Incidence and characteristics of invasive fungal diseases in allogeneic hematopoietic stem cell transplant recipients: a retrospective cohort study

Ethical approval for this study was obtained from the local Ethics Committee of the Medical University of Vienna (No. 982/2011). All adult patients receiving an allogeneic HSCT at the Bone Marrow Transplant Unit of the University Hospital of Vienna from January 2009 to December 2013 were enrolled. Their medical charts were reviewed retrospectively and follow-up data including information on mortality was obtained in January 2015. For those patients who received multiple transplants during the study period, only the most recent HSCT was included in the analysis. The patients’ demographic characteristics, details about the transplantation including donor, type of graft, conditioning regimen, and the post-transplant follow-up including the severity of GvHD [17], post-transplant complications as well as the use of prophylaxis and antifungal therapy were entered into a database. The transplantation risk for each patient was calculated using the European Society for Blood and Marrow Transplantation (EBMT) risk score which includes age at HSCT, disease stage, time from diagnosis to transplantation, donor type and donor recipient sex combination [18].

Patients received peripheral blood stem cells (PBSC), bone marrow or cord blood as graft from HLA-matched related donors, matched unrelated donors (12/12 alleles) or mismatched unrelated donors (11/12 alleles). The myeloablative regimen consisted of cyclophosphamide and total body irradiation, while several different reduced-intensity conditioning (RIC) regimens were used. Acute GvHD prophylaxis included cyclosporine A (CSA) and methotrexate after myeloablative conditioning and CSA and mycophenolate mofetil after RIC. All patients were isolated in negative pressure rooms until sufficient engraftment was achieved. Systemic antifungal prophylaxis was not used generally for all patients during aplasia, but according to their risk profile (e.g. previous IFD) and to the attending clinical physician’s evaluation. Patients who developed fever during aplasia were treated empirically with antifungals if the fever persisted for more than 48 h despite antibiotic therapy.

All patients were screened for invasive aspergillosis using serum galactomannan before HSCT and twice weekly after HSCT until they were discharged. During the follow-up patients were screened with serum galactomannan every two weeks until day +100. In case of fever the standard procedures included blood cultures at two different time points and a multiplex PCR which is capable to detect five different Candida species and Aspergillus fumigatus. Patients presenting with respiratory symptoms were first examined by chest X-ray and in case of infiltrates or worsening gas exchange followed-up with a computed tomography (CT) scan of the lungs. If the CT scan showed pulmonary infiltrates, bronchoscopy was performed and fungal culture, galactomannan and PCR for fungal pathogens including Pneumocystis jirovecii were conducted from the bronchoalveolar lavage fluid [19]. Transbronchial biopsy during bronchoscopy or CT-guided biopsy of suspected liver lesions was performed in some cases. Patients with neurological symptoms received magnetic resonance imaging (MRI) of the brain and lumbar puncture including culture and PCR for different fungal pathogens.

All microbiological specimens were analysed for fungal pathogens by the department of clinical microbiology of the Medical University of Vienna. Blood cultures were incubated in the BacTAlert® (BioMerieux, France) for fourteen days and if a positive signal was obtained, a gram stain and a subculture on Sabouraud Glucose Agar (SAB) and Chromagar Candida® (Becton Dickinson, Heidelberg, Germany) were performed. Further identification of yeasts was done using either the Vitek® system or ATB Candida ID32C® (both BioMerieux, France). Also, rice-extract agar was inoculated and incubated at 28 °C for the formation and examination of micromorphology. Other specimens such as sputum or bronchoalveolar lavage fluid were inoculated on Sabouraud Glucose Agar (SAB) and Chromagar Candida® and incubated at 35 °C and 21 °C for seven days. In addition a SAB-broth was inoculated for enrichment. Identification of moulds was performed by examining macro- and micromorphology. In case identification was impossible using conventional methods, sequence analysis was suspended. In addition, the multiplex PCR Septifast (Roche, USA) was used for more rapid detection of certain Candida species and Aspergillus fumigatus. Aspergillus antigen was determined by using the serum galactomannan ELISA assay (Bio-Rad, Hercules, CA, USA) with a cut-off value for the optical density index of ≥0.5 for serum and ≥0.8 for BAL [9, 20]. For the identification of Pneumocystis jirovecii real-time PCR from BAL was performed [21, 22]. In some cases fungi were identified during autopsy or histological examination of tissues by direct microscopy by the department of pathology.

The primary end-point for this study was the one-year incidence for proven or probable invasive fungal diseases. IFDs were classified as probable or proven according to the revised definitions of the EORTC/MSG consensus group [9]. The secondary end-points for this study were the analysis of risk factors for IFD, the associated mortality and the impact of prophylaxis in preventing IFD.

Statistical analysis was performed using SPSS 22 (IBM, Armonk US). All end-points were analysed using descriptive statistics: values were expressed as median and range for continuous variables and as percentages for categorical variables. Differences in patient’s characteristics were compared between groups with and without IFD and with and without aspergillosis. To explore possible risk factors for IFDs univariate and multivariate logistic regression analyses were performed. In addition, a sub-analysis for invasive aspergillosis was conducted excluding other IFDs. Risk factors with a p-value ≤ 0.05 in the univariate analysis were included in the multivariate model using the “Forward Wald method”. For the multivariate model a two-tailed p-value ≤ 0.05 was considered to be significant. Survival analysis was performed with Kaplan-Meier survival curves and the log-rank test.

During the study period a total of 242 patients received 255 HSCTs – 11 patients had two and one patient 3 transplants. The median age was 46 years (range 19; 70 years) at the time of HSCT with 130 males (53.7 %) and 112 females (46.3 %). Underlying diseases were acute leukemia (71.9 %), chronic leukemia (6.2 %), lymphoma (11.2 %), myelodysplastic syndrome (4.1 %), aplastic anaemia (2.1 %), multiple myeloma (2.1 %) and other haematological diseases (2.4 %). Details concerning the EBMT risk score of patients, type of grafts, donors and conditioning regimens are included in Table 1.

During the study period a total of 28 patients experienced an IFD (11.6 %), 25 of them (89.3 %) during the first year after HSCT. Therefore, the one-year incidence for IFD was 10.3 % (25/242), with peaks in the years 2010 (6/42, 14.3 %) and 2012 (6/44, 13.6 %), an average incidence in the years 2009 (5/53, 9.4 %) and 2013 (6/61, 9.8 %) and the lowest incidence in the year 2011 (2/42, 4.8 %).

Among IFDs invasive aspergillosis was predominant (18/28, 64.3 %) with 17 pulmonary and one cerebral aspergillosis. Second came invasive candidiasis (7/28, 25 %) and third pneumocystis pneumonia (3/28, 10.7 %). Eight IFDs (28.6 %) were proven infections and the other 20 IFDs (71.4 %) were classified as probable. Among the proven IFDs the following fungal species were identified: Candida albicans (n = 1), Candida glabrata complex (n = 2), Candida krusei (n = 1), Candida species (n = 1), Aspergillus section fumigati (n = 1) and Aspergillus species (n = 2). Pathogens of probable IFDs included Candida albicans (n = 2), Pneumocystis jirovecii (n = 3), Aspergillus section fumigati (n = 1), Aspergillus section nigri (n = 1), Aspergillus species (n = 1) and 12 cases of invasive aspergillosis diagnosed on basis of a suspicious CT-scan and positive test for galactomannan antigen.

As shown in Fig. 1, the median time from HSCT to the diagnosis of IFD was 8 days for invasive candidiasis, 36 days for invasive aspergillosis and 319 days for pneumocystis pneumonia. Twelve percent of patients (29/242) had already experienced a documented IFD before HSCT and three of those patients also developed an IFD after HSCT. But only in a single case a recurrence of a previous invasive aspergillosis can be assumed.

The univariate logistic regression analyses for all IFDs as well as the sub-analysis for invasive aspergillosis are summarized in Table 1. Among the pre-transplant risk factors, only female sex was predictive for a higher risk for IFD (odds ratio (OR) 2.3, p = 0.05) and lymphoma as underlying disease was associated with a higher risk for invasive aspergillosis (OR 3.4, p = 0.03), whereas an EBMT-score of > 2 was associated with a lower risk for IFD (OR 0.4, p = 0.03). However, all three parameters did not reach significance (p ≤ 0.05) in the multivariate analysis. Intensified salvage therapy for severe GvHD (high-dose corticosteroids (≥1 mg/kg body weight) and basiliximab or etanercept) was a highly significant risk factor for IFD (OR 5.2, p = 0.001) and for invasive aspergillosis (OR 8.7, p < 0.001). Among the post-transplant complications transplant-associated microangiopathy (TMA) was significantly associated with IFD (OR 5.0, p = 0.004) and with invasive aspergillosis (OR 9.2, p < 0.001). Intensified GvHD therapy and TMA were moderately correlated risk factors (Spearman’s rho 0.3, p < 0.0001). Prophylaxis proved only to be significantly protective if it was given during aplasia as well as during post-engraftment (OR 0.3, p = 0.03 for IFD and OR 0.2, p = 0.05 for invasive aspergillosis). In the multivariate model only intensified GvHD therapy and TMA remained significantly associated with an increased risk for IFD and for invasive aspergillosis, while antifungal prophylaxis given during aplasia and post-engraftment was associated with a decreased risk (see Table 2).

The median survival during our study period was 677 days (range 2; 2185) for patients without IFD, 525 days for patients with IFD (range 14; 2030) and 235 days for patients with invasive aspergillosis (range 14; 1699) (see Fig. 2). Thus, the presence of an IFD was associated with a higher mortality rate compared to patients without IFD (48 % vs. 25.3 %, p = 0.02) during the first year after HSCT. The higher mortality rate was even more pronounced for patients with invasive aspergillosis (62.5 %, p = 0.003). Patients with IFD were also admitted to an ICU more often (18/28, 64.3 %) than patients without IFD (26/214, 12.1 %, p < 0.0001).

Systemic antifungal prophylaxis was given to 43.3 % (105/242) of patients during aplasia, with the most commonly used antifungals being posaconazole (52/105, 49.5 %), voriconazole (28/105, 26.7 %) and fluconazole (17/105, 16.2 %). The use of prophylaxis during aplasia increased during our study period from 15.1 % in 2009 to 68.9 % in 2013. Sixty-five percent (158/242) of patients received prophylaxis during post-engraftment.

Thirty-six percent (88/242) of patients received empirical antifungal therapy during aplasia, with caspofungin (44/88, 50 %) as the first choice followed by liposomal amphotericin B (18/88, 20.5 %). In total, 69 % (167/242) of patients received an antifungal agent as either prophylaxis or empirical therapy during aplasia.

Of 25 IFD patients with either invasive aspergillosis or Candida infection, 56 % (14/25) were without prophylaxis at the time of IFD diagnosis. Sixteen percent (4/25) developed an invasive aspergillosis under fluconazole and 28 % (7/25) had mould-active prophylaxis but developed a fungal “breakthrough infection”. Of these 7 cases, 6 patients received posaconazole and suffered from invasive aspergillosis (n = 5) and invasive candidiasis (n = 1), and a single patient developed invasive candidiasis while receiving caspofungin. Overall, 5.3 % (6/113) of patients who received posaconazole prophylaxis at any time during aplasia or post-engraftment developed a “breakthrough” IFD compared to 6.3 % (4/64) of patients under fluconazole prophylaxis.