Available data on the incidence and outcome of invasive fungal diseases (IFD) in children treated for a hematological malignancy or undergoing allogeneic hematopoietic stem cell transplantation (HSCT) are mostly based on single site, retrospective studies or on studies performed prior to the availability of newer compounds such as broad-spectrum triazoles or echinocandins. Single site studies have a high likelihood to be biased by factors such as local epidemiology and local strategies to use available diagnostic tools and to initiate antifungal agents, and therefore, data and conclusions may not be inferred and applied to other centers. In addition, the introduction of newer diagnostic tests and new antifungal agents, as well as the refinement of algorithms in the supportive care in the pediatric setting may have changed the epidemiology and outcome of IFDs over the last decade (Groll et al., 2014; Lehrnbecher et al., 2017a,b). Unfortunately, data from adult studies cannot be transferred to the pediatric population, as children have different underlying malignancies with unique biology, treatments and outcomes, and lack most of the age-dependent comorbidities observed in adults (Sung et al., 2011).

For these reasons, we initiated a prospective study in three major pediatric cancer centers to obtain contemporary data of incidence and outcome of IFDs in children treated for a hematological malignancy or undergoing allogeneic HSCT. We also included specific data on diagnostics and the use of antifungal agents, as they have major impact on the study endpoints.

All children and adolescents less than 18 years of age who were diagnosed between April 1, 2014 and March 31, 2016 with de novo acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), relapse of acute leukemia, non-Hodgkin lymphoma (NHL), or undergoing allogeneic HSCT were included in this prospective observational study (DRKS00006341). Patients were treated according to Berlin-Frankfurt-Münster (BFM)-based protocols (e.g., AIEOP-BFM ALL 2009, AML-BFM 2012 registry, ALL-REZ BFM registry, or B-NHL 2013) at the University Children’s Hospitals of Frankfurt and Münster, Germany, or at the St. Anna Children’s Hospital, Vienna, Austria. Patients could be enrolled several times in the study if they received different treatments within the study period [e.g., a patient who received chemotherapeutic treatment (usually consisting of several chemotherapy cycles) for de novo ALL, followed by relapse chemotherapy and HSCT could potentially be included three times]. The study was reviewed and approved by the local Ethical committees of all three participating centers (Frankfurt: 348/13; Münster: 2014-048-b-S; Vienna: 17-6-2014).

According to current pediatric guidelines, all centers routinely performed diagnostics such as imaging studies in children at highest risk for IFD, e.g., persistently febrile neutropenic patients not responding to broad-spectrum antibiotics after 96 h or patients with any clinical sign or symptom consistent with IFD (Groll et al., 2014; Lehrnbecher et al., 2017b). These patients also received empirical or pre-emptive mold-active antifungal therapy, respectively. Regarding antifungal prophylaxis, no common policy was adopted by the participating centers, and prophylaxis was instituted according to local standard operating procedures.

Data collection was performed using an electronic database (secuTrial^®), and included demographic data, disease characteristics, data on laboratory diagnostic studies and imaging as well as information on antifungal drug use. Data were collected for each cycle of intensive chemotherapy, defined by the first day of chemotherapy until the start of the next cycle of chemotherapy, and for patients undergoing HSCT from the start of the conditioning regimen until at least day +100. Last follow-up was after 12 months after the end of intensive chemotherapy or the date of allogeneic HSCT and included the current status on relapse/disease-free survival, death (including cause of death) and, in case of the occurrence of an IFD, the outcome of this infection.

Contemporary data on epidemiology, treatment and outcome of IFD in children with hematological malignancies or undergoing allogeneic HSCT are important as they may help to improve antifungal management (Pana et al., 2017). Whereas our data on the incidence of IFDs in the different subgroups corroborate the results of a recent multicenter analysis (Cesaro et al., 2017), several other studies reported on higher incidences (Hovi et al., 2000; Castagnola et al., 2006; Sung et al., 2007; Hale et al., 2010). These differences may be explained not only by local epidemiology, but also by the fact that studies often included “suspected” fungal infections in their analysis. We considered only the 19 patients with proven and probable IFDs for the calculation of incidence rates, but excluded the 23 additional patients with possible IFDs. The relatively high percentage of possible IFD in our analysis as compared to previous data may be due to the fact that rigorous imaging studies were performed if signs or symptoms were indicating the potential presence of an IFD, although microbiological tests remained negative. The information on the use of diagnostic tools for the analysis of the epidemiology of IFD is important, which is supported by the observation that the number of CT scans performed in each center positively correlated with the rate of possible IFD. Similarly, the information whether patients received antifungal prophylaxis or not is essential for the interpretation of reported incidence rates of IFDs, but is unfortunately not reported by all studies.

Although patients with ALL are at a lower risk for IFDs compared to children with AML or leukemia relapse, it is important to analyze this patient population in more detail for several reasons. First, in pediatric cancer patients, this entity comprises the largest group of patients at risk for IFDs, and second, the largest absolute number of IFDs is diagnosed in pediatric ALL patients (Cesaro et al., 2017). Whereas most studies do not discriminate between the different risk groups of children with ALL who receive different treatment intensities which influence the risk for IFDs, Hale et al. (2010) reported on a considerably lower incidence of IFDs in “low-risk” ALL, and no invasive mold infection occurred in these “low-risk” ALL patients. In contrast, we found 3 out of 52 “low-risk” ALL patients with invasive mold infection, who also comprised half of the 6 ALL-patients in whom this infection was diagnosed. Notably, significant differences were observed in ALL-SR and ALL-MR patients in the initiation and duration of antifungal prophylaxis and in the choice of the antifungal compound. For example, as compared to Vienna, antifungal prophylaxis was initiated in significantly less ALL-SR and ALL-MR patients treated in Frankfurt, but, on the other hand, the duration of antifungal prophylaxis was significantly longer in Frankfurt as compared to Vienna. Therefore, one is prompted to speculate that both indication and duration of antifungal prophylaxis could be optimized. Unfortunately, it has not fully been clarified how to predict the individual risk for IFDs in ALL-SR and ALL-MR patients. However, corroborating the findings of a recent meta-analysis (Fisher et al., 2017), our study demonstrates that patients with invasive mold infection were significant older than patients without IFDs, and that all IFDs occurred during phases of chemotherapy phases where patients had profound and prolonged neutropenia or received prolonged high-dose corticosteroid therapy. Based on these findings, the site in Frankfurt has recently modified its antifungal strategy and administers antifungal prophylaxis during the phases of induction and re-induction to all ALL-SR and ALL-MR patients that are older than 10 years. Whether this will decrease the rates of IFDs and will be cost effective is the focus of a planned audit. The wide variety between the centers in the choice of the antifungal compounds reflects the uncertainties in the pediatric setting, in particular in children with ALL. In these patients, the use of broad-spectrum triazoles such as voriconazole (approved for children older than 2 years) or posaconazole (no pediatric label within the European Union) is limited due to the interaction with vinca-alkaloids, which are a cornerstone in the treatment of ALL. On the other hand, amphotericin B formulations and the new class of echinocandins, which are not approved for anti-mold prophylaxis, have to be administered intravenously on a daily basis. Whether intermittent dosing of these compounds may become a suitable option needs to be explored in future studies (Bochennek et al., 2011, 2015).

Our analysis demonstrated a favorable response (CR or PR) in 15 patients (80%), whereas 3 deaths were caused by IFD (15.8%), all of them occurring in the pre-engraftment period of patients undergoing HSCT. The substantially lower mortality relative to the reports of previous, older studies is most likely due to the improvement of supportive care, including newer diagnostic tools and the availability of new, versatile and potent antifungal compounds (Hovi et al., 2000; Castagnola et al., 2006).

We recognize that our study has several limitations, including the lack of uniform operating procedures for diagnostics and management which, however, is inherent to any epidemiological study. Due to the relatively small number of patients with proven and probable IFD we did not perform a multivariate analysis of risk factors. On the other hand, our analysis has unique and important strengths. For example, the prospectively collected data of three major pediatric cancer centers provide information on the current incidence and outcome of IFD in2 the real-life setting outside prospective randomized trials on antifungal management. In addition, different to previous studies, our analysis includes detailed data on the use of diagnostic tests and the use of prophylactic antifungal agents, which is a prerequisite for interpreting the risk of IFDs. Finally, differences between the centers in both the initiation and cessation of antifungal prophylaxis identify an important research gap, and allow a preliminary comparative analysis, although the conclusions might be limited by not pre-defined strategies.

In conclusion, this prospective multicenter study confirms that children treated for ALL, AML or undergoing HSCT have a significant risk for IFDs, and the risk is individually determined by factors such as age or treatment course. IFD-related mortality was low, in particular in patients receiving chemotherapy. Differences between the sites in the use of imaging diagnostics and antifungal prophylaxis may help to identify approaches to improve the antifungal management in particular of children with ALL, the largest at risk population in pediatric cancer medicine.

The Ethical committees of each participating center (Frankfurt, Münster, Vienna) have reviewed and approved the study (approval numbers 348/13 for Frankfurt, 2014-048-b-S for Münster, and 17-6-201 for Vienna). Patients were included in the analysis if they and/or their caregivers had given written informed consent.

All authors have significantly contributed to the manuscript by designing the study (TL, SS, AA, and AG), collecting the data (FP, JG, AB, and KG), and analyzing the data (all authors). All authors have reviewed and agreed upon the manuscript content.

TL has received research grants from Gilead Sciences, is a consultant to Astellas, Basilea, Gilead Sciences, and Merck/MSD, and served at the speaker’s bureau of Astellas, Gilead Sciences, Merck/MSD, and Pfizer. AA has a consulting and advisory role for Jazz Pharmaceuticals and received travel and accommodations expenses from Jazz Pharmaceuticals, Amgen, and Pfizer. AG has received grants from Gilead, Merck, Sharp and Dohme, Pfizer, and Schering-Plough; is a consultant to Amplyx, Astellas, Basilea, Gilead, Merck, Sharp and Dohme, and Schering-Plough, and served at the speakers’ bureau of Astellas, Basilea, Gilead, Merck, Sharp and Dohme, Pfizer, Schering-Plough, and Zeneus/Cephalon. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.