Detection and Management of Invasive Mold Disease in Pediatric Hematological Cancer Patients

Introduction

Infectious complications still remain a major challenge in the treatment of children and adolescents treated for hematological malignancies. Infections are associated with significant morbidity, decrease the quality of life of both patients and their families, and, although the mortality has significantly decreased over time, patients potentially die of these complications. Infectious complications are also a limiting factor for the intensification of antineoplastic therapy, which, however, may be necessary for cure. In that respect, it has been demonstrated in a recent analysis that in current treatment protocols relapses and therapy-related toxicity equally contribute to poor outcome in children with high-risk acute lymphoblastic leukemia (ALL), indicating that further increasing dose-intensity might be counter-productive and ultimately result in lower overall survival.¹

Pediatric patients receiving intensive conventional therapy for cancer are at particular risk for bacterial and invasive fungal infections, whereas in the setting of hematopoietic cell transplantation (HCT), patients also often suffer from severe viral infections.² Invasive fungal infections are often difficult to diagnose, in particular in the early stage of infection. Compared to adults, diagnostic approaches may perform differently in children. However, over the last decades, the diagnostic tools as well as the antifungal armamentarium have significantly be expanded, all of which might ultimately impact on outcome. This review will focus on invasive infections with molds, such as Aspergillus spp and Mucormycetes, and will critically highlight the current status, the latest development as well as research gaps in diagnosis and therapeutic strategies in the pediatric setting.

Significance, Epidemiology and Risk Factors for Invasive Mold Infection

Due to the complex impairment of their immune system by the underlying malignancy as well as by the therapy (eg, chemotherapy, radiation), children and adolescents with cancer are at risk for invasive fungal disease.^3,4 A retrospective cohort study found a total of 666 cases of invasive aspergillosis among 152,231 immunocompromised children.⁵ Invasive fungal infection significantly increased in-hospital mortality, independently of whether the children were treated for a hematological malignancy or a solid tumor, or underwent HCT. A recent analysis of a prospective multi-national trial in children with ALL found an overall incidence of invasive fungal disease of 3.8%, with a 12-week mortality of 11.2%.⁶ Similar data have been reported in a systematic review and meta-analysis of studies on children aged 0–17 years and treated for ALL after 2000.⁷ Although the mortality rates have significantly improved compared to previous analyses,⁸ invasive fungal disease was described as an independent risk factor for relapse and poor outcome in pediatric ALL.⁶ Therefore, better diagnostics and therapeutic strategies for invasive fungal disease might also result in an improved overall outcome in these children.

Solid data on the epidemiology of mold infections are scarce for several reasons. First, the diagnosis of invasive mold infection is difficult, and incidence rates depend on the strictness of diagnostic procedures, which might be challenging in the pediatric setting (see below, “diagnostic considerations”). In addition, the definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSG) have been updated and modified several times,⁹ which might bias the reported data. Last, exact data of populations at risk are limited by the restriction of analyses to individual centers, by differences in population denominators, and by fungal pathogens included. Although the epidemiology of invasive fungal disease is known to differ geographically, a preponderance of infections with molds versus yeast as well as of infections with Aspergillus versus non-Aspergillus molds has been found in a number of analyses.^10–13 This corroborates our results from a prospective multi-international study of more than 6000 children treated for de novo ALL,⁶ in which we also found that invasive aspergillosis was mostly caused by A. fumigatus, less often by A. niger and A. flavus (manuscript submitted). In addition, this analysis demonstrated that almost 5% of patients had a co-infection of Aspergillus with other molds or yeast, which might have important implications for the choice of therapy.

The natural incidence of invasive fungal disease, which is defined as the incidence of the infection without prophylaxis, has been reported in a number of early studies and is estimated to be at least 10% in children receiving conventional therapy for acute myeloid leukemia (AML), high-risk ALL, and relapsed acute leukemia, and for those undergoing allogeneic HCT (Table 1).¹⁴ However, it is a major challenge to estimate the natural incidence of fungal infections in present studies for two reasons: first, currently, most centers administer antifungal prophylaxis to high-risk patients, which has been strongly recommended in pediatric-specific guidelines by the panel of the European Conference on Infections in Leukemia (ECIL) (grade A recommendation, level of evidence IIt).¹⁴ Second, the increasing use of novel agents including genetically generated antibodies such as blinatumomab or inhibitors such as venetoclax may have an important impact on the risk of invasive fungal disease, but the extent of this impact is mostly unknown to date. In this respect, a single center analysis reported that only one out of 27 patients who received their first CAR-T infusion for ALL had probable aspergillosis, but it is important to note, that all patients were on mold-active prophylaxis.¹⁵

Table 1 Risk Groups and Individual Risk Factors for Invasive Mold Infection

A systematic review including 22 pediatric studies demonstrated that individual factors such as prolonged neutropenia, high-dose steroid exposure, and acute and chronic graft-versus-host disease (GvHD) were associated with invasive fungal disease (Table 1).¹⁶ This review also suggested older age as a risk factor, which had been confirmed in a recent study of children with ALL.⁶ Another analysis which included both children and adults found respiratory viral infection as a risk factor for invasive aspergillosis.¹⁷ Importantly, the clinical experience demonstrates that the risk for invasive fungal disease is not static, and an interesting report suggested to use the Sankey approach which provides a dynamic figure to understand the risk of invasive aspergillosis over time for various patient populations.¹⁸ This internet-based tool could provide pop-up highlights for different incidence rates of invasive fungal disease, thus guiding the use of antifungal prophylaxis.

Diagnostic Considerations

At an early stage of invasive mold disease, clinical signs and symptoms are often absent or unspecific (eg, isolated fever) (Figure 1 and Table 2). Less often, and mostly at a later stage, patients may present with dyspnea, chest pain and cough or with neurological symptoms such as headache or convulsions.¹⁹ Therefore, clinicians have to be highly aware of invasive mold infection, as early diagnosis and treatment are associated with better outcome. Unfortunately, despite better diagnostic tools, early diagnosis of invasive mold infection is challenging, and diagnosis mainly is based on imaging and microbiological evaluation, such as biomarkers.²⁰ However, it has to be emphasized that for both strategies important differences between children and adults have to be considered.

Table 2 Diagnostic Approaches for Invasive Mold Infection in Children

Figure 1 Clinical symptoms of invasive mold infection leading to diagnostic procedures in immunocompromised children. High awareness is needed in these patients as clinical symptoms of the infection may be subtle or even absent. Diagnostic tools not well validated in the pediatric setting are indicated in italics. The arrows in the imaging studies indicate the lesions of invasive aspergillosis. The arrows show the fungal infiltrates in the lung (upper image) and in the brain (lower image). Abbreviations: CT, computerized tomography; MRI, magnetic resonance imaging; PET, positron emission tomography; CNS, central nervous system.

Imaging

In febrile neutropenic adults, it has been demonstrated that systematic computerized tomography (CT) scans result in earlier diagnosis of invasive pulmonary aspergillosis, and early treatment is associated with better outcome (Figure 1 and Table 2).²¹ Compared to conventional chest X-ray, CT scans detect invasive aspergillosis of the lung by 5 days earlier and have a higher sensitivity.²² Pulmonary nodules, in particular, nodules with halo sign, air crescent sign and cavitation are typical CT findings for fungal pneumonia in adults, even they are not specific.²³ Importantly, the appearance of these findings depends on time of imaging, with the halo sign as an early finding, emphasizing the value of early CT scans.

Due to radiation exposure, frequent or even serial CT scans in pediatric cancer patients are problematic. A recent study estimated the lifetime incidence of radiation-induced cancers by evaluating the distribution of CT examinations and associated organ-specific radiation doses scaled to the US population based on the number of examinations in 2023.²⁴ Using current data on CT utilization, these examinations were projected to result in more than 100,000 cancers over the lifetime of the exposed patients, accounting for approximately 5% of all new cancer diagnoses per year. Importantly, children and adolescents had a higher radiation-induced cancer risk than adults. Similar data had been reported from a European study, in which a significant dose-response relationship between CT-related radiation exposure and brain cancer was observed, which might be important for the diagnostic approach in mucormycosis that often affecting the sinus.²⁵

In the pediatric setting, an early study demonstrated pulmonary infiltrates in 59% of children with invasive aspergillosis.²⁶ Importantly, the authors reported that the halo sign was present in only 6.4% of the patients, suggesting that the CT scans were performed rather late. Unspecific findings were seen in particular in children younger than 5 years. In contrast, another study reported that in 138 children with febrile neutropenia unresponsive to broad-spectrum antibiotics and pulmonary symptoms (among them 14 patients with pulmonary aspergillosis), the halo sign was present in 57% of the patients, corroborating results of an analysis demonstrating the halo sign in 40.7% of neutropenic febrile pediatric cancer patients without invasive pulmonary aspergillosis.^27,28 Similarly, other pulmonary findings such as nodules, consolidations or the air crescent sign may be seen in children with cancer and febrile neutropenia or pulmonary symptoms without aspergillosis, indicating that these abnormalities are common and not specific.^27,28 Taken together, CT scans for the detection of pulmonary aspergillosis in children with cancer has limited specificity, which might be improved by techniques such as CT pulmonary angiography imaging for angio-invasive molds.²⁹ Similarly, in adults, the presence of a reversed halo sign on chest CT is considered a suggestive imaging feature of pulmonary mucormycosis, but pediatric studies have shown that the reversed halo sign is only occasionally observed and has a low specificity.³⁰ As findings of pulmonary involvement of fungal infection may be subtle, an experienced pediatric radiologist is required for correct interpretation.³¹

In the current pediatric-specific guidelines by the ECIL group, CT imaging of the lung is strongly recommended in children at high risk of invasive fungal disease if fever during neutropenia persists for more than 96 hours despite broad-spectrum antibiotics or in patients with focal clinical findings (grade A recommendation, level of evidence II).¹⁴ However, based on the knowledge that typical signs of invasive mold disease might be missing in a high percentage of children, even non-typical pulmonary infiltrates might lead to mold-active antifungal treatment and further diagnostic investigation (grade A recommendation, level of evidence II).

It is important to note that in children with proven or probable invasive pulmonary aspergillosis, cranial magnetic resonance imaging (MRI) is strongly recommended even in the absence of neurological symptoms (Table 2).¹⁴ This recommendation is based, at least in part, on the results of a retrospective study in 25 children with cancer and proven or probable invasive mold infection of the central nervous system (CNS); nine of these patients were neurologically asymptomatic and the infection was detected by pre-emptive imaging.³² Although this investigation might have important consequences for therapy, eg, the choice of the antifungal compound, it is important to note that cranial MRI often needs anesthesia which might be a limiting factor in very sick children.

Coupling CT/MRI imaging with positron emission tomography (PET) using the Aspergillus-specific monoclonal antibody (mAb) JF5 might be a promising strategy to improve specificity and sensitivity.³³ The antibody has been humanized for diagnostic use in patients with cancer, and preliminary results in nine prospectively enrolled patients with high suspicion of invasive Aspergillus-infection are promising in detecting and localizing pulmonary and cerebral aspergillosis.³³ Notably, highly specific mAbs are now also available for other important fungal pathogens such as Fusarium, Scedosporium, and Lomentospora.³⁴

Microbiological Evaluation – Culture Based Evaluation

Culture and direct detection of fungal elements by microscopy are still considered as the “gold-standard” of diagnosing invasive fungal disease, although these methods have limited sensitivity (Table 2).¹⁹ Samples obtained from invasive diagnostic procedures such as bronchoalveloar lavage (BAL) should always be microscopically examined by an experienced microbiologist, which may give preliminary information on whether yeast or a mold is present, and in some instances information on the type of mold causing the infection. Direct microscopy might differentiate between fungi with septate hyphae (eg, Aspergillus, Scedosporium, Fusarium) and aseptate hyphae (eg, Mucormycetes), and hyphae may be either non-pigmented (hyaline) or pigmented (often black/brown).³⁵ Direct microscopic examinations may use calcofluor white stain (binding to chitin), Gram-stain (often inadequate for staining hyphae) or India ink stain (binding to the extracellular polysaccharide capsule) and potassium hydroxide preparations (to clear organic background).³⁵ For surgical samples or biopsies, histopathology should be performed, as it plays a key role in the diagnosis of invasive infection and may demonstrate inflammation and fungal tissue invasion. For histopathology, the most sensitive stains for the detection of fungi and their characterization are the periodic acid–Schiff, the Grocott-Gomori methenamine silver, and the Gridley fungus stains.³⁶

Although the results of a culture are not rapidly available and are sometimes confusing with respect to contamination, this diagnostic tool can identify the fungus and may allow susceptibility testing (Table 2).³⁷ Although most fungi grow on standard culture media, such as blood and chocolate agar, it is important to use also specific media for fungi such as Sabouraud glucose agar cloheximide, potato dextrose agar, malt extract dextrose agar, cornmeal agar, and brain heart infusion agar.³⁷ Cycloheximide may be added to prevent the overgrowth of rapidly growing saprophytic fungi, and antibiotics to inhibit bacterial growth. For most clinically important fungi, the growth temperature is 30°C, and the duration of incubation depends from the organism but may need up to several weeks.³⁷

Microbiological Evaluation – Non-Culture Based Evaluation

Importantly, non-culture based tests such as the detection of galactomannan are included in the current updated consensus definitions of invasive fungal disease from the EORTC/MSG, and may be helpful in clinical decision-making (Figure 1 and Table 2).⁹

The detection of galactomannan, which is a polysaccharide cell-wall component released by most Aspergillus spp. during hyphal growth, is a useful tool in the early diagnosis of invasive aspergillosis.¹⁹ Most commonly used is an FDA-approved enzyme immunoassay that uses EB-A2 rat mAb (Platelia™ Aspergillus Enzyme Immunoassay, Bio-Rad), but other tests have been developed to improve the performance of the assay. For example, the lateral flow device (LFD) is based on a mouse mAb called JF5 that binds to a protein epitope on an extracellular glycoprotein antigen released by Aspergillus.³⁸ Importantly, the LFD device may be used as a point of care test, which increases the practicability in daily clinical work. Galactomannan can be detected in serum, BAL and cerebrospinal fluid (CSF), suggesting invasive aspergillosis with an optical density index of ≥0.5 as threshold in serum and ≥1 in BAL and CSF, respectively.¹⁴ Whereas mold-active prophylaxis may result in false-negative test results, a number of reasons may account for false positivity, eg, cross-reactivity with other fungi such as Penicillium or Fusarium, some batches of beta-lactam antibiotics or blood products, respectively.¹⁴ Based on a systematic review and meta-analysis evaluating the assessment of serum galactomannan as a screening or as a diagnostic tool, pediatric-specific guidelines recommend the use of this assay as follows:^14,39 Prospective monitoring of galactomannan (“screening”) is recommended for early diagnosis of invasive aspergillosis in pediatric patients at high risk of invasive fungal disease not receiving mold-active prophylaxis (grade A recommendation, level of evidence II), but is discouraged in patients on mold-active prophylaxis (grade D recommendation, level of evidence IIt). Screening should be performed twice weekly, as circulation of galactomannan in serum is transient. In contrast, the galactomannan assay is strongly recommended as a diagnostic tool in children at high risk of invasive fungal disease with signs or symptoms suggestive of invasive fungal disease, including prolonged fever during neutropenia and/or infiltrates detected in pulmonary CT imaging (grade A recommendation, level of evidence II).¹⁴ Evaluating galactomannan in BAL and CSF is recommended as an adjunctive tool for diagnosis of invasive pulmonary or CNS aspergillosis (grade A recommendation, level of evidence IIt and II, respectively). Notably, the galactomannan assay has not been validated in non-neutropenic patients. In contrast to the galactomannan assay, the ECIL panel does not recommend beta-D-glucan testing for screening or for diagnostic use in the serum of children with cancer.¹⁴ The assay detects a wide variety of fungi such as Candida spp., Pneumocystis jiroveci, Aspergillus spp., Acremonium spp., and Mucorales, but the data in children are scarce, indicate poor positive predictive values, and the optimal threshold for positivity of beta-D-glucan testing in children is unknown.³⁸

In addition to diagnosing invasive aspergillosis, galactomannan is currently being evaluated as a prognostic factor and for therapy response evaluation, which could have an important impact on clinical studies investigating antifungal compounds. For example, in a study in immunocompromised adults (median age, 59 years) with invasive aspergillosis, peak serum and BAL fluid galactomannan levels were strongly associated with poor clinical outcomes (P < 0.01), and dual-source galactomannan positivity was linked to reduced treatment response (22% versus 43%, P = 0.01) and higher mortality (52% vs 27%, P = 0.002).⁴⁰ Similarly, a number of studies in adults correlated changes in the serum galactomannan index from baseline with outcome criteria at 6 and 12 weeks, and mortality at 12 weeks, respectively.⁴¹ To this end, kinetics of serum galactomannan may become an important adjunctive tool in guiding treatment, but current data are preliminary and have to be validated in children.

Microbiological Evaluation – New Techniques

Over the past decades, there has been an impressing development in molecular techniques detecting fungal nucleic acids. As a consequence, standardized PCR-based diagnostic methods for blood or BAL fluid have been included in the revised and updated consensus definitions of invasive fungal disease from the EORTC/MSG (Table 2).⁹ The ECIL panel further recommends the diagnostic use of fungal nucleic acids detection in plasma, serum, or whole blood as well as in BAL, CSF, body fluids, and tissue specimen whenever these specimens are available (grade B and A recommendation, respectively, level of evidence IIt).¹⁴ A recent meta-analysis has demonstrated that both sensitivity and specificity of mucormycosis diagnosis is considerably enhanced by PCR compared to conventional methods, although this observation needs validation in children.⁴² In addition to the use of PCR as a diagnostic tool, which may help to characterize fungi up to species level, PCR-based resistance testing can be performed even in culture negative samples, which was reported to have a clinical impact on samples with triazole resistance.⁴³

Whereas pathogen-specific PCR-based methods may fail to detect rare or unexpected pathogens, the development of next-generation sequencing (NGS) of a DNA or RNA specimen followed by bioinformatic analysis allows the detection and identification of any pathogen from a single sample. Preliminary analyses have shown sensitive and accurate species-level diagnosis of invasive fungal infections in immunocompromised patients using plasma, BAL, or CSF samples.^44,45

As invasive diagnostics are a major limitation in severely ill patients, particularly in the pediatric setting, the use of cell-free DNA, also referred to as “liquid biopsy”, seems to be an important complement to the diagnostic toolbox. To date, a number of studies evaluated the use of cell-free plasma DNA to detect invasive fungal infection in immunocompromised patients, and reported promising results regarding sensitivity and specificity.^46,47 In addition, fungal plasma cell-free DNA concentrations may correlate with extra-pulmonary spread and mortality,⁴⁸ and the baseline cycle threshold may correlate with survival.⁴⁹ However, these data have to be validated both in the real-life as well as in the pediatric setting.

There are a number of other diagnostic tests under development (Table 2), which, however, are currently not used in clinical practice, such as universal digital high-resolution melting (U-dHRM) to detect mold pathogens in BAL,⁵⁰ plasma metabolomic profiling integrated with an AI-derived fungal secondary metabolite database,⁵¹ or ELISPOT assays using Aspergillus-specific T cells.⁵² These test systems have to be validated in immunocompromised children and adults, tested in the routine clinical care for diagnosis and response to therapy, and need to be compared to the established diagnostic tools.

Antifungal Strategies

Prophylaxis

According to the pediatric-specific ECIL-8 guidelines, primary antifungal prophylaxis is strongly recommended for children and adolescents at high risk of invasive fungal disease, which is defined as at least 10% estimated natural incidence.¹⁴ Based on various analyses, high-risk patients include children with AML, children with high-risk ALL and relapsed leukemia, and those undergoing allogeneic HCT in the pre-engraftment and in the post-engraftment phase until immune reconstitution. In addition, prophylaxis should be considered in special circumstances, such as in prolonged administration of therapeutic doses of glucocorticosteroids.¹⁶ While the administration of a mold-active agent for prophylaxis is strongly recommended, the local epidemiology has always to be considered for the choice of the antifungal compound.⁵³ One recent large, prospective randomized trial in children treated for AML demonstrated that, compared to fluconazole, daily administration of caspofungin during neutropenia significantly reduced the risk of both invasive fungal disease overall and invasive aspergillosis, but did not have a significant impact on mortality.⁵⁴ As both echinocandins and amphotericin B formulations have to be administered intravenously, there is growing interest in extended dosing regimens, but to date, no randomized trial has evaluated the optimal dosage of potential agents such as micafungin or liposomal amphotericin B in this setting.^55–58 On the other hand, the use of azoles, which are available both orally and intravenously, is limited by the fact that their concomitant use with a number of drugs is contraindicated, in particular with vincristine, which is a cornerstone in the treatment of ALL.

Empirical and Pre-Emptive Therapy

As invasive mold infection is difficult to diagnose at an early stage, but early therapy is associated with improved outcome, empirical antifungal therapy is the longstanding standard of care in patients at high risk for invasive fungal disease such as patients with AML, high-risk ALL or relapsed leukemia. In this setting, empirical antifungals are instituted in patients with prolonged severe neutropenia (defined as an absolute neutrophil count of less than 500/µL for at least 10 days) who have persistent fever for at least 3 days or experience recurrent fever despite broad-spectrum antibiotics.^14,59 Both liposomal amphotericin B and caspofungin are approved for this indication in children and are recommended by the ECIL panel (grade A recommendation, level of evidence I).¹⁴ The pre-emptive or diagnostic-driven therapeutic approach has the rapid availability of a CT scan of the lung and galactomannan assay results as prerequisite and has a grade B recommendation with a level of evidence I, based on a randomized study in children.^14,60

Therapy of Established Invasive Mold Infections

According to pediatric-specific guidelines, recommendations for initial antifungal therapy of invasive aspergillosis include intravenous voriconazole for children 2 years and older (grade A recommendation, level of evidence IIt; therapeutic drug monitoring!), liposomal amphotericin B (grade B recommendation, level of evidence IIt), and amphotericin B lipid complex (grade C recommendation, level of evidence II).¹⁴ Isavuconazole has a provisional grade A, IIt recommendation depending on pediatric approval (see below). Whereas, based on a randomized study in adults, a marginal support for the combination of voriconazole plus anidulafungin is given (grade C recommendation, level of evidence IIt), no data support any other antifungal combination therapy.^14,61

For treatment of mucormycosis, pediatric-specific guidelines recommend liposomal amphotericin B (grade A recommendation, level of evidence IIt) and, with lesser strength, amphotericin B lipid complex (grade B recommendation, level of evidence II).¹⁴ Isavuconazole has a provisional grade A, IIt recommendation (see below). The combination of lipid amphotericin B with either caspofungin or posaconazole has a grade C recommendation, with a level of evidence III. Whereas the use of deferasirox is not recommended (grade D recommendation, level of evidence IIt), no recommendation for or against the use of hyperbaric oxygen can be made.¹⁴

For both aspergillosis and mucormycosis, the treatment duration of therapy is not well defined, but will be determined by the resolution of all signs and symptoms of the infection and the resolution of the immunodeficiency.

In addition to the prompt initiation of antifungal compounds, the general principles of the management of invasive mold infections, such as the control of predisposing conditions (eg, reduction of immunosuppressive therapy, particularly discontinuation or tapering of glucocorticosteroids, whenever possible) have to be considered. It is important to note that both for pulmonary aspergillosis and rhino-orbital mucormycosis, feasibility, timing and extent of surgery has to be discussed on a case-to-case basis within a multi-disciplinary team, as for both settings, data suggest a clear benefit of a surgical intervention.^62,63 Notably, in mucormycosis, achieving microscopically clear resection margins is ideal and has been associated with improved outcomes.

For further reading, existing international guidelines of the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium that include pediatric-specific considerations are recommended.^64,65

Novel Antifungal Agents and New Antifungal Strategies

Isavuconazole is a new broad-spectrum triazole, which has been approved for the pediatric setting by the European Medical Agency (EMA) in 2024 as first line therapy for invasive infection with Aspergillus spp and with Mucorales. The fact that the compound had already been approved for adults in 2015 underlines the continuing problem that the approval of most antifungal compounds for children lags years behind that for adults. Notably, based on efficacy data in adults, the ECIL group issued in 2021 a provisional recommendation for isavuconazole as first-line treatment for aspergillosis (grade A recommendation, level of evidence IIt) and mucormycosis (grade B recommendation, level of evidence IIt), pending regulatory approval for pediatric patients.^14,66,67 Compared to other clinically relevant broad-spectrum azoles, in particular to voriconazole, isavuconazole exhibits less CYP450-dependent drug-drug interactions.⁶⁸ In addition, the compound is the only triazole effective against both Aspergillus and Mucormycetes that penetrates the CNS at clinically relevant concentrations.⁶⁹ Favorable data on both safety and efficacy of isavuconazole, which is available as oral and intravenous formulation and is approved for children older than 1 year, have been reported in a number of retrospective analyses and case series in children.^70,71

Rezafungin is a novel echinocandin with high stability and long-acting pharmacokinetics, which allows once-weekly dosing.⁷² The compound is approved since 2023 for invasive candidiasis in adults. The spectrum of antifungal activity is similar to that of the other echinocandins, and higher survival rates in animals challenged with A. fumigatus, as well as a decreased kidney burden of Candida albicans and Pneumocystis after rezafungin prophylaxis have been demonstrated.⁷³ Whereas the Pediatric Investigational Plan with a first study defining an age-appropriate dosage is currently on hold, a Phase 3, multicenter, randomized, double-blind study of the efficacy and safety of rezafungin versus the standard antimicrobial regimen [posaconazole or fluconazole plus oral trimethoprim/sulfamethoxazole (TMP/SMX)] to prevent invasive fungal diseases in adults undergoing allogeneic HCT (NCT04368559) is ongoing. With favorable results in this study, rezafungin would be an ideal candidate for antifungal prophylaxis in pediatric patients treated for ALL, as azoles are contraindicated due to the concurrent administration of vincristine, which is a cornerstone in this setting.

Ibrexafungerp is a semi-synthetic triterpenoid, which inhibits the beta 1,3 glucan synthase, but is structurally distinct from echinocandins.⁷⁴ The compound is currently approved for the treatment of refractory vulvovaginal candidiasis in adults. Ibrexafungerp has a broad activity against Candida spp including most echinocandin-resistant strains and is also active against C. auris. In addition, the compound is effective against Aspergillus spp, including triazole-resistant isolates. In contrast to the echinocandins, ibrexafungerp has oral bioavailability and is therefore attractive as an oral step-down option for resistant Candida spp., and has a potential role in invasive aspergillosis and in prophylaxis.

Fosmanogepix is the prodrug of manogepix and exhibits its effect by the inhibition of glycosylphosphatidylinositol (GPI) anchored wall transfer protein 1.⁷⁵ Both intravenous and oral formulation show a wide tissue distribution and penetrate the brain, eyes, and intra-abdominal viscera. With its broad spectrum of activity against yeasts and molds, Fosmanogepix is a potential treatment for refractory or resistant infections affecting the CNS and eye, or as an oral step-down option. Relevant data for the pediatric setting are missing.

Olorofim is a first-in-class orotomide antifungal compound that inhibits dihydroorotate dehydrogenase (DHODH) and disrupts fungal cell wall synthesis. The activity includes Aspergillus spp. and multi-drug-resistant molds, including Scedosporium spp, Lomentospora prolificans, and Scopulariopsis, but has no activity against Candida, Cryptococcus, or Mucorales.^72,76 To date, the drug has not been approved for pediatric or adult use. A report on the compassionate use of olorofim in a pediatric patients with X-linked Chronic Granulomatous Disease (CGD) and pan-azole–resistant pulmonary aspergillosis post-lobectomy demonstrated full remission, and a Phase 2 study in patients aged 16 years and older (NCT03583164) has been completed.⁷⁷

Opelconazole is a novel long-acting triazole designed for inhalation.⁷⁸ The inhaled route offers high lung concentrations with minimal systemic absorption, and therefore, the agent has a low potential for systemic toxicities and drug–drug interaction. As the compound is active against Candida spp (including C. auris and C. krusei) and Aspergillus spp and has also some activity against Mucorales, it may have a potential for prophylaxis in high-risk groups or for combination therapy with systemic antifungals for refractory disease. There are no relevant data published for children.

For more than two decades, there has been an ongoing interest in the development of immunotherapeutic strategies. For example, the pro-inflammatory molecule interferon-gamma (IFN-y) enhances the antimicrobial activity of phagocytes and may be used as adjunctive immunotherapy for invasive fungal disease, in particular in immunocompromised patients.⁷⁹ Notably, preclinical studies demonstrated that the cytokine has also synergistic effects when administered in combination with antifungal compounds such as amphotericin B or azoles.⁷⁹ Another approach to treat invasive aspergillosis is the adoptive administration of immune cells,⁸⁰ but the generation of antifungal T cells is complex and cost- and/or time-consuming, both of which limit their wide clinical implementation. Recently, a two-step protocol was suggested that offers the early administration (after 24 hours) of magnetically enriched interferon-gamma (IFN-g) secreting anti-Aspergillus T cells, followed by the administration of short-term in vitro expanded anti-Aspergillus T cells from the same donor after 12 days.⁸¹ Another strategy is the generation of CAR-T cells targeting fungal ß-glucan, which have demonstrated anti-Aspergillus activity in vitro and in mouse models, even after administration of glucocorticosteroids.^82,83 Another option for immunotherapy are Natural Killer (NK) cells, which can also be genetically modified with CARs or be used as cell-line (eg, NK-92).⁸⁴ Natural Killer cells are attractive as they are effective against a wide spectrum of fungi and are rapidly available as a standardized cell product.^85,86 However, larger clinical studies are definitely needed before antifungal immunotherapy can be used in routine clinical practice.

Conclusions and Perspectives

Over the last decades, the anti-infective strategies in the pediatric setting significantly improved. First, it was recognized that there is a need for pediatric specific guidelines,⁸⁷ and based on a number of systematic reviews (eg,^16,39,88), evidence-based guidelines for children and adolescents were created and are regularly updated (eg,^{14,59,89–91}). However, newer therapeutic anti-cancer strategies such as bispecific antibodies, inhibitors, or immunotherapy with CAR-T cells, are changing the risk for infectious complications, including the risk of invasive mold disease, making it a moving target. We therefore have to analyze data of ongoing clinical trials in order to exactly quantify the risk for infection, which allows us to target highest-risk populations with prophylactic antifungal measures, but withhold this strategy in low-risk patients. Ongoing challenges also include validating new diagnostic measures, such as NGS or ImmunoPET in the pediatric population, analyzing how these methods perform in clinical practice, and determining whether they are able to improve the early and specific diagnosis of invasive mold infection. In addition, further studies need to better define the impact of genetic polymorphisms, such as those affecting tumor-necrosis-factor-receptor 2 (TNFR2), which may contribute to the risk to invasive fungal disease as well as may modulate treatment response.⁷⁹ A better understanding of these polymorphisms and biomarkers may improve the personalized management and ultimately improve outcome. Major efforts have to be undertaken to make new and promising antifungal compounds promptly available to children. In this regard, these compounds have to be analyzed regarding pharmacokinetics/pharmacodynamics in the different age groups, which is also a prerequisite for the pediatric approval. Finally, it will be exciting to further develop antifungal immunotherapies with genetically modified T or NK cells, which can complement or potentially replace conventional antifungal compounds.