Monitoring SARS-CoV-2 variant transitions using differences in diagnostic cycle threshold values of target genes (preprint)

Monitoring the emergence of new SARS-CoV-2 variants is important to detect potential risks of increased transmission or disease severity. We investigated the identification of SARS-CoV-2 variants from real-time reverse transcriptase polymerase chain reaction (RT-PCR) routine diagnostics data. Cycle threshold (Ct) values of positive samples were collected from April 2021 to January 2022 in the Northern Metropolitan Area of Barcelona (n = 15,254). Viral lineage identification from whole-genome sequencing (WGS) was available for 4,618 (30.3%) of these samples. Pairwise differences in the Ct values between gene targets (ΔCt) were analyzed for variants of concern or interest circulating in our area. A specific delay in the Ct of the N-gene compared to the RdRp-gene (ΔCtNR) was observed for Alpha, Delta, Eta and Omicron. Temporal differences in ΔCtNR correlated with the dynamics of viral replacement of Alpha by Delta and of Delta by Omicron according to WGS results. Using ΔCtNR, prediction of new variants of concern at early stages of circulation was achieved with high sensitivity and specificity (91.1% and 97.8% for Delta; 98.5% and 90.8% for Omicron). Thus, tracking population-wide trends in ΔCt values obtained from routine diagnostics testing in combination with WGS could be useful for real-time management and response to local epidemics.

Array-based dynamic allele specific hybridization (Array-DASH): Optimization-free microarray processing for multiple simultaneous genomic assays.

We report proof-of-principle experiments regarding a dynamic microarray protocol enabling accurate and semi-quantitative DNA analysis for re-sequencing, fingerprinting and genotyping. Single-stranded target molecules hybridise to surface-bound probes during initial gradual cooling with high-fidelity. Real-time tracking of target denaturation (via fluorescence) during a 'dynamic' gradual heating phase permits 'melt-curve' analysis. The probe most closely matching the target sequence is identified based on the highest melting temperature. We demonstrated a >99% re-sequencing accuracy and a potential detection rate of 1% for SNPs. Experiments employing Hypericum ribosomal ITS regions and HIV genomes illustrated a reliable detection level of 5% plus simultaneous re-sequencing and genotyping. Such performance suggests a range of potential real-world applications involving rapid sequence interrogation, for example, in the Covid-19 pandemic. Guidance is offered towards the development of a commercial platform and dedicated software required to bring this technique into mainstream science.

Bronchoscopy in critically ill COVID-19 Patients: microbiological profile and factors related to nosocomial respiratory infection (preprint)

Background: Nosocomial co-infections are a cause of morbidity and mortality in Intensive Care Units (ICU). Objectives: Our aim was to describe bronchoscopy findings and analyse co-infection through bronchial aspirate (BA) samples in patients with COVID-19 pneumonia requiring ICU admission. Methods: We conducted a retrospective observational study, analysing the BA samples collected from intubated patients with COVID-19 to diagnose nosocomial respiratory infection. Results: One-hundred and fifty-five consecutive BA samples were collected from 75 patients. Of them, 90 (58%) were positive cultures for different microorganisms, 11 (7.1%) were polymicrobial, and 37 (23.7%) contained resistant microorganisms. There was a statistically significant association between increased days of orotracheal intubation (OTI) and positive BA (18.9 days versus 10.9 days, p<0.01), polymicrobial infection (22.11 versus 13.54, p<0.01) and isolation of resistant microorganisms (18.88 versus 10.94, p<0.01). In 88% of the cases a change in antibiotic treatment was made. Conclusion: Nosocomial respiratory infection in intubated COVID-19 patients seems to be higher than in non-epidemic periods. The longer the intubation period, the greater the probability of co-infection, isolation of resistant microorganisms and polymicrobial infection. Microbiological sampling through BA is an essential tool to manage these patients appropriately