off (pg/mL)|
|Desmet, 2009 ||16 12||HIV Non-HIV||1496 3779||16 12||HIV Non-HIV||100||96.4||PCR + IFS/microscopy||Fungitell||100|
|Watanabe, 2009 ||111||HIV||175||425||NS||96.4||87.8||Microscopy||Fungitec G||23.2|
|Del Bono, 2009 ||16||Various||423||15 11||Various None||–||–||Clinical diagnosis||Fungitell||80|
|Nakamura, 2009 ||19 16||HIV Non-HIV||300 85.4||24||Bacterial pneumonia||100 88||NS||Microscopy + PCR||Fungitec G||20|
|Persat, 2008 ||16 4||HIV Non-HIV||945||40 120||None Risk of IFI||100||NS||Microscopy||Fungitell||80|
|Marty, 2007 ||16||Various||>500||None||–||93.8||–||IFS||Fungitell||NS|
|Tasaka, 2007 ||57||Various||NS||222||NS||92.3||86.1||Microscopy||β-Glucan test (WAKO)||31.1|
|Fujii, 2007 ||28||HIV||147||None||–||96.8||–||NS||NS||5|
|Iikuni, 2006 ||21||CTD||87.9||45||Various||77.8||76.9||PCR||NS||NS|
|Shimizu, 2005 ||15||CTD||NS||None||–||86.7||–||Microscopy and/or PCR||Fungitec G||20|
We therefore conducted a retrospective study on 50 patients with PJP and 50 control patients to evaluate the diagnostic performance of serum BG measurement, with a special focus on its prognostic relevance.
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We have conducted a retrospective study with the Fungitell assay to evaluate the usefulness of serum BG measurement for the diagnosis of PJP. Our results show an excellent diagnostic performance, with a sensitivity of 98% and a specificity of 94%, and thereby confirm the data of previous studies. The positive predictive value was only 64.7%, possibly because BG is influenced by a number of as yet unknown confounding factors, and because BG is elevated not only in PJP but also in most other invasive fungal infections. As a consequence, BG levels alone cannot prove the existence of PJP, and they must not be interpreted without considering the clinical findings. However, with a negative predictive value of 99.8%, a BG level ≤85 pg/mL almost rules out PJP, and this could be extremely helpful in patients who cannot undergo bronchoscopy or in patients where the clinical suspicion of PJP is low.
Usually, there is a significant delay between the onset of symptoms and the diagnosis of PJP. Median intervals are reported to be 21–28 days in HIV-positive patients . In our study, BG levels were already strongly elevated at an average of 5 days before microbiological diagnosis. For the majority of patients, there was only one pre-existing serum available, and the median of serum sampling was at day 7. Therefore, it is unclear exactly when BG levels exceeded the cut-off. The average time-span of 5 days between BG positivity and microbiological diagnosis is most likely an underestimation caused by a lack of samples dating further back. So far, our data suggest that BG levels are elevated up to 21 days before microbiological diagnosis, and timely BG measurement could allow for the earlier initiation of effective treatment (Fig. 1).
Figure 1. (13)-β-d-Glucan (BG) kinetics of a patient with Pneumocystis jirovecii (carinii) pneumonia in whom timely measurement of BG could have prevented a significant delay in initiation of effective therapy. A 26-year-old female with acute lymphoblastic leukaemia underwent haematopoietic stem cell transplantation in December 2005. On 29 May 2006 (day −5), she presented with fever, dyspnoea and non-productive cough. The patient was on trimethoprim–sulphamethoxazole (SXT) prophylaxis. Chest X-ray showed bilateral infiltrates suggestive of atypical pneumonia. Serum Aspergillus galactomannan testing gave negative findings. Therapy with clarithromycin was initiated but showed no effect. The patient was re-admitted to the hospital on day −3. A computed tomography scan showed progression of the infiltrates, and therapy was changed to meropenem, voriconazole and foscavir. Bronchoscopy was performed on day 0, and P. jirovecii cysts were detected by specific immunofluorescence and PCR. Intravenous trimethoprim–sulphamethoxazole was started, with good treatment response. The subsequent clinical course was uneventful, and the patient was discharged on day 23. Retrospectively, BG levels were already highly elevated (836 pg/mL) on day −5, and a steady decrease in BG levels reflected a good clinical response. The red line indicates the cut-off (85 pg/mL). BAL, bronchoalveolar lavage; CL, clarithromycin; F, foscavir; M, meropenem; Pj, P. jirovecii; V, voriconazole.
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Data on the correlation between BG levels and the outcomes of patients are limited, and the results are inconclusive. Two studies reported that high levels of BG are associated with poor prognosis [5,6], whereas two other studies could not find a significant difference in BG levels between survivors and non-survivors [7,8]. The analysis of our data showed no significant difference in the mortality or severity of PJP between patients with high and those with low BG levels. These findings are supported by our observation that there was no correlation between pulmonary P. jirovecii burden, as detected by quantitative real-time PCR, and serum BG levels. However, the results of real-time PCR on BAL fluids are strongly dependent on the quality of the material, and we cannot rule out the possibility that suboptimal sampling affected the data quality.
Although the absolute BG level at diagnosis and the maximum BG level during hospitalization do not seem to have prognostic relevance, this might be different for BG kinetics. It has been shown in individual cases that a favourable outcome can be paralleled by decreasing BG levels , an observation that is supported by our own experience. However, further work is clearly necessary to confirm these reports in larger series.
The severity and mortality of PJP, as well as BG levels, have been reported to be lower in HIV-positive patients than in non-HIV patients . We did not observe a significant difference in the BG levels between those two groups of patients. However, subgroup analysis of patients with different underlying diseases showed significantly higher BG levels in organ transplant recipients. At the same time, organ transplant recipients and patients with high BG levels in general showed higher creatinine levels, whereas liver enzyme levels were not elevated. The route of BG elimination is still unknown. In rabbit models, 80% of the organ-associated BG was found in the liver and 10% in the kidney. BG was detected in organs, blood and urine at 23%, 18% and 10%, respectively . Together with the observation that elimination of BG in dialysis patients takes much longer than in healthy subjects, this indicates that renal clearance seems to play some role . However, another study found that the median plasma half-life of BG in dialysis patients was approximately 20 h (range 3.1–181.3 h) and was not affected by renal or hepatic impairment . Therefore, a direct correlation between high BG levels and impaired renal function is hypothetical, and the reason for our observation remains unclear.
In accordance with the findings of previous studies, the diagnostic potential of serum PCR—irrespective of whether touchdown PCR or real-time PCR was used—is poor, and it cannot be recommended for the diagnosis of PJP.
In summary, serum BG levels in patients with PJP are strongly elevated and the negative predictive value is high. Therefore, measurement of BG could be used as a preliminary test for patients with suspected PJP before BAL is performed. Neither the outcome nor the pulmonary P. jirovecii burden seems to correlate with the serum BG level at diagnosis or with the peak BG level. Accordingly, the prognostic value of single BG measurements in PJP is low. BG levels start to increase up to 21 days before microbiological diagnosis, and this could be helpful in reducing the time until adequate treatment is initiated.