Fast, cheap and sensitive: Homogenizer-based RNA extraction free method for SARS-CoV-2 detection by RT-qPCR.

Background: The SARS-CoV-2 gold standard detection method is an RT-qPCR with a previous step of viral RNA extraction from the patient sample either by using commercial automatized or manual extraction kits. This RNA extraction step is expensive and time demanding. Objective: The aim of our study was to evaluate the clinical performance of a simple SARS-CoV-2 detection protocol based on a fast and intense sample homogenization followed by direct RT-qPCR. Results: 388 nasopharyngeal swabs were analyzed in this study. 222 of them tested positive for SARS-CoV-2 by the gold standard RNA extraction and RT-qPCR method, while 166 tested negative. 197 of those 222 positive samples were also positive for the homogenization protocol, yielding a sensitivity of 88.74% (95% IC; 83.83 - 92.58). 166 of those negative samples were also negative for the homogenization protocol, so the specificity obtained was 97% (95% IC; 93.11 - 99.01). For Ct values below 30, meaning a viral load of 103 copies/uL, only 4 SARS-CoV-2 positive samples failed for the RNA extraction free method; for that limit of detection, the homogenizer-based method had a sensitivity of 97.92% (95% CI; 96.01 - 99.83). Conclusions: Our results show that this fast and cheap homogenization method for the SARS-CoV-2 detection by RT-qPCR is a reliable alternative of high sensitivity for potentially infectious SARS-CoV-2 positive patients. This RNA extraction free protocol would help to reduce diagnosis time and cost, and to overcome the RNA extraction kits shortage experienced during COVID-19 pandemic.

Analytical and clinical evaluation of a heat shock SARS-CoV-2 detection method without RNA extraction for N and E genes RT-qPCR.

BACKGROUND: The COVID-19 pandemic has caused significant supply shortages worldwide for SARS-CoV-2 molecular diagnosis, like RNA extraction kits. OBJECTIVE: The aim of our study was to evaluate the clinical performance and analytical sensitivity of a simple SARS-CoV-2 diagnosis protocol based on heat shock without RNA extraction using both "CDC" (N gene) and "Charite" (E gene) RT-qPCR protocols. RESULTS: 1,036 nasopharyngeal samples, 543 of them SARS-CoV-2 positive, were analyzed. The heat shock method correctly identified 68.8% (232/337) and 89.4% (202/226) of SARS-CoV-2 positive samples for N gene and E gene, respectively. Analytical sensitivity was assessed for heat shock method using the CDC RT-qPCR protocol, obtaining sensitivity values of 98.6%, 93.3% and 84.8% for limit of detection of 100.000, 50.000 and 20.000 viral RNA copies/mL of sample. CONCLUSIONS: Our findings show that a simple heat shock SARS-CoV-2 RT-qPCR diagnosis method without RNA extraction is a reliable alternative for potentially infectious SARS-CoV-2 positive patients at the time of testing. This affordable protocol can help overcome the cost and supply shortages for SARS-CoV-2 diagnosis, especially in developing countries. In Ecuador, it has been used already by laboratories in the public health system for more than 100.000 specimens.

Crucial contribution of the universities to SARS-CoV-2 surveillance in Ecuador: Lessons for developing countries.

COVID-19 pandemic has challenged public health systems worldwide, particularly affecting developing countries in Latin America like Ecuador. In this report, we exposed the fundamental role of the Ecuadorian universities to improve COVID-19 surveillance in the country, with an overall contribution over 15% of the total SARS-CoV-2 RT-PCR tests done. We highlight the role of our university during the first semester of the COVID-19 pandemic, contributing to a massive free SARS-CoV-2 testing up to almost 10% of the total diagnosis completed in the country, mainly focus on underserved urban, rural and indigenous communities. Finally, we described our contribution to a high quality and low-cost SARS-CoV-2 RT-PCR diagnostic in Ecuador.

Methicillin resistant Staphylococcus aureus carriage among guinea pigs raised as livestock in Ecuador.

Antimicrobial resistance is a growing problem in human and veterinary medicine. Here we show that 6.25% of the guinea pigs studied in Ecuador, raised as livestock, carry methicillin resistant Staphylococcus aureus (MRSA) in the nasopharynx and therefore may potentially play a role in the transmission of MRSA in the Andean Region of South America.

CRISPR-Cas9: A Precise Approach to Genome Engineering.

In the last few decades, genomic manipulation has made significant progress as a result of the development of recombinant DNA technologies; however, more often than not, these techniques have been costly and labor intensive. In contrast, recently developed next-generation sequencing (NGS) technologies have provided a cheaper, faster, and easier process to study genomics. In particular, an NGS technique emerged from bacterial CRISPR-associated protein-9 nuclease (Cas9) as a revolutionary method to modify, regulate, or mark specific genomic sequences on virtually any organism. A later adaptation of this bacterial defense mechanism that successfully and permanently edits dysfunctional genes and corrects missing proteins has resulted in a new era for disease genetic engineering. Clinical trials using this technique are already being performed, and the applicability of CRISPR-Cas9 techniques is actively being investigated using in vivo studies. However, the concept of genome correction poses great concerns from a regulatory perspective, especially in terms of security, so principles for the regulation of these methodologies are being established. We delved into CRISPR-Cas9 from its natural and ortholog origins to its engineered variants and behaviors to present its notable and diverse applications in the fields of biotechnology and human therapeutics.

WtsE, an AvrE-family effector protein from Pantoea stewartii subsp. stewartii, causes disease-associated cell death in corn and requires a chaperone protein for stability.

The pathogenicity of Pantoea stewartii subsp. stewartii to sweet corn and maize requires a Hrp type III secretion system. In this study, we genetically and functionally characterized a disease-specific (Dsp) effector locus, composed of wtsE and wtsF, that is adjacent to the hrp gene cluster. WtsE, a member of the AvrE family of effector proteins, was essential for pathogenesis on corn and was complemented by DspA/E from Erwinia amylovora. An intact C-terminus of WtsE, which contained a putative endoplasmic reticulum membrane retention signal, was important for function of WtsE. Delivery of WtsE into sweet corn leaves by an Escherichia coli strain carrying the hrp cluster of Erwinia chrysanthemi caused water-soaking and necrosis. WtsE-induced cell death was not inhibited by cycloheximide treatment, unlike the hypersensitive response caused by a known Avr protein, AvrRxol. WtsF, the putative chaperone of WtsE, was not required for secretion of WtsE from P. stewartii, and the virulence of wtsF mutants was reduced only at low inoculum concentrations. However, WtsF was required for full accumulation of WtsE within the bacteria at low temperatures. In contrast, WtsF was needed for efficient delivery of WtsE from E. coli via the Erwinia chrysanthemi Hrp system.