gms | German Medical Science

Background: Invasive Candida infection (IC) is the fourth most common bloodstream infection. With a sensitivity of 21–71% blood cultures are the current gold standard diagnostic method, however false negatives remain a clinical challenge. In deep organ IC histologic proof is often contraindicated. Indirect serologic tests cannot discriminate between Candida spp. We developed a novel technique measuring Candida reactive T cells. In a pilot study, we examined healthy donors and 3 patient cohorts with either proven IC, suspected IC, or high risk of IC.

Material/methods: Candida cells were lysed mechanically by gentleMACS^® dissociator (Miltenyi Biotec, Germany) and protein concentration was determined by BCA Bradford assay. Peripheral blood mononuclear cells (PBMC) of patients and healthy donors were isolated by density gravitation. Cultured cells were stimulated with CD28 pure antibodies and co-incubated with lysate of either C. albicans, C. glabrata, C. parapsilosis, C. tropicalis or C. krusei in presence of CD40 antibodies for 5h in 5% CO₂. Missing challenge with fungal lysate serves as negative control, Staphylococcal Enterotoxin B as positive control. PBMCs were stained with CD4FITC, CD154PE, CD69APC, CD8PerCP, CD14PerCP, CD20PerCP and 7AAD and measured on MACSQuant^® flow cytometer (all Miltenyi Biotec, Germany). Candida reactive CD4⁺ T cells were detected based on the upregulation of CD69 and CD154 (CD40L). Positivity was defined as 0.4% CD69⁺/CD154⁺ cells among CD4⁺ with a parallel 3x fold increase compared to unstimulated CD4⁺ T cells.

Results: We determined the performance of Candida reactive T cells in 25 patients, including 14 proven IC (blood culture [11], tissue culture [3]) and 2 with either probable or possible hepatosplenic candidiasis. Nine haematological high risk patients served as disease control and 14 healthy donors as negative control. Due to autofluorescence of cells we excluded 3 candidaemia patients from analysis. Ten of 11 patients with proven IC and 2 of 2 patients with either probable or possible IC had elevated yields of Candida reactive CD4⁺ T cells. In 2 of 13 patients, T cells reacted with C. albicans, C. tropicalis and C. parapsilosis. In 8 of 11 proven IC, T cell reaction matched the Candida species identified by culture. Disease and healthy control patients had no elevated Candida directed T cells counts.

Conclusions: The Candida reactive T cell assay correctly identified the majority of IC patients. Autofluorescence of cells is a limiting factor. Cross reactivity towards Candida spp may represent mixed infection, but currently remains unexplained. The Candida reactive T cell assay has potential to complement current diagnostic assays for invasive Candida infection.