Methodological Comparison Of Aspergillus Spp. Diagnostics And The Potential Clinical Application Of The Aspergillus Fumigatus Surface Protein Cal Family

Background : Filamentous fungi are important pathogens causing superficial and invasive infections and allergic reactions in humans.In particular,the diagnosis of invasive fungal diseases caused by Aspergillus spp.,although progress has been made,is still difficult and the current rate of resistance to antifungal drugs is increasing,so there is an urgent need to find rapid and accurate diagnostic methods as well as preventive measures.The current application of immunoproteomics technologies provides new targets for the diagnosis,treatment and vaccine development of Aspergillus spp.diseases and is a major direction for future research.Objectives:(1)To explore Aspergillus spp.infections in patients with lung infections from November 2020 to March 2021 and compare the sensitivity and specificity of different assays for Aspergillus spp.detection.(2)This study assessed the antigenicity and immunogenicity of Cal A and Cal B to evaluate their feasibility as antigenic diagnostic methods and vaccine candidate antigens.Materials and methods:(1)Alveolar lavage fluid was collected from November 2020 to March 2021,and nine Aspergillus spp.infections were identified from 127 alveolar lavage fluid by molecular diagnostic techniques,which were combined with MALDITOF MS automated biomass spectrometry identification system,laboratory fungal culture,GM and BDG tests to analyze the sensitivity and specificity of their diagnostic methods.(2)After expression of recombinant Cal A and Cal B proteins using the E.coli expression system,the proteins were immunized against mice after purification on NiNTA agarose affinity columns.The levels of Cal A and Cal B-specific Ig G antibodies and their Ig G subtypes in the sera of 25 patients with A.fumigatus infection and a group of immunized mice were measured by enzyme-linked immunosorbent assay(ELISA).The antigen-specific immune responses of splenocytes from mice with Cal A and Cal B immunization were identified.The FACS technique was used to detect peripheral blood/splenocyte subsets and Th1/Th2 type cytokine patterns in mice,all together to explore the antigenicity and immunogenicity of Cal A and Cal B.Results:(1)Patients with Aspergillus spp infections were predominantly male and predominantly aged 65 years or older.(2)In comparison,culture methods were more sensitive and specific,antigenic methods were less specific and microscopic methods were the least sensitive.(3)Molecular diagnostic methods have a high sensitivity.(4)Patients with Aspergillus fumigatus infection have specific Cal A and Cal B antibodies in mice immunised with Cal A and Cal B,of which Ig G1 is the predominant Ig G isoform.(5)Cytokines of Th1,Th2 and Th17 types were secreted in Cal A or Cal B immunized mice.Conclusion:The results of in vitro and animal experiments indicate that Cal A and Cal B,which have good antigenicity and immunogenicity,can induce Th1,Th2 and Th17 type responsess and high levels of Ig G1 antibody production,suggesting that Cal A and Cal B have potential as diagnostic markers and vaccine candidates.