High SARS-CoV-2 incidence and asymptomatic fraction during Delta and Omicron BA.1 waves in The Gambia.

Little is known about SARS-CoV-2 infection risk in African countries with high levels of infection-driven immunity and low vaccine coverage. We conducted a prospective cohort study of 349 participants from 52 households in The Gambia between March 2021 and June 2022, with routine weekly SARS-CoV-2 RT-PCR and 6-monthly SARS-CoV-2 serology. Attack rates of 45% and 57% were seen during Delta and Omicron BA.1 waves respectively. Eighty-four percent of RT-PCR-positive infections were asymptomatic. Children under 5-years had a lower incidence of infection than 18-49-year-olds. One prior SARS-CoV-2 infection reduced infection risk during the Delta wave only, with immunity from ≥2 prior infections required to reduce the risk of infection with early Omicron lineage viruses. In an African population with high levels of infection-driven immunity and low vaccine coverage, we find high attack rates during SARS-CoV-2 waves, with a high proportion of asymptomatic infections and young children remaining relatively protected from infection.

Transforming Growth Factor Beta and Alveolar Rhabdomyosarcoma: A Challenge of Tumor Differentiation and Chemotherapy Response.

Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.

C1q is essential for myelination in the central nervous system (CNS).

Myelin sheath in the central nervous system (CNS) is essential for efficient action potential conduction. Microglia, the macrophages in the CNS, are suggested to regulate myelin development. However, the specific involvement of microglia in initial myelination is yet to be elucidated. Here, first, by culturing neural stem cells, we demonstrated that myelin sheath formation only occurred in the presence of a microglia-conditioned medium. Furthermore, the absence of C1q, a microglia-derived factor, resulted in myelination failure in the neural stem cell culture. Additionally, adding native human C1q protein was sufficient to induce myelination in vitro. Finally, in the C1q conditional knockout mouse model (C1qaFL/FL: Cx3cr1CreER), C1q deficiency prior to the onset of myelination led to reduced myelin thickness and elevated g-ratio during initial myelination. This study uncovers the pivotal role of microglia-derived C1q in developmental myelination and could potentially pave the way for new therapeutic strategies for treating demyelinating diseases.

Rhabdomyosarcoma: Current Therapy, Challenges, and Future Approaches to Treatment Strategies.

Rhabdomyosarcoma is a rare cancer arising in skeletal muscle that typically impacts children and young adults. It is a worldwide challenge in child health as treatment outcomes for metastatic and recurrent disease still pose a major concern for both basic and clinical scientists. The treatment strategies for rhabdomyosarcoma include multi-agent chemotherapies after surgical resection with or without ionization radiotherapy. In this comprehensive review, we first provide a detailed clinical understanding of rhabdomyosarcoma including its classification and subtypes, diagnosis, and treatment strategies. Later, we focus on chemotherapy strategies for this childhood sarcoma and discuss the impact of three mechanisms that are involved in the chemotherapy response including apoptosis, macro-autophagy, and the unfolded protein response. Finally, we discuss in vivo mouse and zebrafish models and in vitro three-dimensional bioengineering models of rhabdomyosarcoma to screen future therapeutic approaches and promote muscle regeneration.

Medial Ankle Instability: Review of Anatomy, Evaluation, and Treatment.

The medial ankle ligamentous complex, which includes the deltoid, talocalcaneal, and calcaneonavicular ligaments, functions to provide stability to the medial ankle. Injuries to the deltoid ligament can lead to medial-sided ankle pain, subsequent instability, and posttraumatic osteoarthritis given the altered biomechanics of the ankle joint. After completing a thorough physical examination, imaging modalities such as stress radiographs and magnetic resonance imaging can be used to confirm the diagnosis. Acute injuries to the deltoid ligament should be managed conservatively with a short course of immobilization. For patients with continued pain and instability following a regimen of nonoperative management, surgical intervention can be considered. Primary repair using suture anchor fixation to the medial malleolus can be utilized if sufficient tissue remains. However, if reconstruction is necessitated, autograft or allograft can be utilized in several described techniques.Levels of Evidence: Therapeutic.

Uncovering the spectrum of adult zebrafish neural stem cell cycle regulators.

Adult neural stem and progenitor cells (aNSPCs) persist lifelong in teleost models in diverse stem cell niches of the brain and spinal cord. Fish maintain developmental stem cell populations throughout life, including both neuro-epithelial cells (NECs) and radial-glial cells (RGCs). Within stem cell domains of the brain, RGCs persist in a cycling or quiescent state, whereas NECs continuously divide. Heterogeneous populations of RGCs also sit adjacent the central canal of the spinal cord, showing infrequent proliferative activity under homeostasis. With the rise of the zebrafish (Danio rerio) model to study adult neurogenesis and neuroregeneration in the central nervous system (CNS), it has become evident that aNSPC proliferation is regulated by a wealth of stimuli that may be coupled with biological function. Growing evidence suggests that aNSPCs are sensitive to environmental cues, social interactions, nutrient availability, and neurotrauma for example, and that distinct stem and progenitor cell populations alter their cell cycle activity accordingly. Such stimuli appear to act as triggers to either turn on normally dormant aNSPCs or modulate constitutive rates of niche-specific cell cycle behaviour. Defining the various forms of stimuli that influence RGC and NEC proliferation, and identifying the molecular regulators responsible, will strengthen our understanding of the connection between aNSPC activity and their biological significance. In this review, we aim to bring together the current state of knowledge on aNSPCs from studies investigating the zebrafish CNS, while highlighting emerging cell cycle regulators and outstanding questions that will help to advance this fascinating field of stem cell biology.

Altered subgenomic RNA abundance provides unique insight into SARS-CoV-2 B.1.1.7/Alpha variant infections.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

A Direct Posterior Approach for Subtalar Distraction Arthrodesis.

SUMMARY: Because of soft tissue constraints, definitive management of hindfoot trauma occasionally requires a direct posterior approach via a coronal split of the Achilles tendon. This article and the accompanying video demonstrate a direct posterior approach for subtalar distraction arthrodesis and review treatment decisions involved with management of these complex cases.

Erratum: Prior upregulation of interferon pathways in the nasopharynx impacts viral shedding following live attenuated influenza vaccine challenge in children.

[This corrects the article DOI: 10.1016/j.xcrm.2021.100465.].

Characterising within-hospitalSARS-CoV-2 transmission events using epidemiological and viral genomic data across two pandemic waves.

Hospital outbreaks of COVID19 result in considerable mortality and disruption to healthcare services and yet little is known about transmission within this setting. We characterise within hospital transmission by combining viral genomic and epidemiological data using Bayesian modelling amongst 2181 patients and healthcare workers from a large UK NHS Trust. Transmission events were compared between Wave 1 (1st March to 25th J'uly 2020) and Wave 2 (30th November 2020 to 24th January 2021). We show that staff-to-staff transmissions reduced from 31.6% to 12.9% of all infections. Patient-to-patient transmissions increased from 27.1% to 52.1%. 40%-50% of hospital-onset patient cases resulted in onward transmission compared to 4% of community-acquired cases. Control measures introduced during the pandemic likely reduced transmissions between healthcare workers but were insufficient to prevent increasing numbers of patient-to-patient transmissions. As hospital-acquired cases drive most onward transmission, earlier identification of nosocomial cases will be required to break hospital transmission chains.

Estimation of Seasonal Influenza Attack Rates and Antibody Dynamics in Children Using Cross-Sectional Serological Data.

Directly measuring evidence of influenza infections is difficult, especially in low-surveillance settings such as sub-Saharan Africa. Using a Bayesian model, we estimated unobserved infection times and underlying antibody responses to influenza A/H3N2, using cross-sectional serum antibody responses to 4 strains in children aged 24-60 months. Among the 242 individuals, we estimated a variable seasonal attack rate and found that most children had ≥1 infection before 2 years of age. Our results are consistent with previously published high attack rates in children. The modeling approach highlights how cross-sectional serological data can be used to estimate epidemiological dynamics.

The effect of live attenuated influenza vaccine on pneumococcal colonisation densities among children aged 24-59 months in The Gambia: a phase 4, open label, randomised, controlled trial.

BACKGROUND: Influenza and other respiratory viruses promote Streptococcus pneumoniae proliferation in the upper respiratory tract. We sought to investigate for what we believe is the first time, the effect of intranasal live attenuated influenza vaccine (LAIV) on nasopharyngeal S pneumoniae density in a low-income to middle-income country population with high pneumococcal carriage rates. METHODS: In an open-label, randomised, controlled trial in The Gambia, 330 healthy children aged 24-59 months were randomly assigned 2:1 to receive one trivalent LAIV dose at enrolment (day 0, intervention) or at the end of active follow-up (day 21, control). The investigator team were initially masked to block size and randomisation sequence to avoid allocation bias. Group allocation was later revealed to the investigator team. The primary outcome was PCR-quantified day 7 and 21 pneumococcal density. Asymptomatic respiratory viral infection at baseline and LAIV strain shedding were included as covariates in generalised mixed-effects models, to assess the effect of LAIV and other variables on pneumococcal densities. The study is registered at ClinicalTrials.gov, NCT02972957, and is closed to recruitment. FINDINGS: Between Feb 8 and April 12, 2017, and Jan 15 and March 28, 2018, of 343 children assessed for eligibility, 213 in the intervention group and 108 in the control group completed the study and were included in the final analysis. Although no significant differences were seen in pneumococcal carriage or density at each timepoint when comparing groups, changes from baseline were observed in the LAIV group. The baseline S pneumoniae carriage prevalence was high in both LAIV and control groups (75%) and increased by day 21 in the LAIV group (85%, p=0·0037), but not in the control group (79%, p=0·44). An increase in pneumococcal density from day 0 amounts was seen in the LAIV group at day 7 (+0·207 log10 copies per µL, SE 0·105, p=0·050) and day 21 (+0·280 log10 copies per µL, SE 0·105, p=0·0082), but not in the control group. Older age was associated with lower pneumococcal density (-0·015 log10 copies per µL, SE 0·005, p=0·0030), with the presence of asymptomatic respiratory viruses at baseline (+0·259 log10 copies per µL, SE 0·097, p=0·017), and greater LAIV shedding at day 7 (+0·380 log10 copies per µL, SE 0·167, p=0·024) associated with higher pneumococcal density. A significant increase in rhinorrhoea was reported in the LAIV group compared with the control group children during the first 7 days of the study (103 [48%] of 213, compared with 25 [23%] of 108, p<0·0001), and between day 7 and 21 (108 [51%] of 213, compared with 28 [26%] of 108, p<0·0001). INTERPRETATION: LAIV was associated with a modest increase in nasopharyngeal pneumococcal carriage and density in the 21 days following vaccination, with the increase in density lower in magnitude than previously described in the UK. This increase was accelerated when LAIV was administered in the presence of pre-existing asymptomatic respiratory viruses, suggesting that nasopharyngeal S pneumoniae proliferation is driven by cumulative mixed-viral co-infections. The effect of LAIV on pneumococcal density is probably similar to other respiratory viral infections in children. Our findings provide reassurance for the use of LAIV to expand influenza vaccine programmes in low-income to middle-income country populations with high pneumococcal carriage. FUNDING: Wellcome Trust.

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.

Genomic and Epidemiological Analysis of SARS-CoV-2 Viruses in Sri Lanka.

Background: In order to understand the molecular epidemiology of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Sri Lanka, since March 2020, we carried out genomic sequencing overlaid on available epidemiological data until April 2021. Methods: Whole genome sequencing was carried out on diagnostic sputum or nasopharyngeal swabs from 373 patients with COVID-19. Molecular clock phylogenetic analysis was undertaken to further explore dominant lineages. Results: The B.1.411 lineage was most prevalent, which was established in Sri Lanka and caused outbreaks throughout the country until March 2021. The estimated time of the most recent common ancestor (tMRCA) of this lineage was June 1, 2020 (with 95% lower and upper bounds March 30 to July 27) suggesting cryptic transmission may have occurred, prior to a large epidemic starting in October 2020. Returning travellers were identified with infections caused by lineage B.1.258, as well as the more transmissible B.1.1.7 lineage, which has replaced B.1.411 to fuel the ongoing large outbreak in the country. Conclusions: The large outbreak that started in early October, is due to spread of a single virus lineage, B.1.411 until the end of March 2021, when B.1.1.7 emerged and became the dominant lineage.

Generation of a Novel SARS-CoV-2 Sub-genomic RNA Due to the R203K/G204R Variant in Nucleocapsid: Homologous Recombination has Potential to Change SARS-CoV-2 at Both Protein and RNA Level.

BACKGROUND: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host's anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. METHODS: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. CONCLUSIONS: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host.

Risk factors for SARS-CoV-2 seroprevalence following the first pandemic wave in UK healthcare workers in a large NHS Foundation Trust.

BACKGROUND: We aimed to measure SARS-CoV-2 seroprevalence in a cohort of healthcare workers (HCWs) during the first UK wave of the COVID-19 pandemic, explore risk factors associated with infection, and investigate the impact of antibody titres on assay sensitivity. METHODS: HCWs at Sheffield Teaching Hospitals NHS Foundation Trust (STH) were prospectively enrolled and sampled at two time points. SARS-CoV-2 antibodies were tested using an in-house assay for IgG and IgA reactivity against Spike and Nucleoprotein (sensitivity 99·47%, specificity 99·56%). Data were analysed using three statistical models: a seroprevalence model, an antibody kinetics model, and a heterogeneous sensitivity model. FINDINGS: As of 12th June 2020, 24·4% (n=311/1275) HCWs were seropositive. Of these, 39·2% (n=122/311) were asymptomatic. The highest adjusted seroprevalence was measured in HCWs on the Acute Medical Unit (41·1%, 95% CrI 30·0-52·9) and in Physiotherapists and Occupational Therapists (39·2%, 95% CrI 24·4-56·5). Older age groups showed overall higher median antibody titres. Further modelling suggests that, for a serological assay with an overall sensitivity of 80%, antibody titres may be markedly affected by differences in age, with sensitivity estimates of 89% in those over 60 years but 61% in those ≤30 years. INTERPRETATION: HCWs in acute medical units working closely with COVID-19 patients were at highest risk of infection, though whether these are infections acquired from patients or other staff is unknown. Current serological assays may underestimate seroprevalence in younger age groups if validated using sera from older and/or more symptomatic individuals. RESEARCH IN CONTEXT: Evidence before this study: We searched PubMed for studies published up to March 6th 2021, using the terms "COVID", "SARS-CoV-2", "seroprevalence", and "healthcare workers", and in addition for articles of antibody titres in different age groups against coronaviruses using "coronavirus", "SARS-CoV-2, "antibody", "antibody tires", "COVID" and "age". We included studies that used serology to estimate prevalence in healthcare workers. SARS-CoV-2 seroprevalence has been shown to be greater in healthcare workers working on acute medical units or within domestic services. Antibody levels against seasonal coronaviruses, SARS-CoV and SARS-CoV-2 were found to be higher in older adults, and patients who were hospitalised.Added value of this study: In this healthcare worker seroprevalence modelling study at a large NHS foundation trust, we confirm that those working on acute medical units, COVID-19 "Red Zones" and within domestic services are most likely to be seropositive. Furthermore, we show that physiotherapists and occupational therapists have an increased risk of COVID-19 infection. We also confirm that antibody titres are greater in older individuals, even in the context of non-hospitalised cases. Importantly, we demonstrate that this can result in age-specific sensitivity in serological assays, where lower antibody titres in younger individuals results in lower assay sensitivity.Implications of all the available evidence: There are distinct occupational roles and locations in hospitals where the risk of COVID-19 infection to healthcare workers is greatest, and this knowledge should be used to prioritise infection prevention control and other measures to protect healthcare workers. Serological assays may have different sensitivity profiles across different age groups, especially if assay validation was undertaken using samples from older and/or hospitalised patients, who tend to have higher antibody titres. Future seroprevalence studies should consider adjusting for age-specific assay sensitivities to estimate true seroprevalence rates.

Percutaneous Fixation of a Fifth Metatarsal Base Fracture.

SUMMARY: Owing to a vascular watershed, zone II and III fifth metatarsal base fractures commonly progress to nonunion without operative intervention. This article and the accompanying video demonstrate the use of intramedullary screw fixation for a fifth metatarsal base fracture and review treatment decisions involved with management of these injuries.

Rapid feedback on hospital onset SARS-CoV-2 infections combining epidemiological and sequencing data.

Background: Rapid identification and investigation of healthcare-associated infections (HCAIs) is important for suppression of SARS-CoV-2, but the infection source for hospital onset COVID-19 infections (HOCIs) cannot always be readily identified based only on epidemiological data. Viral sequencing data provides additional information regarding potential transmission clusters, but the low mutation rate of SARS-CoV-2 can make interpretation using standard phylogenetic methods difficult. Methods: We developed a novel statistical method and sequence reporting tool (SRT) that combines epidemiological and sequence data in order to provide a rapid assessment of the probability of HCAI among HOCI cases (defined as first positive test >48 hr following admission) and to identify infections that could plausibly constitute outbreak events. The method is designed for prospective use, but was validated using retrospective datasets from hospitals in Glasgow and Sheffield collected February-May 2020. Results: We analysed data from 326 HOCIs. Among HOCIs with time from admission ≥8 days, the SRT algorithm identified close sequence matches from the same ward for 160/244 (65.6%) and in the remainder 68/84 (81.0%) had at least one similar sequence elsewhere in the hospital, resulting in high estimated probabilities of within-ward and within-hospital transmission. For HOCIs with time from admission 3-7 days, the SRT probability of healthcare acquisition was >0.5 in 33/82 (40.2%). Conclusions: The methodology developed can provide rapid feedback on HOCIs that could be useful for infection prevention and control teams, and warrants further prospective evaluation. The integration of epidemiological and sequence data is important given the low mutation rate of SARS-CoV-2 and its variable incubation period. Funding: COG-UK HOCI funded by COG-UK consortium, supported by funding from UK Research and Innovation, National Institute of Health Research and Wellcome Sanger Institute.

Generation of a novel SARS-CoV-2 sub-genomic RNA due to the R203K/G204R variant in nucleocapsid: homologous recombination has potential to change SARS-CoV-2 at both protein and RNA level.

BACKGROUND: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host’s anti-viral immune response, in turn affecting the frequency of variants over-time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. METHODS: Deep sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. RESULTS: Sequence analysis suggests that the three adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence (CS) of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. CONCLUSIONS: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans resulting in both coding changes and novel sub-genomic RNA transcripts suggests this as a mechanism for diversification and adaptation within its new host.