Direct microscopic examination of clinical materials is one of the most straightforward methods, simple and useful for the diagnosis of fungal infection of the respiratory tract and skin lesions, as reported in patients with fusariosis.46 Advantages of this method include the low cost and short turn-around time for providing results, but it may have a sensitivity lower than 50%, especially in patients already receiving systemic antifungal agents.44

Cultures play a fundamental role in the diagnosis of fungal infections, providing an accurate characterization of the agent. However, sensitivity is low (<50%) and final results may take many days pending on the pathogen. The volume of the biologic sample may substantially impact the sensitivity of the test what should be considered along the collection of the samples according with the bottle used (neonates <4 kg: 1 mL; pediatric: 4–13.9 kg: 3 mL, 14–24.9 kg: 10 ml and adults or >25 kg: 20 mL).47 Automated blood culture systems, such as Bactec™ (BD Instruments, USA) and BacT/Alert® (bioMérieux, Brazil) have sensitivity around 50% for invasive candidiasis.48

The identification of fungi at species level is mandatory for the adequate characterization of the IFD epidemiology in each center, as well as to allow the adequate selection of strategies for prophylaxis, control and treatment of the infection. The identification of filamentous fungi is usually based on the analysis of the micro-morphological characteristics of the reproductive mycelium of the mold pathogen isolated in culture. Accurate species identification of molds is highly dependent on the combination of data provided by colony micromorphology examination and sequencing of specific DNA target regions, a strategy that may allow discrimination of genetic closely related species. More recently, commercial systems based on proteomic analysis performed by Matrix Associated Laser Desorption-Ionization — Time of Flight (MALDI-TOF) have been integrated to the routine of clinical laboratories providing accurate automated identification of yeast and molds within few minutes.49–51

The gold standard for the diagnostic of IFD caused by a mold is the histopathologic demonstration of tissue invasion by a filamentous fungi complemented by culture of the biologic sample with accurate identification of the pathogen. However, tissue biopsies are not frequently performed in hematologic patients due to the coexistence of coagulopathies, thrombocytopenia, neutropenia and clinical instabilities, such as respiratory failure. When available, the tissue sample should be cultured and sent for histopathological evaluation with hematoxylin and eosin (HE) and specific staining for fungi (periodic acid Shiff (PAS) and/or Grocottstain (methenamine-silver).44

GM is a polysaccharide present in large amounts in the cell wall of the Aspergillus spp. It is released into infected host tissues during the hyphae growth. Despite its relevance for the diagnosis of invasive aspergillosis, this antigen is also present in other fungal agents and can generate positive results in infections caused by Fusarium sp and Histoplasma capsulatum, among others.53

In Brazil, the sandwich-type immunoenzymatic assay (ELISA, Platelia™ Aspergillus, RioRad, France) is commercially available for detecting this antigen. Final results may be obtained after approximately three hours, much faster than tissue culture. Galactomannan detection in biologic samples from high-risk patients has been used with great success to aid in the early diagnosis of IA.54

In adults, in a significant number of patients, circulating GM may be detected at a median of five to eight days before clinical and radiological manifestations of aspergillosis. Based on several studies, GM positivity in serum, bronchoalveolar lavage fluid, or cerebrospinal fluid has been accepted as mycological criteria of IA in the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and by the National Institute of Allergy and Infectious Diseases Mycoses Study Group — EORTC/MSG.55 False-positive and false-negative results can occur for several reasons (Table 1).53 Lower sensitivity rates are documented in non-neutropenic patients, without onco-hematological diseases, as well as in patients on anti-mold antifungal prophylaxis.57,58

A recent systematic review addressing the accuracy of GM in pediatric patients analyzed results of 10 studies in which GM was used for screening of fungal infections in high-risk patients and eight studies evaluating GM as a diagnostic tool in the presence of specific clinical/radiological findings. Excluding cases of possible IFD, only five studies for each one of the settings (screening versus diagnosis) remained in the pooled analysis of specificity and sensitivity. In the cohort where GM assay was used for screening IA, specificity and sensitivity values were 91% [95% confidence interval (CI): 86–94%] and 68% (95% CI: 51–81%), respectively. Otherwise, in the setting where GM was requested only at the moment of diagnosis, specificity and sensitivity values were 85% (95% CI: 51–97%) and 89% (95% CI: 79–95%), respectively.59

The positive predictive values (PPVs) in children were rather low and ranged between 0 and 100% in each setting, while the negative predictive values (NPVs) were considerably higher, ranging from 85 to 100% and from 70 to 100% in the screening and diagnostic setting, respectively.59

Data on GM in pediatric patients are still controversial to support a general recommendations for the clinical use of this biomarker for screening of aspergillosis in high-risk patients.60 Some medical centers started to use serum GM monitorization of high-risk patients as part of their strategy for preemptive treatment of aspergillosis.61 Experience with GM screening of pediatric patients is still scarce to provide any general recommendation.

Two consecutive serum samples exhibiting galactomannan index ≥0.5 or one result ≥0.7 are suggestive of IA. For bronchoalveolar lavage (BAL) samples, the cutoff it is still controversial: the manufacturer considers an index ≥0.5 in the BAL as positive, but most meta-analyses and pediatric studies suggest that a positive result requires GM index ≥0.8 or 1.0 in the BAL.1,51,52,53

BDG is a polysaccharide found in large quantities in the cell wall of several pathogenic fungi including Candida spp., Fusarium spp, Aspergillus spp, and agents of endemic mycoses. The presence of BDG in blood or other biological fluids can be a serological marker of fungal sepsis in patients with candidemia, aspergillosis, fusariosis, or infections caused by Pneumocystis jiroveci, Acremonium spp. or Saccharomyces spp. It is important to mention that this test is negative in infections caused by Cryptococcus and all Mucorales.44,63

A limitation of this test is the large number of conditions that may generate false-positive results and the small number of studies addressing the relevance of this test in pediatric populations.59,64

In Brazil, ANVISA approved the Fungitell® commercial test (Associates of Cape Cod, Inc.) for clinical use. In adults, results below 60 pg/ml are considered negative, values between 60−80 pg/ml inconclusive (suggesting the repetition in a new sample) and >80 pg/ml is considered positive. In patients at risk of IFD, different authors recommend serum collection twice a week.

Recently the Dynamiker Fungus (1-3)-B-d-Glucan Assay (Dinamiker Biotechnology (Tianjin) CO., LTD, Popular Republic of China) has been approved by ANVISA for IFI diagnosis. Although its performance is equivalent to Fungitel, it is seldom used in Brazil.65

Cutoff values of (1-3)-B-d-Glucan commercial tests for diagnosing fungal infections in pediatric patients are not yet well validated.64,66

According to the 2017 guidelines on clinical management of children with febrile neutropenia, imaging studies, including chest CT scan or adequate imaging of the symptomatic organ/system, should be performed in the evaluation of children considered to be at high-risk for developing IFD, who remain febrile and neutropenic for more than 96 h despite adequate antibiotic coverage.10,60

There is little data on the evaluation of children suspected of sinonasal IFD. Notably, children with less than two years of age do not have sufficient pneumatization of the sinus cavities, and thus, sinus imaging is rarely informative in this age range. The role of routine abdominal imaging is uncertain in patients with persistent febrile neutropenia, and imaging of abdominal lesions may generate falsely negative results.60

Imaging findings in infants with pulmonary involvement can differ from those in older children and adults.

Candida involvement of the lungs is rare in this setting of patients. The few cases reported in this population are typically secondary to hematogenous dissemination and lung lesions are usually represented by infarctions, homogeneous consolidations such as bacterial lobar pneumonia or presence of mass-like rounded images with or without central necrosis, as occur with Aspergillus.69

The image spectrum of pulmonary aspergilosis may include ground-glass appearance, small nodules, larger nodules to segmental or lobar consolidations, and invasion of the rib cage. Hilar lymph node enlargement and small pleural effusions may occur.70

The presence of small nodules or consolidations with surrounding ground-glass area, known as the halo sign, resulting from perilesional hemorrhage, secondary to angio-invasion, suggests angio-invasive aspergillosis in the neutropenic patient. The halo sign is most commonly reported in pulmonary aspergillosis but is not pathognomonic of this fungal infection and may occur secondary to other conditions. Cavitation and air crescent formation might occur more frequently in older children and adults with aspergillosis, when compared to younger children.68,71,72

The largest series reporting aspergillosis in children was collect by Burgos et al. in 2008, addressing 188 pulmonary radiographic findings generated with 110 patients with pulmonary disease where nodules were documented in 59% of the cases. Nodules were less frequently seen in the youngest children compared to the older age groups: nodules were seen in 38.7% (12 of 31) of the 0- to 5-year-olds compared with 71.8% (28 of 39) and 62.5% (25 of 40) of the 6- to 12-year-old and 13-year-old groups, respectively. Only three (2.2%) of 110 patients showed the air crescent sign, and none of them was reported within the group with 0- to 5-year-olds.4,72

In terms of obtaining accurate diagnosis of fungal infections, it is mandatory to correlate radiological findings with clinical presentation, the host immunity status, results of fungal biomarkers, and data provided by conventional microbiology assays.10,44

There is currently great concern regarding the radiation dose of the radiological exams, as these patients undergo multiple examinations throughout the follow-up period.

CDC is a persistent Candida infection of the liver, spleen, and others organs that may follow candidemia in neutropenic patients after recovery of neutropenia.73 When restricted to the liver or spleen, this entity is often referred to as hepatosplenic candidiasis (HSC).44

Ultrasonography (USG) remains a useful tool for detecting and monitoring CDC.74 The main advantage of this method is the lack of nephrotoxicity and radiation exposure to perform the exam, but its main limitation is that USG might not detect small lesions. The use of contrast with USG might improve the yield of USG for detecting fungal lesions that are represented by disseminated hypoecogenic small lesions. A typical finding of CDC lesions in the liver is the bull’s eye or target pattern with peripheral hypoecogenic halo encircling a central hyperechogenic core.73

MRI seems to be superior to CT in identifying liver lesions associated with CDC; its sensitivity is 100% and specificity is 96% when the appropriate techniques are used.75,76 The major drawback of MRI use is cost and availability. In this regard, contrast-enhanced biphasic CT or USG remain credible imaging modalities for patients with suspected CDC.77

It is important to emphasize that CT imaging tests cannot demonstrate the paranasal sinuses involvement in patients with early-stage Aspergillus rhinosinusitis, whose disease is restricted to the nasal cavity.

Acute Aspergillus infection of the paranasal sinuses appears in CT images as nonspecific dense opacification of the airspaces. Bone erosion is often absent or present only at late stage of infection and should not be considered a necessary finding for early diagnosis of acute Aspergillus sinusitis. Ethmoidal sinusitis with involvement of an orbit can occur in the absence of bony erosion of the lamina papyracea. Also consistent with this observation is that ethmoidal sinus aspergillosis can involve the medial rectus muscle of the orbit while the lamina papyracea remains intact. Increased diameter on a CT scan and elevated signal intensity on T2-weighted imaging will delineate the medial rectus infection. Similarly, aspergillosis of the frontal sinuses can be associated with infection of the frontal lobes without erosion of the walls of the frontal bone. Maxillary sinus aspergillosis can invade the palatine vessels and result in necrosis of the hard palate.66,78

The edema signal of the paranasal sinuses mucosa corresponds to the STIR hypersignal and T1 hyposignal. When this signal is modified to STIR hyposignal and T1 hypersignal (heavy fungi material), it is diagnostic of IFD. The sign of the fat of the posterior wall of maxillary sinus should also to be evaluated to characterize the extent of the disease.79

In the pediatric cancer population, Aspergillus spp may infect the CNS via hematogenous dissemination to the brain parenchyma, seeding of CSF, or by direct extension from a sinus fungal infection, resulting in a clinical presentation that includes cerebritis, abscess, meningitis, and ventriculitis.80,81

Patterns of CNS aspergillosis identified in neuroimaging in immunocompromised patients include (1) multiple areas of hypodensity (CT) or hyperintensity (T2-weighted MRI) in the cortex and/or subcortical white matter corresponding to thromboembolic infarction with or without hemorrhage; (2) multiple intracerebral ring-enhancing lesions consistent with abscesses with a low signal (T2-weighted MRI); and (3) dural enhancement associated with enhancing lesions in the adjacent paranasal sinus structure or calvaria or dural enhancement of the optic sheath with associated optic nerve and intraorbital fat enhancement.82

In a recent review of 29 children submitted to hematologic stem cell transplantation who developed IFD by mold, it was found that 20% of the patients did not present any neurological symptoms at the time imaging results suggested the involvement of CNS by the fungal infection. Notably, all patients in this series presented at least one site of fungal infection besides the CNS, mostly the lungs. Based on their experience the authors suggested that facing a severely immunocompromised patient with diagnosis of IMD at any site we should consider a diagnostic work up including images of CNS.83 Further studies are necessary to confirm if this strategy is really cost-effective in clinical practice.

Voriconazole has been considered by several guidelines as the treatment of choice for this condition, but monitoring of drug plasma levels is recommended to optimize antifungal therapy.10,84,85,91 In order to avoid toxicity and optimize clinical results, the target plasmatic concentration of voriconazole should remain is the range of 1–5 mg/L.92 Liposomal amphotericin B, initially at 3 mg/kg/day, is an acceptable alternative for patients aged less than two years old, as well as facing any contraindication for voriconazole use.10

There is no evidence to support the use of combination antifungal therapy for treating children with aspergillosis. Recently, Isavuconazole was found to be equally efficient and less toxic than voriconazole in a randomized clinical trial performed with adult patients.93 So far, there are no clinical studies addressing the use of isavuconzole in children with IA.

The genus Fusarium contains many species and is widely distributed in nature. Human fusariosis manifests as superficial infections (dermatomycosis and onychomycosis), localized subcutaneous infections and, in severely immunosuppressed patients, sinus, lung or disseminated disease.40,94 Therapeutic outcomes are poor due to underlying severe immunosuppression and suboptimal susceptibility of the pathogen to almost all antifungals. The European Society of Clinical Microbiology and Infectious Diseases/European Confederation of Medical Mycology guidelines for treatment of rare mould infections recommends the use of voriconazole as the best alternative for fusariosis, being lipid formulations of AMB accepted as alternative choices.95,96 Considering the concerns with voriconazole plasma levels in children, combination therapy of amphotericin B lipid formulation with voriconazole has been extensively used by several medical centers.42,43,94

The optimal treatment of mucormycosis has not been defined, due to the lack of appropriate prospective and randomized clinical trials. Amphotericin B deoxycholate (AmB-d) at maximum tolerable doses (1–1.5 mg/kg/day) was historically the antifungal treatment of choice prior to the availability of less nephrotoxic lipid formulations of amphotericin B. Lipid formulations of AMB, starting at 5 mg/kg/day, are now preferred as first-line therapy, with a higher initial dose of 10 mg/kg/day recommended in cases of CNS disease by some investigators.97

Based on observational data, posaconazole may have a role in patients who require ongoing maintenance therapy.97 Maintenance therapy is indicated in patients with residual tissue-based infection or with risk factors that are not readily modifiable. In these patients, therapeutic drug monitoring should be performed where possible. Given the paucity of high-quality evidence and the uncertain efficacy and safety of this strategy, combination polyene-echinocandin therapy cannot currently be recommended. Similar to adults, surgery combined with antifungal therapy is a major factor to increase survival.97

Patients should be initially treated with echinocandins as suggested by most guidelines published recently.98–101 Considering the lack of data on the safety and efficacy of anidulafungin in pediatric patients, caspofungin and micafungin represent the most appropriate options for children among the echinocandins. Formulations of AMB are also considered as alternative, especially in patients with CNS infections or endocarditis.99–101

After initial course of therapy with broad spectrum antifungals, step-down therapy with fluconazole may be considered after clinical stabilization and facing the correct identification of the pathogen. Treatment with fluconazole is not recommended for infections by Candida krusei and Candida glabrata, since C. krusei is inherently resistant, and C. glabrata has low susceptibility to this agent (Tables 2–5).99,100

Patients with hepatosplenic candidiasis usually requires several weeks or months of therapy what usually demands induction with echinocandins followed by long periods of fluconazole up to complete resolution of lesions.99–101