A modified in situ RT-PCR method for localizing fungal-specific gene expression in Candida-infected mice renal cells

Abstract

In situ Reverse Transcriptase PCR (in situ RT-PCR) can amplify mRNA and localize gene expression in cells. However, this method is not feasible in fungi as the thick fungal cell wall constitutes a barrier to this procedure. We developed a two step in situ RT-PCR procedure which enabled the detection and localization of Candida tropicalis mRNA expression in formalin-fixed, paraffin-embedded (FFPE) mouse kidney sections. This in situ hybridization study revealed the first direct evidence for deposition of Candida tropicalis secreted aspartic proteinase 2 (CtSAP2) in the tip of pseudohyphae and its involvement in acute systemic candidiasis. We conclude that in situ RT-PCR can be successfully applied to FFPE tissues and will offer new perspectives in studying gene expression in Candida species.

Candida Tropicalis renal microabscesses in a child with leukemia confirmed using nucleic acid amplification and recovery after prolonged antifungal and corticosteroid treatment

“…PCR appears to have a more rapid identification and higher diagnostic sensitivity for candidemia in blood samples compared to conventional culture methods (Ashrafi et al, 2015;Rickerts et al, 2013). However, the utility of PCR for detection of Candidal infections in visceral organs remains unclear since the thick cell wall of fungi within human tissues may theoretically create a barrier that interferes with binding and amplification of intracellular nucleic acid material (Ashrafi et al, 2015;Yong et al, 2009). A recent study using a twostep in situ RT-PCR procedure in a murine model enabled localization and detection of Candida tropicalis in kidney sections (Yong et al, 2009).…”

“…However, the utility of PCR for detection of Candidal infections in visceral organs remains unclear since the thick cell wall of fungi within human tissues may theoretically create a barrier that interferes with binding and amplification of intracellular nucleic acid material (Ashrafi et al, 2015;Yong et al, 2009). A recent study using a twostep in situ RT-PCR procedure in a murine model enabled localization and detection of Candida tropicalis in kidney sections (Yong et al, 2009). To the best of our knowledge, our patient is the first case of human Candida tropicalis microabscesses occurring within visceral organs detected by PCR.…”