Abstract
Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy.
Highlights
Fungal pathogens belonging to the genus Candida can cause various infections ranging from superficial mucocutaneous infections to devastating invasive candidiasis in humans [1]
The Secreted aspartyl proteinase 2 (Sap2) gene fragment was PCR amplified from genomic DNA of the C. parapsilosis strain (ATCC 22019, Genbank Z11918.1) using Sap2 gene-specific primers designed for the study (Table 2)
Using a Sap2-specific enzyme-linked immunosorbent assay (ELISA) assay, which detects specific Ig, immunoglobulin G (IgG) and IgM antibodies against the recombinant Sap2 protein, we evaluated the diagnostic potential of the Sap2 protein by comparing serum reactivity in candidiasis patients and healthy controls
Summary
Fungal pathogens belonging to the genus Candida can cause various infections ranging from superficial mucocutaneous infections to devastating invasive candidiasis in humans [1]. Systemic candidiasis is one of the most common bloodstream infections in hospitalized patients worldwide [1,2] and is associated with a 40–70% mortality rate, even in the presence of antifungal therapies [3,4,5]. The increasing incidence of candidiasis worldwide over recent years, resulting in high morbidity and mortality, is a cause for concern. Candida species are ranked as the fourth most common cause of nosocomial bloodstream infections in the United States and seventh in Europe [6,7]. A change in the epidemiology towards non-albicans Candida (NAC) species has been observed over the past few decades [10,11]. Epidemiological studies in SARS-CoV2 infections reported that secondary invasive candidiasis infections are associated with considerable morbidity and mortality [19]
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