The Omicron variant of concern (VOC) has surged in many countries and replaced the previously reported VOC. To identify different Omicron strains/sublineages on a rapid, convenient, and precise ...platform, we report a novel multiplex real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) method in one tube based on the Omicron lineage sequence variants' information. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) subvariants were used in a PCR‐based assay for rapid identification of Omicron sublineage genotyping in 1000 clinical samples. Several characteristic mutations were analyzed using specific primers and probes for the spike gene, del69–70, and F486V. To distinguish Omicron sublineages (BA.2, BA.4, and BA.5), the NSP1:141–143del in the ORF1a region and D3N mutation in membrane protein occurring outside the spike protein region were analyzed. Results from the real‐time PCR assay for one‐tube accuracy were compared to those of whole genome sequencing. The developed PCR assay was used to analyze 400 SARS‐CoV‐2 positive samples. Ten samples determined as BA.4 were positive for NSP1:141–143del, del69–70, and F486V mutations; 160 BA.5 samples were positive for D3N, del69–70, and F486V mutations, and 230 BA.2 samples were without del69–70. Screening these samples allowed the identification of epidemic trends at different time intervals. Our novel one‐tube multiplex PCR assay was effective in identifying Omicron sublineages.
•Our findings suggest that viral load is positively correlated with disease severity when symptomatic individuals compared to asymptomatic individuals.•The length of hospital stays was directly ...proportional to viral load, particularly for those in the severe group.•The Cobas SARS-CoV-2 Duo assay provides a commutable unitage IU/mL for interlaboratory interpretations.
The viral load (VL) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals is critical for improving clinical treatment strategies, care, and decisions. Several studies have reported that the initial SARS-CoV-2 VL is associated with disease severity and mortality. Cycle threshold (Ct) values and/or copies/mL are often used to quantify VL. However, a multitude of platforms, primer/probe sets of different SARS-CoV-2 target genes, and reference material manufacturers may cause inconsistent interlaboratory interpretations. The first International Standard for SARS-CoV-2 RNA quantitative assays has allowed diagnostic laboratories to transition SARS-CoV-2 VL results into international units per milliliter (IU/mL). The Cobas SARS-CoV-2 Duo quantitative assay provides VL results expressed in IU/mL.
We enrolled 145 and 50 SARS-CoV-2-positive, hospitalized and 50-negative individuals at the Tri-Service General Hospital, Taiwan from January to May 2022. Each participant’s electronic medical record was reviewed to determine asymptomatic, mild, moderate, and severe cases. Nasopharyngeal swabs were collected using universal transport medium. We investigated the association of SARS-CoV-2 VL with disease severity using the Cobas SARS-CoV-2 Duo quantitative assay and its functionality in clinical assessment and decision making to further improve clinical treatment strategies. Limit of detection (LOD) was assessed.
All 50 SARS-CoV-2-negative samples confirmed negative for SARS-CoV-2, demonstrating 100 % specificity of the Cobas SARS-CoV-2 Duo assay. Patients with severe symptoms had longer hospital stays, and the length of hospital stay (30.56 days on average) positively correlated with the VL (8.22 ± 1.21 log10 IU/mL). Asymptomatic patients had the lowest VL (5.54 ± 2.06 log10 IU/mL) at admission and the shortest hospital stay (14.1 days on average).
VL is associated with disease severity and duration of hospitalization; therefore, its quantification should be considered when making clinical care decisions and treatment strategies. The Cobas SARS-CoV-2 Duo assay provides a commutable unitage IU/mL for interlaboratory interpretations.
•The assay could separate Delta, Omicron, Alpha, Beta, and Gamma variants in one tube.•The variants could be identified within 2.5 hours using the developed assay.•The assay showed good concordance ...with whole-genome sequencing and a commercial kit test.•The assay offers a rapid high-throughput and is suitable for the sample-to-result platform.
We have established a novel 5-in-1 VOC assay to rapidly detect SARS-CoV-2 and immediately distinguish whether positive samples represent variants of concern (VOCs).
This assay could distinguish among five VOCs: Alpha, Beta, Gamma, Delta, and Omicron, in a single reaction tube. The five variants exhibit different single nucleotide polymorphisms (SNPs) in their viral genome, which can be used to distinguish them. We selected target SNPs in the spike gene, including N501Y, P681R, K417N, and deletion H69/V70 for the assay.
The limit of detection of each gene locus was 80 copies per polymerase chain reaction. We observed a high consistency among the results when comparing the performance of our 5-in-1 VOC assay, whole gene sequencing, and the Roche VirSNiP SARS-CoV-2 test in retrospectively analyzing 150 clinical SARS-CoV-2 variant positive samples. The 5-in-1 VOC assay offers an alternative and rapid high-throughput test for most diagnostic laboratories in a flexible sample-to-result platform.
The assay can also be applied in a commercial platform with the completion of the SARS-CoV-2 confirmation test and identification of its variant within 2.5 hours.
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have ...increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants.
analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H).
COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies.
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major healthcare threat worldwide. Since it was first identified in November 2021, ...the Omicron (B.1.1.529) variant of SARS-CoV-2 has evolved into several lineages, including BA.1, BA.2-BA.4, and BA.5. SARS-CoV-2 variants might increase transmissibility, pathogenicity, and resistance to vaccine-induced immunity. Thus, the epidemiological surveillance of circulating lineages using variant phenotyping is essential. The aim of the current study was to characterize the clinical outcome of Omicron BA.2 infections among hospitalized COVID-19 patients and to perform an immunological assessment of such cases against SARS-CoV-2.
We evaluated the analytical and clinical performance of the BioIC SARS-CoV-2 immunoglobulin (Ig)M/IgG detection kit, which was used for detecting antibodies against SARS-CoV-2 in 257 patients infected with the Omicron variant.
Poor prognosis was noted in 38 patients, including eight deaths in patients characterized by comorbidities predisposing them to severe COVID-19. The variant-of-concern (VOC) typing and serological analysis identified time-dependent epidemic trends of BA.2 variants emerging in the outbreak of the fourth wave in Taiwan. Of the 257 specimens analyzed, 108 (42%) and 24 (9.3%) were positive for anti-N IgM and IgG respectively.
The VOC typing of these samples allowed for the identification of epidemic trends by time intervals, including the B.1.1.529 variant replacing the B.1.617.2 variant. Moreover, antibody testing might serve as a complementary method for COVID-19 diagnosis. The combination of serological testing results with the reverse transcription-polymerase chain reaction cycle threshold value has potential value in disease prognosis, thereby aiding in epidemic investigations conducted by clinicians or the healthcare department.
Superior capsular reconstruction is a common treatment option for irreparable rotator cuffs. Arthroscopic surgery procedures mostly use anchor-based methods. However, difficulty in preoperative graft ...measurement and intra-articular knot-tying present an obstacle for most sport surgeons. Complementing the known advantages of the transosseous technique in rotator cuff repair, a feasible, economical arthroscopic transosseous superior capsular reconstruction technique is described in this Technical Note. This procedure results not only in similar fixation strength and stability and greater bone stock but also in greater cost effectiveness due to using fewer anchors. This Technical Note describes the procedure in detail and compares it with conventional procedures.
Video 1
Patients undergo surgery in the beach-chair position. A standard posterior portal is created for arthroscopic visualization. Intra-articular management, including debridement or labral reattachment, is performed via the anterior portal. Then, the scope is shifted to the subacromial space from the posterior portal, and bursectomy is undertaken via the anterior portal. The irreparable cuff tear edge is debrided to approach the medial aspect of the superior labrum. Two inlets are created: one anterior inlet near the biceps groove and one 2-cm inlet posterior to the anterior tunnel inlet. The ETHIBOND 5-0 suture needle tip is visible through the posterior portal and is grasped using a curved needle holder introduced via the lateral portal. The needle tip is introduced into the medial row inlet with a needle holder, and the lateral humeral cortex is pierced. The scope is shifted to the subdeltoid space for retrieval of the needle tip using the curved needle holder through the lateral portal. The ETHIBOND suture is left in the tunnel for shuttle purposes. Next, 3.7-mm all-suture anchors are fixed at the superior glenoid. Single-limb sutures are retrieved from 2 anchors outside via the lateral portal, and the proximal allograft edge is pierced. The graft is delivered along two sutures of glenoid-fixed anchors using a knot pusher. Glenoid graft fixation is achieved by tying knots at each all-suture anchor. After corner knots are tied, an extra knot is tied at the center of the proximal graft edge using each of the corner sutures. Suture tunnel entry points are located using the scope to determine the following piercing site on the graft. The graft undergoes intra-articular piercing, and tunnel sutures are relayed on the proximal and distal row using a tissue penetrator and shuttle suture manipulation. Four sutures are passed through 2 suture tunnels, one of which crosses 2 tunnels in the proximal region to form a medial row of footprint sutures. The shoulder is placed in a neutral position to lessen suture tension. The lateral row is tied first on the lateral cortex, simultaneously applying tension to suture tunnel exits and the medial row. The superior limb of the posterior cross-link suture and the inferior limb of the anterior cross-link suture are retrieved and tied together. The inferior limb of the posterior cross-link suture and superior limb of the anterior cross-link suture are tied to finish the cross-link construction. The final “hourglass” shape construction is achieved after tuberosity fixation.
The nephrotoxicity of sofosbuvir (SOF) on human immunodeficiency virus and hepatitis C virus (HIV/HCV)‐coinfected patients receiving antiretroviral therapy (ART) remains controversial. We ...prospectively compared the estimated glomerular filtration rate (eGFR) changes in 167 patients receiving SOF‐based direct‐acting antivirals (DAAs) who also received tenofovir disoproxil fumarate (TFV)‐based (n = 116) and TFV‐free ART (n = 51). The eGFR was assessed by the Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) equation, and the eGFR changes between ART regimens were compared by the generalized estimated equation. During DAA treatment, participants on TFV‐based ART had a higher eGFR decline than those on TFV‐free ART (slope coefficient difference: −0.82 ml/min/1.73 m2/month 95% CI: −1.21 to −0.43; p < 0.001), whereas the eGFR changes did not differ between groups (slope coefficient difference: 0.13 ml/min/1.73 m2/month 95% CI: −0.32 to 0.58; p = 0.42) after discontinuing DAAs. Participants on TFV TDF‐based ART had a higher eGFR decline than those on TFV alafenamide fumarate (TAF)‐based ART (slope coefficient difference: −0.31 ml/min/1.73 m2/month 95% CI: −0.50 to −0.12; p = 0.01). After discontinuing DAAs, the eGFR changes did not differ between groups (slope coefficient difference: 0.06 ml/min/1.73 m2/month 95% CI: −0.98 to 1.10; p = 0.91). In conclusion, HIV/HCV‐coinfected patients on TFV‐based ART had a slight eGFR decline compared to those on TFV‐free ART during SOF‐based DAA therapy. A similar trend between TDF‐based and TAF‐based ART was also observed. Because the differences of eGFR changes are limited, the physicians should not discourage the use of SOF‐based DAAs in HIV‐positive patients on TFV‐based ART.
There is a global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Information on viral genomics is crucial for understanding global dispersion and for providing insight into ...viral pathogenicity and transmission. Here, we characterized the SARS-CoV-2 genomes isolated from five travelers who returned to Taiwan from the United States of America (USA) between March and April 2020.
Haplotype network analysis was performed using genome-wide single-nucleotide variations to trace potential infection routes. To determine the genetic variations and evolutionary trajectory of the isolates, the genomes of isolates were compared to those of global virus strains from GISAID. Pharyngeal specimens were confirmed to be SARS-CoV-2-positive by RT-PCR. Direct whole-genome sequencing was performed, and viral assemblies were subsequently uploaded to GISAID. Comparative genome sequence and single-nucleotide variation analyses were performed.
The D614G mutation was identified in imported cases, which separated into two clusters related to viruses originally detected in the USA. Our findings highlight the risk of spreading SARS-CoV-2 variants through air travel and the need for continued genomic tracing for the epidemiological investigation and surveillance of SARS-CoV-2 using viral genomic data.
Continuous genomic surveillance is warranted to trace virus circulation and evolution in different global settings during future outbreaks.
Mass screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important to prevent the spread of coronavirus disease 2019 (COVID-19). Pooling samples can increase the number of ...tests processed. LabTurbo AIO 48 is an automated platform that allows ribonucleic acid extraction and sample analysis on the same instrument. We created a novel pooling assay on this platform for SARS-CoV-2 detection and demonstrated that the pooling strategy increases testing capacity without affecting accuracy and sensitivity.
Comparative limit of detection (LoD) assessment was performed on the LabTurbo AIO 48 platform and the current standard detection system based on real-time reverse transcription polymerase chain reaction (rRT-PCR) using 55 clinically positive samples. An additional 330 primary clinical samples were assessed.
Six samples pooled into one reaction tube were detected in approximately 2.5 h using the World Health Organization rRT-PCR protocol. LabTurbo AIO 48 also demonstrated a higher throughput than our reference rRT-PCR assay, with an LoD of 1000 copies/mL. The overall percentage agreement between the methods for the 330 samples was 100%.
We created a novel multi-specimen pooling assay using LabTurbo AIO 48 for the robust detection of SARS-CoV-2, allowing high-throughput results; this assay will aid in better control and prevention of COVID-19. The diagnostic assay was cost-effective and time-efficient; thus, the pooling strategy is a practical and effective method for diagnosing large quantities of specimens without compromising precision.
Augmented reality (AR), a technology that superimposes virtual information onto a user's direct view of real-world scenes, is considered one of the next-generation display technologies and has been ...attracting considerable attention. Here, we propose a flat optic AR system that synergistically integrates a polarization-independent metalens with micro light-emitting diodes (LEDs). A key component is a meticulously designed metalens with a numerical aperture of 0.25, providing a simulated focusing efficiency of approximately 76.5% at a wavelength of 532 nm. Furthermore, the laser measurement system substantiates that the fabricated metalens achieves a focusing efficiency of 70.8%. By exploiting the reversibility of light characteristics, the metalens transforms the divergent light from green micro-LEDs into a collimated beam that passes through the pupil and images on the retina. Monochromatic pixels with a size of 5×5 µm
and a pitch of 10 µm can be distinctly resolved with a power efficiency of 50%. This work illustrates the feasibility of integrating the metalens with microdisplays, realizing a high-efficiency AR device without the need for additional optical components and showcasing great potential for the development of near-eye display applications.
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