Optimization of the SARS-CoV-2 ARTIC network V4 primers and whole genome sequencing protocol

Introduction: The ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. An update to the V3 primer set was released on 18th June 2021 to address amplicon drop-off observed among the Delta variant of concern. Here, we report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs. Methods: We utilized a matched set of 43 clinical samples and serially diluted positive controls that were amplified by ARTIC V3, V4 and optimized V4 primers and sequenced using GridION from the Oxford Nanopore Technologies'. Results: We observed a 0.5% to 46% increase in genome recovery in 67% of the samples when using the original V4 pooling strategy compared to the V3 primers. Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. When using the optimized protocol, we observed a 60% improvement in genome recovery across all samples and an increase in the average depth in amplicon 23 and 90. Consequently, ≥95% of the genome was recovered in 72% (n = 31) of the samples. However, only 60–70% of the genomes could be recovered in samples that had 0.05) correlation between Ct value and genome recovery. Conclusion: Utilizing the ARTIC V4 primers, while increasing the primer concentrations for amplicons with drop-offs or low average read-depth, greatly improves genome recovery of Alpha, Beta, Delta, Eta and non-VOC/non-VOI SARS-CoV-2 variants

Detection of SARS-CoV-2 variant 501Y.V2 in Comoros Islands in January 2021 [version 1; peer review: 2 approved]

Background. Genomic data is key in understanding the spread and evolution of SARS-CoV-2 pandemic and informing the design and evaluation of interventions. However, SARS-CoV-2 genomic data remains scarce across Africa, with no reports yet from the Indian Ocean islands. Methods. We genome sequenced six SARS-CoV-2 positive samples from the first major infection wave in the Union of Comoros in January 2021 and undertook detailed phylogenetic analysis. Results. All the recovered six genomes classified within the 501Y.V2 variant of concern (also known as lineage B.1.351) and appeared to be from 2 sub-clusters with the most recent common ancestor dated 30th Oct-2020 (95% Credibility Interval: 06th Sep-2020 to 10th Dec-2020). Comparison of the Comoros genomes with those of 501Y.V2 variant of concern from other countries deposited into the GISAID database revealed their close association with viruses identified in France and Mayotte (part of the Comoros archipelago and a France, Overseas Department). Conclusions. The recovered genomes, albeit few, confirmed local transmission following probably multiple introductions of the SARS-CoV-2 501Y.V2 variant of concern during the Comoros’s first major COVID-19 wave. These findings demonstrate the importance of genomic surveillance and have implications for ongoing control strategies on the islands

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Prospective, multicentre study of screening, investigation and management of hyponatraemia after subarachnoid haemorrhage in the UK and Ireland

Background: Hyponatraemia often occurs after subarachnoid haemorrhage (SAH). However, its clinical significance and optimal management are uncertain. We audited the screening, investigation and management of hyponatraemia after SAH. Methods: We prospectively identified consecutive patients with spontaneous SAH admitted to neurosurgical units in the United Kingdom or Ireland. We reviewed medical records daily from admission to discharge, 21 days or death and extracted all measurements of serum sodium to identify hyponatraemia (<135 mmol/L). Main outcomes were death/dependency at discharge or 21 days and admission duration >10 days. Associations of hyponatraemia with outcome were assessed using logistic regression with adjustment for predictors of outcome after SAH and admission duration. We assessed hyponatraemia-free survival using multivariable Cox regression. Results: 175/407 (43%) patients admitted to 24 neurosurgical units developed hyponatraemia. 5976 serum sodium measurements were made. Serum osmolality, urine osmolality and urine sodium were measured in 30/166 (18%) hyponatraemic patients with complete data. The most frequently target daily fluid intake was >3 L and this did not differ during hyponatraemic or non-hyponatraemic episodes. 26% (n/N=42/164) patients with hyponatraemia received sodium supplementation. 133 (35%) patients were dead or dependent within the study period and 240 (68%) patients had hospital admission for over 10 days. In the multivariable analyses, hyponatraemia was associated with less dependency (adjusted OR (aOR)=0.35 (95% CI 0.17 to 0.69)) but longer admissions (aOR=3.2 (1.8 to 5.7)). World Federation of Neurosurgical Societies grade I–III, modified Fisher 2–4 and posterior circulation aneurysms were associated with greater hazards of hyponatraemia. Conclusions: In this comprehensive multicentre prospective-adjusted analysis of patients with SAH, hyponatraemia was investigated inconsistently and, for most patients, was not associated with changes in management or clinical outcome. This work establishes a basis for the development of evidence-based SAH-specific guidance for targeted screening, investigation and management of high-risk patients to minimise the impact of hyponatraemia on admission duration and to improve consistency of patient care

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Abstract 5444: Real-time RT-PCR of terminal deoxynucleotidyl transferase (TdT) is a useful tool for identification of minimal leukemia in cerebrospinal fluid

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. The detection of cerebrospinal fluid (CSF) infiltration by leukemic cells as performed by morphological assessment is subjective and unable to detect low levels of involvement. Therefore, newer and more accurate techniques are required to more efficiently detect minimal leukemic cell involvement. For this reason, a quantitative, Real-time RT-PCR targeting the TdT molecule was developed and tested to allow an objective and sensitive methodology for detecting CNS leukemia. 35 diagnostic CSF samples were subjected to the qRT-PCR assay for TdT. Only 4/35 samples were positive by cytomorphological detection. Of these 4 samples, one sample was diagnosed with CNS disease (CNS 3) and three samples had lower numbers of leukemic blasts (CNS 2). When compared to morphology results, as the gold standard, we found 24 out of the 35 samples to be positive by qRT-PCR. 20 CSF samples that were negative by morphology had detectable TdT by RT-PCR, possibly indicating leukemic infiltration below the level of cytomorphological detection. Serial samples for the 4 CNS-positive patients were tested by qRT-PCR and showed persistent positivity with gradual reduction in quantity of TdT with chemotherapy administration. 11 samples had no detectable TdT by RT-PCR and were found to be negative by cytomorphological assessment as well. In patients with no cytomorphologically detected blasts, TdT positivity was not associated with the level of peripheral blood WBC count. This methodology provides an objective, quantitative and sensitive tool for the detection of leukemic blasts in the CSF. This allows us to detect molecular CNS involvement in CNS negative patients. Although the outcome for all CNS negative patients is good, this has been achieved with fairly intensive prophylactic CNS-directed therapy, reduction in CNS-directed therapy may be possible in molecularly negative patients

Metacognitive Awareness Perceptions of Students with High and Low Scores on TIMSS-Like Science Tests

The current study explores the differences in metacognitive awareness perceptions of students who had high and low scores on TIMSS-like science tests. The sample consisted of 937 Omani students, 478 in Grade Five and 459 in Grade Nine. TIMSS-like tests were specially designed for both grade levels, and students also completed a metacognitive awareness perceptions inventory which explored their use of four main skills: planning, information management strategies, debugging strategies and evaluation. MANOVA was used to analyze the data. The findings indicated that students with high scores in the TIMSS-like test out-performed students with low scores in the test on all four metacognitive skills surveyed. This was true for all three performance areas analysed: performance in the TIMSS-like test as a whole, performance in lower-level test questions and performance in higher-level test questions. These findings highlight the extent to which students’ metacognitive skills influence their performance in science tests. The study recommends that students be trained to improve their metacognitive skills, reviews several methods for doing this, and suggests that such training might better prepare them for taking science tests. However, it also notes that further research is needed to explore the impact of metacognitive training on student performance in specific science examinations such as TIMSS

Workplace Violence Against Doctors in Khartoum State, Sudan, 2020

Background: Workplace violence against healthcare workers and especially doctors are now clearly taken as a rapidly rising health problem in Sudan with great political and legal concerns. The current study was aimed at exploring the prevalence of verbal and physical workplace violence, the major factors associated with it, and its consequences reported by medical staff in Khartoum, Sudan government hospitals in 2020. Methods: This cross-sectional study utilized self-administered questionnaires to collect data on aspects of workplace violence against doctors working in the government hospitals of Khartoum, Sudan. A nine-item self-reported workplace violence scale was used. An online survey of self-reported work-related violence exposure during the preceding 12 months was sent to conveniently selected doctors. In total, 387 doctors completed the questionnaire in 2020. Data were analyzed using the SPSS version 26. Results: One hundred and ninety five (50%) respondents reported they were victims of violence in the previous 12 months; 92% of them experienced nonphysical violence; while female doctors were more likely to experience this type of violence (67%), younger respondents <30 years of age were more likely to encounter violent episodes. Results show an association between exposure to physical violence and working at an emergency department. The assailants were mostly males in the age group of 19–35 years (70.2%), most of them were under no influence (60.8%). Conclusion: Approximately one in every two doctors had experienced some degree of violence, either physical or nonphysical or both, and it was negatively reflected on their psychological status as well as their work performance

Effect of Inorganic and Organic Nitrogen Sources and Biofertilizer on Murcott Mandarin Fruit Quality

Mandarin ‘Murcott’ (Citrus reticulata Blanco) trees aged five years that were grafted onto lemon ‘Volkamer’ (Citrus volkameriana) rootstock and grown in sandy soil under a drip irrigation system were used in this study during the growing seasons of 2018 and 2019. Ten different fertilization treatments combining inorganic, organic, and biofertilization in a completely randomized block were performed. The results revealed that fertilizing ‘Murcott’ mandarin trees with 75% of the recommended dose (RD) of nitrogen as inorganic nitrogen (33.5% N) in the form of NH4NO3 + 25% of RD as organic nitrogen in the form of chicken manure (3% N) per tree per year without or with a biofertilizer (Effective Microorganisms, EM1) at 150 mL/tree increased the weight, size, pulp, and peels of mandarin fruit, as well as the fruit juice volume, juice volume/fruit, and vitamin C, but reduced the total acidity in both seasons. However, fertilizing ‘Murcott’ mandarin trees with 100% of RD as inorganic nitrogen increased the pulp/fruit ratio, and fertilizing with 25% of RD as inorganic nitrogen + 75% of RD as organic nitrogen + biofertilizer EM1 increased the peel/fruit ratio, peel thickness, and fruit firmness. Fertilizing ‘Murcott’ mandarin trees with 100% organic nitrogen + biofertilizer EM1 increased total soluble solids (TSS) and total sugar contents while producing the lowest nitrate (NO3) percentage in ‘Murcott’ mandarin fruit compared with trees fertilized with inorganic nitrogen only. The fruit produced by ‘Murcott’ mandarin trees fertilized with 100% of RD as organic nitrogen with or without biofertilizer EM1 contained higher TSS, total carbohydrates, and sugars and lower nitrate percentages than those fertilized with inorganic nitrogen and biofertilizer EM1. This study contributes to reducing the use of inorganic fertilizers by adding a percentage of an organic fertilizer to obtain a healthy product that contains a lower percentage of NO3, which affects the health of the consumer, and is of high quality and suitable for export