PMC9755863_01
White blood cell (WBC) count analysis revealed abnormal cell masses and a microscopic morphology. (A) The WBC differential channel of the Sysmex XN-3000  Automated Hematology Analyzer suggests the presence of abnormal cells. The area shown in the circle originally belonged to mature granulocytes, but the scatters were not concentrated, and the apparatus defined these cells as eosinophils (shown in red). (B) A peripheral blood smear revealed scattered phagocytic phenomena in fungal spores and mature granulocytes, with oval fungal spores, small nuclei, a purple color, deviation to one side, pale blue plasma, and the entire profile resembling corn, with a morphology consistent with Talaromyces marneffei.
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The initial blood cell analysis suggested the presence of abnormal cells, which were identified as mature granulocytes engulfing fungal spores ( Figure 1 ). 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.
Talaromyces marneffei identified by metagenomic next-generation sequencing and Sanger sequencing. (A) The pathogen sequences (N = 564 reads) identified in the patient's plasma included Talaromyces marneffei (N = 539; 86.80%), Torque teno virus 10, Haemophilus parainfluenzae, and Staphylococcus warneri. (B) After staining the blood culture isolate at 25 C with lactophenol cotton blue, the morphology of branching septate hyphae and typical brushlike cladings could be observed. The isolate was sequenced by Sanger, and the genbank number was OP744583.
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Metagenomic next-generation sequencing (mNGS) and Sanger sequencing were used to identify infectious pathogens for further confirmation ( Figure 2 ). The 3D structure of the patient's CARD9 protein differed from the wild-type protein ( Supplementary Figure S2 ).
The peripheral blood culture revealed an infection with Talaromyces marneffei. (A) Peripheral blood culture-positive samples were inoculated in Sabouraud medium at 37 C, and the colonies were bacterial-like, glabrous, colorless, initially dull, and later gyral, without diffused pigments. (B) At 25 C, Talaromyces marneffei grew rapidly, with broom-like branches and spore chains unique to Penicillium, indicating a hyphal phase. The characteristic water-soluble wine-red pigment was produced after 2 to 3 days, staining the medium or colonies red.
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TM infection ( Figure 3 ). On the fourth day of administration, the abnormal cell clusters in the patient's blood routine scatter diagram disappeared ( Supplementary Figure S3 ).
Family investigation of CD40LG and CARD9. (A) c.346G > A p.Gly116Ser in the CD40LG exon 3 is an acquired novel variant in this patient. (B) c.820dup (p.Asp274GlyfsTer61) in CARD9 exon 6 was inherited from the patient 's mother.
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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. 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 ).