Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations.

Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.

A RT-qPCR system using a degenerate probe for specific identification and differentiation of SARS-CoV-2 Omicron (B.1.1.529) variants of concern.

Fast surveillance strategies are needed to control the spread of new emerging SARS-CoV-2 variants and gain time for evaluation of their pathogenic potential. This was essential for the Omicron variant (B.1.1.529) that replaced the Delta variant (B.1.617.2) and is currently the dominant SARS-CoV-2 variant circulating worldwide. RT-qPCR strategies complement whole genome sequencing, especially in resource lean countries, but mutations in the targeting primer and probe sequences of new emerging variants can lead to a failure of the existing RT-qPCRs. Here, we introduced an RT-qPCR platform for detecting the Delta- and the Omicron variant simultaneously using a degenerate probe targeting the key ΔH69/V70 mutation in the spike protein. By inclusion of the L452R mutation into the RT-qPCR platform, we could detect not only the Delta and the Omicron variants, but also the Omicron sub-lineages BA.1, BA.2 and BA.4/BA.5. The RT-qPCR platform was validated in small- and large-scale. It can easily be incorporated for continued monitoring of Omicron sub-lineages, and offers a fast adaption strategy of existing RT-qPCRs to detect new emerging SARS-CoV-2 variants using degenerate probes.

Posterior tilt in nondisplaced femoral neck fractures increases the risk of reoperations after osteosynthesis. A systematic review and meta-analysis.

OBJECTIVE: To conduct a systematic review on the effect of posterior tilt on reoperations, patient reported outcome measures (PROM) and functional outcome following osteosynthesis of undisplaced FNFs (uFNF). MATERIAL AND METHODS: A search string was developed with the aid of a scientific librarian and the search was performed in PubMed, CINAHL and Embase. The studies were screened independently by two authors using Covidence. Data were extracted by two authors and quality assessment was performed using Robins-I tool. The meta-analyses were performed in STATA IC 16 using Risk Ratio as the primary effect estimate. RESULTS: In total, 617 studies were screened and ten studies were included with a total of 3,131 patients. The mean age ranged from 68.3 to 85.0 years and the prevalence of posterior tilt ≥20° in the studies ranged from 4.5% to 27.6%. There were 10.3% reoperations when posterior tilt was <20° whereas there were 24.5% when posterior tilt was ≥20° The meta-analysis therefore demonstrated an overall risk ratio of 0.11 (95% confidence interval; 0.04-0.18). Only one study investigated functional outcome, using ADL as measurement, but found no significant difference. No studies investigated PROM. In general, the studies were assessed to be of poor quality mainly due possible bias and confounding. CONCLUSION: A posterior tilt ≥20° leads to a higher risk of reoperations in uFNF. Even though the studies were of poor quality, the results suggest that we should include the measurement of posterior tilt in national guidelines.