Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR

BACKGROUND: Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts. RESULTS: The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable) as follows: ACT1 (β-actin)/PMA1 (adenosine triphosphatase), RIP (ubiquinol cytochrome-c reductase complex component), RPP2B (cytosolic ribosomal acidic protein P2B), LSC2 (succinyl-CoA synthetase β-subunit fragment), IMH3 (inosine-5'-monophosphate dehydrogenase fragment), CPA1 (carbamoyl-phosphate synthethase small subunit) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control), for 24 hours. CONCLUSION: In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2) for normalization, when analysing differences in gene expression levels between C. albicans biofilm cells and planktonic cells. Validation of the normalization strategies described above showed that the ALS1 gene is overexpressed and the ALS3 gene is underexpressed in C. albicans biofilms grown on silicone in the CDC reactor for 24 hours

Monitoring ALS1 and ALS3 Gene Expression During In Vitro Candida albicans Biofilm Formation Under Continuous Flow Conditions

Abstract: ALS1 and ALS3 encode cell-surface associated glycoproteins that are considered to be important for Candida albicans biofilm formation. The main goal of the present study was to monitor ALS1 and ALS3 gene expression during C. albicans biofilm formation (on silicone) under continuous flow conditions, using the Centers for Disease Control biofilm reactor (CDC reactor). For ALS1, we found few changes in gene expression until later stages of biofilm formation (72 and 96 h) when this gene appeared to be downregulated relative to the gene expression level in the start culture. We observed an induction of ALS3 gene expression in the initial stages of biofilm formation (0.5, 1, and 6 h), whereas at later stages, this gene was also downregulated relative to the gene expression level in the start culture. We also found that biofilms of an als3/als3 deletion mutant contained less filaments at several time points (1, 6, 24, and 48 h), although filamentation as such was not affected in this strain. Together, our data indicate an important role for ALS3 in the early phases of biofilm formation in the CDC reactor, probably related to adhesion of filaments, while the role of ALS1 is less clear

Real-time PCR expression profiling of genes encoding potential virulence factors in <it>Candida albicans </it>biofilms: identification of model-dependent and -independent gene expression

<p>Abstract</p> <p>Background</p> <p><it>Candida albicans </it>infections are often associated with biofilm formation. Previous work demonstrated that the expression of <it>HWP1 </it>(hyphal wall protein) and of genes belonging to the <it>ALS </it>(agglutinin-like sequence), <it>SAP </it>(secreted aspartyl protease), <it>PLB </it>(phospholipase B) and <it>LIP </it>(lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, <it>C. albicans </it>biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.</p> <p>Results</p> <p><it>HWP1 </it>and genes belonging to the <it>ALS</it>, <it>SAP</it>, <it>PLB </it>and <it>LIP </it>gene families were constitutively expressed in <it>C. albicans </it>biofilms. <it>ALS1-5 </it>were upregulated in all model systems, while <it>ALS9 </it>was mostly downregulated. <it>ALS6 </it>and <it>HWP1 </it>were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of <it>SAP1 </it>were more pronounced in both in vitro models, while those of <it>SAP2</it>, <it>SAP4 </it>and <it>SAP6 </it>were higher in the in vivo model. Furthermore, <it>SAP5 </it>was highly upregulated in the in vivo and RHE models. For <it>SAP9 </it>and <it>SAP10 </it>similar gene expression levels were observed in all model systems. <it>PLB </it>genes were not considerably upregulated in biofilms, while <it>LIP1-3, LIP5-7 </it>and <it>LIP9</it>-<it>10 </it>were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model.</p> <p>Conclusions</p> <p>Our findings show that <it>HWP1 </it>and most of the genes belonging to the <it>ALS, SAP </it>and <it>LIP </it>gene families are upregulated in <it>C. albicans </it>biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in <it>C. albicans </it>biofilms is highlighted.</p

Vulvovaginal candidiasis in a Flemish patient population

Increased resistance to fluconazole has been reported in oral, oesophageal and urinary Candida isolates, but this has not been observed commonly in genital tract isolates. The rate of isolation of Candida spp. and their susceptibility to amphotericin B, flucytosine and azoles were determined in a number of clinical practices in the city of Ghent, Belgium. Patients with symptomatic vulvovaginal candidiasis (VVC) were treated with fluconazole, and the mycological and clinical outcomes were evaluated. Isolates were identified as Candida albicans (78.6%), Candida guilliermondii (17.3%), Candida glabrata (2.6%) and Candida dubliniensis (1.3%). The rates of mycological and clinical cures were 79.5% and 100%, respectively. Women with recurrent VVC were infected more frequently by non-albicans Candida spp., but no association was found between the use of antifungal agents and the presence of non-albicans spp. In-vitro resistance to fluconazole was not detected, even among subsequent Candida isolates from nine patients for whom mycological cure was not achieved

Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (10, 10 and 10 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions