Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing.

Congenital heart disease (CHD) can be complicated by pulmonary arterial hypertension (PAH). Cardiopulmonary bypass (CPB) for corrective surgery may cause endothelial dysfunction, involving endothelin-1 (ET-1), circulating endothelial cells (CECs), and endothelial progenitor cells (EPCs). These markers can gauge disease severity, but their levels in children’s peripheral blood still lack consensus for prognostic value. The aim of our study was to investigate changes in ET-1, cytokines, and the absolute numbers (Ɲ) of CECs and EPCs in children 24 h before and 48 h after CPB surgery to identify high-risk patients of complications. A cohort of 56 children was included: 41 cases with CHD-PAH (22 with high pulmonary flow and 19 with low pulmonary flow) and 15 control cases. We observed that Ɲ-CECs increased in both CHD groups and that Ɲ-EPCs decreased in the immediate post-surgical period, and there was a strong negative correlation between ET-1 and CEC before surgery, along with significant changes in ET-1, IL8, IL6, and CEC levels. Our findings support the understanding of endothelial cell precursors’ role in endogenous repair and contribute to knowledge about endothelial dysfunction in CHD.

Patients with end-stage renal disease (ESRD) are at increased risk of cardiovascular disease (CVD), such as myocardial infarction (MI). Uremic toxins and endothelial dysfunction are central to this process. In this exploratory study, we used the Affymetrix GeneChip microarray to investigate the gene expression profile in uremic serum-induced human coronary arterial endothelial cells (HCAECs) from ESRD patients with and without MI (UWI and UWOI groups) as an approach to its underlying mechanism. We also explored which pathways are involved in this process. We found 100 differentially expressed genes (DEGs) among the conditions of interest by supervised principal component analysis and hierarchical cluster analysis. The expressions of four major DEGs were validated by quantitative RT-PCR. Pathway analysis and molecular network were used to analyze the interaction and expression patterns. Ten pathways were identified as the main enriched metabolic pathways according to the transcriptome profiling analysis, which were, among others, positive regulation of inflammatory response, positive regulation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) cascade, cardiac muscle cell development, highlighting positive regulation of mitogen-activated protein kinase (MAPK) activity (p = 0.00016). Up- and down-regulation of genes from HCAECs exposed to uremic serum could contribute to increased endothelial dysfunction and CVD in ESRD patients. Our study suggests that inflammation and the ERK-MAPK pathway are highly enriched in kidney disease patients with MI, suggesting their role in ESRD pathology. Further studies and approaches based on MAPK pathway interfering strategies are needed to confirm these data.