Clinical features of invasive fungal disease in children with no underlying disease

There is limited research into Invasive fungal disease (IFD) in children with no underlying disease. We undertook a retrospective study of children with IFD who did not suffer from another underlying disease, from June 2010 to March 2018 in Changsha, China. Nine children were identified. Eosinophil counts were elevated in six cases. The level of procalcitonin (PCT) was elevated in six cases. Fungal culture was positive in all patients, including eight cases of Cryptococcus neoformans and one case of Candida parapsilosis. 8.33 days following antifungal treatment, the body temperature of the eight patients affected by cryptococcal disease had returned to normal. Our study indicates that the primary pathogen in IFD was Cryptococcus neoformans in children who had no other underlying disease. Eosinophils can be considered to be indicators of cryptococcal infection. IFD in children with no other underlying disease has a satisfactory prognosis.

Laboratory and microbiological examination. Six cases had elevated peripheral blood eosinophil levels in the range of (1.6-18.8) × 10 9 /L. CRP levels (90.37 ± 64.71 mg/L, range 24.5-257) were elevated in all patients. PCT levels were greater than 0.5 ng/ml in six cases, with a median PCT of 2.82 (± 3.29) ng/ml (range 0.59-8.34). The (1-3)-β-D-glucan (BG) and galactomannan (GM)BG and GM tests were performed in nine patients, with one being positive. Cell immune function and T lymphocyte immune function were normal in all patients. Fungi were cultured in the biopsy specimens (including blood, bone marrow, cerebrospinal fluid, lymph node and skin) of all patients. Significantly, bacterial culture was negative in all patients. Cryptococcus neoformans were isolated in eight cases, Candida parapsilosis in one. The most common infected organ was the lung (8), followed by the brain (6) (Fig. 1), liver (5) (Fig. 2), lymph nodes (5), marrow (4), spleen (4) and skin (2). The evidence of infection comes from imaging, culture and biopsy. Treatment and outcome. One child infected with Candida parapsilosis refused treatment after diagnosis. The remaining patients underwent successful antifungal treatment. Eight patients infected with Cryptococcus neoformans received intravenous antifungal therapy as soon as the diagnosis of IFD was established. This treatment decision was made according to guidelines published by the Infectious Diseases Society of America in 2010 8 . Average time to fever remission following inception of antifungal therapy was 8.33 (± 4.8) days (range [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. It was remarkably noted, when the fungal infection complicating CNS diseases, the treatment process should be monitored whether existed side effect. Actually, during their hospitalization, all patients were applied antifungal therapy with amphotericin B liposome and fluorocytosine, or additionally provided with neurotrophic drug including ganglioside needle, mouse nerve growth factor to protect CNS. Furthermore, for patients with muscle weakness, potassium supplementary was given. During the administration of amphotericin B liposome, the antifungal effect was remarkable, but renal injury needed to be prevented. One of patients represented high levels of uric acid after treatment with amphotericin B liposome 40 mg/d for 1 month, then it was replaced with fluconazole 200 mg/d, but this also led to gastrointestinal reactions, then we stopped it and applied fluconazole capsules orally for discharge. Taken together, all these patients continue received antifungal therapy with fluconazole orally at least 1 months. At the one-year follow-up, the peripheral blood eosinophil count in six of the patients had returned to normal. The eight cases remained under control and no one had relapsed.

Discussion
The global burden of fungal diseases is enormous. The damage caused by IFD can be extensive. Fungi are universally distributed in our surrounding environment, including in the soil and air, and especially in warm and humid climates. These fungi can cause opportunistic infections, whether in immunocompromised people or immunocompetent ones, and are associated with high mortality and high financial cost 9,10 . Identifying IFD where there is no underlying disease can be difficult, because of the non-specific clinical signs and symptoms. An isolated fever is usually the only characteristic symptom 11 . However, IFD can be accompanied by other uncharacteristic symptoms, such as skin lesions, coughing, vomiting, abdominal pain, fatigue and headache. The main clinical sites for IFD are the lung, central nervous system, liver, spleen, lymph nodes, bone marrow and skin. The lung and central nervous system are the most commonly infected organs 11 . The symptoms and Scientific Reports | (2022) 12:208 | https://doi.org/10.1038/s41598-021-03099-w www.nature.com/scientificreports/ signs of infection of the central nervous system in patients with normal immune function were less severe than those in patients with immunosuppression 12,13 . Elevated eosinophil counts usually occur with allergic diseases and parasitic infections. But only 67% of patients infected with Cryptococcus neoformans in our study had abnormal eosinophil counts. No similar finding was identified in the one case of Candida parapsilosis. Several in vitro and in vivo experiments suggest that eosinophils are involved in the presentation of Cryptococcal antigens 14,15 . When Cryptococcus enters the human body, it binds to the Fcγ receptors and activates the T-cell immune response, leading to the accumulation of eosinophils 16 . This process is related to the capsular composition on the cell wall of Cryptococcus. It is not clear whether the condition can be replicated in other fungi. We speculated that elevated eosinophil levels could be used as a predictor of an infection of Cryptococcus neoformans.
Clinicians usually look for biomarkers to help identify the details of an infection, such as CRP, PCT, (1-3)-β-D-glucan (BG) and galactomannan (GM). CRP and PCT are important indicators of bacterial infection, however, their roles in fungal disease are controversial 17 . One study shows, for fungal infections, a combination of CRP 100-300 mg/L and PCT < 0.5 μg/L offers positive predictive values of 73%, and negative predictive values of 89% in immunocompromised patients 18 . Another study reported that low CRP and PCT < 0.5 µg/l at the onset of fever may help to distinguish fungal from bacterial infections 19 . Studies looking into the value of CRP and PCT in immunocompetent patients with fungal infections are scarce. In our study, all the cases had medium-to-high CRP levels, and more than half the cases had a moderate PCT value. The obviously anomalous CRP value may relate to the immune response caused by fungi. Many of the children had been sick for a long time before coming to the hospital, and may therefore have been accompanied by a bacterial infection at the time of admission. This phenomenon may also lead to the excessive CRP and PCT levels, but we discovered the bacterial cultures represented negative results, so we did not apply antibacterial treatment. (1-3)-β-D-glucan (BG) and galactomannan (GM) are important components of the fungal cell wall. Testing for GM in serum is recommended in patients at risk for invasive aspergillosis, and a BG test is recommended for invasive candidiasis and aspergillosis 7,20,21 . Other investigators have different opinions regarding the value of BG and GM testing [22][23][24] . False positives may result from the influence of various factors, including protein products, and some drugs. In our study, BG and GM tests in all the children infected with Cryptococcus neoformans were negative. This may be explained by the low content of BG and GM in the cell wall of Cryptococcus neoformans.  www.nature.com/scientificreports/ Tissue culture and biopsy remain the gold standard for diagnosing IFD. To our knowledge, the most common pathogens of IFD are Cryptococcus, Candida, Aspergillus, and Pneumocystis, accounting for more than 90% of reported infections 6 . In our study, Cryptococcus neoformans was the most common pathogen, accounting for 89% of the cases. We therefore speculate that immunocompetent children are more susceptible to Cryptococcus neoformans. Some studies support this hypothesis. They surmise that this unique phenomenon may be attributed to multiple polymorphisms in the genes encoding the Fc-gamma receptor 2B (FCGR2B) in the Han population of China 25 . Further study is needed.
IFD is often associated with high mortality. For children, overall, in-hospital mortality rates of 15.8% were seen for invasive Candida infection 26 . In our study, the outcomes of IFD in children without any underlying disease, were ideal. This may be attributable to the patient's own immune status.
Several limitations affect our study: We did not determine the total number of inpatients during the study period. Especially, 5 of the 8 patients with cryptococcosis had positive culture findings from the CSF, and we just concerned what kind of infection it was, but ignored the detailed parameters of CSF. Maybe we could explore

Conclusions
IFD has no typical performance and can affect various organs (mainly lungs and brain) in children who have no underlying disease. It is extremely dangerous, with a wide range of invasion, and may lead to irreversible organ damage. In patients with unremitting fever and hepatosplenomegaly, fungal disease should be considered. In addition, evaluation of the condition of all organs in the body is necessary. Cryptococcus neoformans is considered to be the main pathogen. While elevated eosinophil counts may support the diagnosis of IFD. A clinical diagnosis of fungal diseases takes both time and money. We hope that the findings of our study help clinicians identify cryptococcal infections early, and so initiate a complete fluid culture or biopsy as soon as possible to shorten the time of diagnosis. The efficacy and outcome of IFD children with no other underlying disease tends to be good.