Rapid and Accurate Detection of the SARS-CoV-2 Omicron Variant with a CRISPR-Cas12a Reaction in the RT-qPCR Pot.

Gene sequencing in back of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the current approach for discriminating infections produced by different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the clinic. However, sequencing is often a time-consuming step, which hinders the deployment of a very fast response during a pandemic. Here, we propose to run a CRISPR-Cas12a reaction after completing the RT-qPCR and in the very same pot to detect with high specificity genetic marks characterizing variants of concern. A crRNA was appropriately designed to detect the S gene of the SARS-CoV-2 Omicron BA.1 variant. A significant response with >20-fold dynamic range was obtained for the Omicron BA.1 S gene, while the Delta S gene did not produce any detectable signal. The sensitivity of the method was analyzed with a series of diluted samples and different Cas12a nucleases. A correlation between the RT-qPCR CT values and the CRISPR-Cas12a reaction signals was observed. Variant discrimination with the CRISPR-Cas12a reaction was possible in some minutes with high accuracy from patient samples. In conclusion, CRISPR-Cas systems seem ready to be exploited in the clinic to boost personalized diagnoses and accelerate epidemiological surveillance in a cost-effective way.

Within-host transition to GES-55 during a GES-6-producing Serratia marcescens outbreak: Emergence of ceftazidime-avibactam resistance and increased susceptibility to carbapenems.

OBJECTIVES: To describe the in vivo emergence of ceftazidime-avibactam resistance in GES-type carbapenemases and to characterize an unusual outbreak of GES-6-producing Serratia marcescens during the COVID-19 pandemic in Spain. METHODS: Retrospective study to describe a GES-CPSM outbreak based on whole genome sequencing and antimicrobial susceptibility testing (AST). Transferability of blaGES-carrying plasmid was assessed by conjugation experiments. RESULTS: In December 2020, we identified a cluster of S. marcescens harbouring blaGES-6 involving 9 patients. Whole-genome sequence analysis revealed a clonal relationship (≤3 SNPs) between the first isolates identified in each of the evolved patients and environmental samples with GES-CPSM detection. Plasmid analysis showed that the blaGES-6 gene was located in an IncQ3-type plasmid. Triparental mating experiments using a helper plasmid demonstrated mobilization of the blaGES-6-carrying plasmid. Our results also demonstrate within-host evolution in S. marcescens isolates, leading to a transition from blaGES-6 to the new blaGES-55, caused by the P162S mutation, in a subsequent infection in one of the affected patients. In blaGES-55 we identified emergence of ceftazidime-avibactam resistance along with an increase of carbapenems susceptibility. This patient had been treated with a 14-day course of ceftazidime-avibactam. AST of the transformants bearing blaGES-6 and blaGES-55 plasmids, confirmed susceptibility variation affecting ceftazidime-avibactam and carbapenems. CONCLUSIONS: We report an unusual outbreak of GES-6 whose incidence is becoming increasing. Transition from GES-6 to GES-55 may readily occur in vivo leading to ceftazidime-avibactam resistance, which brings to the fore the critical need for developing more accurate diagnosis tools for detection of GES ß-lactamases and optimise the use of antimicrobials.

Sex hormones and the total testosterone:estradiol ratio as predictors of severe acute respiratory syndrome coronavirus 2 infection in hospitalized men.

BACKGROUND: The predictive ability of the early determination of sex steroids and the total testosterone:estradiol ratio for the risk of severe coronavirus disease 2019 or the potential existence of a biological gradient in this relationship has not been evaluated. OBJECTIVES: To assess the relationship of sex steroid levels and the total testosterone:estradiol ratio with the risk of severe acute respiratory syndrome coronavirus 2 infection in men, defined as the need for intensive care unit admission or death, and the predictive ability of each biomarker. MATERIALS AND METHODS: This was a prospective observational study. We included all consecutive adult men with severe acute respiratory syndrome coronavirus 2 infections in a single center admitted to a general hospital ward or to the intensive care unit. Sex steroids were evaluated at the centralized laboratory of our hospital. RESULTS: We recruited 98 patients, 54 (55.1%) of whom developed severe coronavirus disease in 2019. Compared to patients with nonsevere coronavirus disease 2019, patients with severe coronavirus disease 2019 had significantly lower serum levels of total testosterone (111 ± 89 vs. 191 ± 143 ng/dL; p < 0.001), dehydroepiandrosterone (1.69 ± 1.26 vs. 2.96 ± 2.64 ng/mL; p < 0.001), and dehydroepiandrosterone sulfate (91.72 ± 76.20 vs. 134.28 ± 98.261 µg/dL; p = 0.009), significantly higher levels of estradiol (64.61 ± 59.35 vs. 33.78 ± 13.78 pg/mL; p = 0.001), and significantly lower total testosterone:estradiol ratio (0.28 ± 0.31 vs. 0.70 ± 0.75; p < 0.001). The lower the serum level of androgen and the lower the total testosterone:estradiol ratio values, the higher the likelihood of developing severe coronavirus disease 2019, with the linear trend in the adjusted analyses being statistically significant for all parameters except for androstenedione (p = 0.064). In the receiver operating characteristic analysis, better predictive performance was shown by the total testosterone:estradiol ratio, with an area under the curve of 0.77 (95% confidence interval 0.68-0.87; p < 0.001). DISCUSSION AND CONCLUSION: Our results suggest that men with severe acute respiratory syndrome coronavirus 2 infection, decreased androgen levels and increased estradiol levels have a higher likelihood of developing an unfavorable outcome. The total testosterone:estradiol ratio showed the best predictive ability.