A 6-year-old boy was admitted to our hospital with a 3-week history of fever and neck mass. He was initially seen in the local infectious diseases hospital after a chest radiography indicated potential acute milia pulmonary tuberculosis; however, the cause of the fever remained unclear after subsequent examinations. After showing minimal response to antibiotic treatment, he was transferred to our hospital. Upon admission, a physical examination revealed scattered bleeding spots on the extremities, swollen eyelids and lips, and thrush in the oral cavity, but no rash, joint swelling, or joint pain. The breathing sounds were slightly coarse in both lungs, and moist rales were evident, but the heart sounds were normal. The patient's abdomen was distended, and a neurological examination showed no significant abnormality. The chest computed tomography (CT) examination revealed enlarged mediastinal lymph nodes, and the abdominal CT showed significant splenomegaly, multiple mesenteric and retroperitoneal lymphadenopathy, and a small amount of pelvic effusion. Gastroenteroscopy revealed multiple ulcers in the rectum and sigmoid colon.
The serum high-sensitivity C-reactive protein level was 88.25 mg/L (reference range: <10 mg/L), and the procalcitonin level was 6.65 ng/ml (reference range: <0.05 ng/ml). The routine full blood count results revealed pancytopenia with a white blood cell count of 1.38 x 109/L (reference range: 4.3-11.3 x 109 cells/L), a hemoglobin level of 86 g/L (reference range: 110-160 g/L), and a platelet count of 86 x 109/L (reference range: 100-300 x 109 cells/L). In addition, the serum ferritin level was 87,741.30 ng/ml (reference range: 22-322 ng/ml), the fibrinogen level was 1.82 g/L (reference range: 2-4 g/L), and the triglyceride level was 2.69 mmol/L (reference range: <1.7 mmol/L). The cellular immune function tests indicated that 52.29% of the cells were CD3+, 27.32% were CD3+CD4+, 23.47% were CD3+CD8+, 41.85% were CD19+, and 2.07% were CD16/56+. B lymphocyte subsets were also detected, of which 95.30% were naive B cells, 0.50% were memory B cells, 3.90% were transitional B cells, and 0.20% were plasmablasts. The immunoglobulin profile revealed an IgG level of 3.85 g/L (reference range: 5.28-21.90 g/L), an IgM level of 1.80 g/L (reference range: 0.48-2.26 g/L), and an IgA level of 0.33 g/L (reference range: 0.41-2.97 g/L). Additional results are found in  Supplementary Table S1 . The patient's HIV serology was negative. The bone marrow smears revealed the presence of hemophagocytosis and fungal spores. In summary, the child had a fever for >7 days, followed by generalized lymphadenopathy, pancytopenia, decreased fibrinogen levels, significantly elevated triglycerides and serum ferritin levels, and an enlarged spleen. According to the HRH-2004 guidelines, the child could be diagnosed with HLH.
The initial blood cell analysis suggested the presence of abnormal cells, which were identified as mature granulocytes engulfing fungal spores ( Figure 1 ). After consulting the medical records, it was suspected that the patient had TM infection. Metagenomic next-generation sequencing (mNGS) and Sanger sequencing were used to identify infectious pathogens for further confirmation ( Figure 2 ). The subsequent mNGS of the bronchoalveolar lavage fluid and blood culture supported TM infection ( Figure 3 ). There was no evidence of autoantibodies, and bone marrow and lymph node biopsies and CD40 expression excluded autoimmunity and a possible hematologic neoplasm. The cellular immune function tests revealed a decrease in the number and activity of NK cells, lower CD107a expression, normal levels of hemophagocytic proteins:such as perforin, granzyme B, SAP, and XIAP:and an sCD25 expression level of 7,630 U/ml (>2,400 U/ml).
With the consent of the patient's family, samples from the child and family members were collected to conduct whole-exome sequencing of potential genetic diseases. Due to the child's fungal infection and immune deficiency, whole-exome sequencing results were analyzed and sorted using Exomiser Software, and CARD9 and CD40LG mutations were screened. A hemizygous missense variant, c.346G > A (p.Gly116Ser), was detected on the patient's CD40LG gene ( Figure 4 ) that was not present in either parent's validation samples. The activated T cells from the patient had 52% CD40L expression as compared to 80.7% expression in a healthy control ( Supplementary Figure S1 ). This finding was supported by abnormal immunoglobulin levels. A heterozygous frameshift variant, c.820dup (p.Asp274GlyfsTer61), was also detected in CARD9 and found to be inherited from the patient's mother ( Figure 4 ). The 3D structure of the patient's CARD9 protein differed from the wild-type protein ( Supplementary Figure S2 ). The patient's family history revealed that all immediate family members were healthy except for the child who had a history of poor immune function. Notably, the child and his two brothers were born prematurely at approximately 33 weeks of gestation. His mother reported that she suffered from mild dermatitis, including tinea pedis, and had generally self-medicated for this condition.
After TM infection was suggested by peripheral blood smear, voriconazole and amphotericin B treatment was administered, followed by chemotherapy for HLH. On the fourth day of administration, the abnormal cell clusters in the patient's blood routine scatter diagram disappeared ( Supplementary Figure S3 ). During treatment, the child had a series of complications caused by severe sepsis, including multiple organ dysfunction syndrome and fungal enterocolitis complicated with massive bleeding. However, due to the early implementation of antimicrobial and symptomatic treatment, the patient was finally cured and discharged after 2 months.