SARS-CoV-2 Genome Sequencing Methods Differ in Their Abilities To Detect Variants from Low-Viral-Load Samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic surveillance has been vital in understanding the spread of coronavirus disease 2019 (COVID-19), the emergence of viral escape mutants, and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low-frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole-genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low-frequency variants. Our analyses reveal that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina Respiratory Viral Oligo panel and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high-quality sequence data from low-viral-load samples for public health and genomic surveillance purposes.

Laboratory diagnosis of severe acute respiratory syndrome coronavirus 2.

The first laboratory confirmed case of Coronavirus disease 2019 (COVID-19) in Australia was in Victoria on 25 January 2020 in a man returning from Wuhan city, Hubei province, the People's Republic of China. This was followed by three cases in New South Wales the following day. The Australian Government activated the Australian Health Sector Emergency Response Plan for Novel Coronavirus on 27 February 2020 in anticipation of a pandemic. Subsequently, the World Health Organization declared COVID-19 to be a Public Health Emergency of International Concern followed by a pandemic on 30 January 2020 and 11 March 2020, respectively. Laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is key in identifying infected persons to guide timely public health actions of contact tracing and patient isolation to limit transmission of infection. This article aims to provide a comprehensive overview of current laboratory diagnostic methods for SARS-CoV-2, including nucleic acid testing, serology, rapid antigen detection and antibody tests, virus isolation and whole genome sequencing. The relative advantages and disadvantages of the different diagnostic tests are presented, as well as their value in different clinical, infection control and public health contexts. We also describe the challenges in the provision of SARS-CoV-2 diagnostics in Australia, a country with a relatively low COVID-19 incidence in the first pandemic wave but in which prevalence could rapidly change.

Changes in Selected Physiological Parameters Following a Training Block of Specific Circuit Training Among National Top-level Basketball Players.

The study aims at measuring the effects of six weeks of specific circuit training (SCT) according to the 15-15 modality, on selected physiological parameters in national top-level basketball players. It was an intervention study, undertaken with 44 senior players randomly assigned to two groups depending on the program: intervention (IG: n = 22; SCT) and control (CG: n = 22; usual content of the defending champion team's), submitted to a six-week training block. The heart rate recovery at 1 (HRR1) and then 2 (HRR2) minutes, the double product (DP) and V̇O2max were assessed prior to and at the end of the training period. As appropriate, the Student t-test on paired or independent samples, was used to compare measures and groups. At the end of the training period, the HRR1 decreased by 14.2% (p = 0.01) and 14.1% (p = 0.03) respectively in IG and CG. The mean HRR2 was higher in IG than in GC (63 ± 8 bpm versus 57 ± 6 bpm, p = 0.003) at the end of the training period. The variation of DP in IG was not significant (p = 0. 42) while it increased by 7.2% (p = 0.0005) in CG. The V̇O2max increased by 6.5% (p < 0. 001) in IG but not in CG (p = 0.50). The specific circuit training block in the 15-15 modality improved heart rate recovery at one minute and V̇O2max, but had no effect on the double product in the basketball players studied.

Interpret with caution: An evaluation of the commercial AusDiagnostics versus in-house developed assays for the detection of SARS-CoV-2 virus.

INTRODUCTION: There is limited data on the analytical performance of commercial nucleic acid tests (NATs) for laboratory confirmation of COVID-19 infection. METHODS: Nasopharyngeal, combined nose and throat swabs, nasopharyngeal aspirates and sputum was collected from persons with suspected SARS-CoV-2 infection, serial dilutions of SARS-CoV-2 viral cultures and synthetic positive controls (gBlocks, Integrated DNA Technologies) were tested using i) AusDiagnostics assay (AusDiagnostics Pty Ltd); ii) in-house developed assays targeting the E and RdRp genes; iii) multiplex PCR assay targeting endemic respiratory viruses. Discrepant SARS-CoV-2 results were resolved by testing the N, ORF1b, ORF1ab and M genes. RESULTS: Of 52 clinical samples collected from 50 persons tested, respiratory viruses were detected in 22 samples (42 %), including SARS CoV-2 (n = 5), rhinovirus (n = 7), enterovirus (n = 5), influenza B (n = 4), hMPV (n = 5), influenza A (n = 2), PIV-2 (n = 1), RSV (n = 2), CoV-NL63 (n = 1) and CoV-229E (n = 1). SARS-CoV-2 was detected in four additional samples by the AusDiagnostics assay. Using the in-house assays as the "gold standard", the sensitivity, specificity, positive and negative predictive values of the AusDiagnostics assay was 100 %, 92.16 %, 55.56 % and 100 % respectively. The Ct values of the real-time in-house-developed PCR assay targeting the E gene was significantly lower than the corresponding RdRp gene assay when applied to clinical samples, viral culture and positive controls (mean 21.75 vs 28.1, p = 0.0031). CONCLUSIONS: The AusDiagnostics assay is not specific for the detection SARS-CoV-2. Any positive results should be confirmed using another NAT or sequencing. The case definition used to investigate persons with suspected COVID-19 infection is not specific.

Adaptive upregulation of FOXD3 and resistance to PLX4032/4720-induced cell death in mutant B-RAF melanoma cells.

Melanoma cells driven by mutant v-raf murine sarcoma viral oncogene homolog B1 (B-RAF) are highly resistant to chemotherapeutic treatments. Recent phase 1 results with PLX4032/RG7204/vemurafenib, which selectively inhibits B-RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)1/2 signaling in mutant B-RAF cells, has given encouragement to this struggling field. Nearly all patients in the phase 1-3 studies saw at least some response and the overall response rates ranged from 48 and 81%. However, despite initial tumor shrinkage, most responders in the trial experienced tumor relapse over time. These findings indicate that both intrinsic and acquired resistance may affect the clinical efficacy of PLX4032. It is critical to optimize PLX4032 activity to improve response rates and understand why some patients with the B-RAF mutation do not respond. We have previously shown that the stemness factor, Forkhead box D3 (FOXD3), is upregulated following inhibition of B-RAF-MEK signaling in mutant B-RAF melanoma cells. Here, we show that upregulation of FOXD3 following treatment with PLX4032 and PLX4720 (the non-clinical tool compound for PLX4032) confers resistance to cell death. Small interfering RNA-mediated knockdown of FOXD3 significantly enhanced the cell death response after PLX4032/4720 treatment in mutant B-RAF melanoma cell lines. Additionally, upregulation of FOXD3 after PLX4720 treatment was attenuated in non-adherent conditions and correlated with enhanced cell death. Ectopic expression of FOXD3 in non-adherent cells significantly reduced cell death in response to PLX4720 treatment. Together, these data indicate that upregulation of FOXD3 is an adaptive response to RAF inhibitors that promotes a state of drug resistance.

Acute hemoperitoneum in children: prevalence of low-attenuation fluid.

BACKGROUND: Recent evidence indicates that acute hemoperitoneum may have lower than expected attenuation values at CT. OBJECTIVE: To characterize the attenuation of acute hemoperitoneum at CT in children following blunt abdominal trauma and to assess the prevalence of low-attenuation fluid. MATERIALS AND METHODS: The CT scans of 19 consecutive children with isolated hepatic or splenic injury and associated peritoneal fluid were retrospectively analyzed. The attenuation value of peritoneal fluid was assessed in all peritoneal spaces. RESULTS: Fluid was noted in 53 peritoneal spaces (27 abdominal, 26 pelvic). Fluid attenuation ranged from 20 to 64 HU. The mean fluid attenuation in pelvic spaces (37.5 +/- 9.4 HU) was significantly lower than in abdominal spaces (444.9 +/- 10.2 HU) (P = 0.008). Fluid in 8/26 (31 %) pelvic spaces and 2/27 (7 %) abdominal spaces had attenuation values < or = 30 HU. Fluid surrounding the site of injury (perihepatic or perisplenic space) was significantly higher in attenuation than fluid at other sites (P < 0.001). There was no correlation between the mean attenuation value of peritoneal fluid in each patient and the admission hematocrit (r = -0.14, P = 0.55). CONCLUSIONS: There is great variability in the attenuation of acute hemoperitoneum. Blood in pelvic spaces has significantly lower attenuation than blood in abdominal spaces. Hemoperitoneum in the pelvis has values of < or = 30 HU in approximately one-third of spaces. The attenuation of acute hemoperitoneum does not correlate with hematocrit.

Hepatic arterial pseudoaneurysm: a rare complication of blunt abdominal trauma in children.

We report a child who developed a hepatic artery pseudoaneurysm following blunt hepatic injury. This is a rare complication of hepatic trauma in children. The imaging evaluation and clinical management of hepatic artery pseudoaneurysms are presented.

Malignant pheochromocytoma of the anterior mediastinum: PET findings with [18F]FDG and 82Rb.

A case of a malignant pheochromocytoma arising from the anterior mediastinum is presented. We report the use of positron emission tomography with 82Rb and [18F]fluorodeoxyglucose to successfully image this neoplasm.