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.

Transcriptomic approach and membrane fatty acid analysis to study the response mechanisms of Escherichia coli to thyme essential oil, carvacrol, 2-(E)-hexanal and citral exposure.

AIMS: The application of essential oils (EOs) and their components as food preservatives is promising but requires a deeper understanding of their mechanisms of action. This study aims to evaluate the effects of thyme EO, carvacrol, citral and 2-(E)-hexenal, on whole-genome gene expression (the transcriptome), as well as the fatty acid (FA) composition of the cell membranes of Escherichia coli K12. METHODS AND RESULTS: Therefore, we studied the response against 1 h of exposure to sublethal concentrations of natural antimicrobials, of exponentially growing E. coli K12, using DNA microarray technology and a gas chromatographic method. The results show that treatment with a sublethal concentration of the antimicrobials strongly affects global gene expression in E. coli for all antimicrobials used. Major changes in the expression of genes involved in metabolic pathways as well as in FA biosynthesis and protection against oxidative stress were evidenced. Moreover, the sublethal treatments resulted in increased levels of unsaturated and cyclic FAs as well as an increase in the chain length compared to the controls. CONCLUSIONS: The down-regulation of genes involved in aerobic metabolism indicates a shift from respiration to fermentative growth. Moreover, the results obtained suggest that the cytoplasmic membrane of E. coli is the major cellular target of EOs and their components. In addition, the key role of membrane unsaturated FAs in the response mechanisms of E. coli to natural antimicrobials has been confirmed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The transcriptomic data obtained signify a further step to understand the mechanisms of action of natural antimicrobials also when sublethal concentrations and short-term exposure. In addition, this research goes in deep correlating the transcriptomic modification with the changes in E. coli FA composition of cell membrane identified as the main target of the natural antimicrobials.

Detecting dispersed duplications in high-throughput sequencing data using a database-free approach.

MOTIVATION: Dispersed duplications (DDs) such as transposon element insertions and copy number variations are ubiquitous in the human genome. They have attracted the interest of biologists as well as medical researchers due to their role in both evolution and disease. The efforts of discovering DDs in high-throughput sequencing data are currently dominated by database-oriented approaches that require pre-existing knowledge of the DD elements to be detected. RESULTS: We present DD_DETECTION, a database-free approach to finding DD events in high-throughput sequencing data. DD_DETECTION is able to detect DDs purely from paired-end read alignments. We show in a comparative study that this method is able to compete with database-oriented approaches in recovering validated transposon insertion events. We also experimentally validate the predictions of DD_DETECTION on a human DNA sample, showing that it can find not only duplicated elements present in common databases but also DDs of novel type. AVAILABILITY AND IMPLEMENTATION: The software presented in this article is open source and available from https://bitbucket.org/mkroon/dd_detection.

Maintenance tocolysis with nifedipine in threatened preterm labour: 2-year follow up of the offspring in the APOSTEL II trial.

OBJECTIVE: To evaluate long-term effects of maintenance tocolysis with nifedipine on neurodevelopmental outcome of the infant. DESIGN, SETTING AND POPULATION: Follow up of infants of women who participated in a multicentre randomised controlled trial on maintenance tocolysis with nifedipine versus placebo. METHODS: Two years after the APOSTEL II trial on maintenance tocolysis with nifedipine versus placebo, we asked participants to complete the Ages and Stages Questionnaire. MAIN OUTCOME MEASURES: Infant development was measured in five domains. Developmental delay was defined as a score of ≤1 SD in one or more developmental domains. We performed exploratory subgroup analysis in women with preterm prolonged rupture of the membranes, and in women with a cervical length <10 mm at study entry. RESULTS: Of the 276 women eligible for follow up, 135 (52.5%) returned the questionnaire, encompassing data of 170 infants. At 2 years of age, infants of women with nifedipine maintenance tocolysis compared with placebo had a higher overall incidence of fine motor problems (22.2 versus 7.6%, OR 3.43, 95% CI 1.29-9.14, P = 0.01), and a lower incidence of poor problem-solving (21.1 versus 29.1%, OR 0.27, 95% CI 0.08-0.95, P = 0.04). CONCLUSIONS: This follow-up study revealed no clear benefit of nifedipine maintenance tocolysis at 2 years of age. As short-term adverse perinatal outcome was not reduced in the original APOSTEL II trial, we conclude that maintenance tocolysis does not appear to be beneficial at this time. TWEETABLE ABSTRACT: No clear benefit of nifedipine maintenance tocolysis in preterm labour on 2-year infant outcome.

Early intervention leads to long-term developmental improvements in very preterm infants, especially infants with bronchopulmonary dysplasia.

AIM: Various early intervention programmes have been developed in response to the high rate of neurodevelopmental problems in very preterm infants. We investigated longitudinal effects of the Infant Behavioral Assessment and Intervention Program on cognitive and motor development of very preterm infants at the corrected ages of six months to five and a half years. METHODS: This randomised controlled trial divided 176 infants with a gestational age <32 weeks or birthweight <1500 g into intervention (n = 86) and control (n = 90) groups. Cognitive development and motor development were assessed with the Bayley Scales of Infant Development at the CAs of six, 12 and 24 months and at five and a half years with the Wechsler Preschool and Primary Scale of Intelligence and the Movement Assessment Battery for Children. RESULTS: We found significant longitudinal intervention effects (0.4 SD, p = 0.006) on motor development, but no significant impact on cognitive development (p = 0.063). Infants with bronchopulmonary dysplasia showed significant longitudinal intervention effects for cognitive (0.7 SD; p = 0.019) and motor (0.9 SD; p = 0.026) outcomes. Maternal education had little effect on intervention effects over time. CONCLUSION: The Infant Behavioral Assessment and Intervention Program led to long-term developmental improvements in the intervention group, especially in infants with BPD.

Increased prevalence of influenza B/Victoria lineage viruses during early stages of the 2015 influenza season in New South Wales, Australia: implications for vaccination and planning.

During the early weeks of the 2015 Australian influenza season, influenza B accounted for 67% (821/1,234) of all positive influenza tests in New South Wales. Of 81 successive influenza B viruses characterised, 33 (41%) were from children aged < 16 years; 23/81 (28%) belonged to the B/Victoria lineage. This lineage is not contained in the southern hemisphere's 2015 trivalent influenza vaccine. The significant B/Victoria lineage activity in the southern hemisphere suggests that the quadrivalent vaccine should be considered for the northern hemisphere.

Vancomycin-associated drug reaction with eosinophilia and systemic symptoms syndrome.

Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a rare but potentially life-threatening multi-system disorder characterised by the delayed onset of fever, rash and internal organ involvement following the administration of a drug. We report three definite cases of vancomycin-associated DRESS syndrome occurring and review the literature regarding this syndrome.

EfaR is a major regulator of Enterococcus faecalis manganese transporters and influences processes involved in host colonization and infection.

Metal ions, in particular manganese, are important modulators of bacterial pathogenicity. However, little is known about the role of manganese-dependent proteins in the nosocomial pathogen Enterococcus faecalis, a major cause of bacterial endocarditis. The present study demonstrates that the DtxR/MntR family metalloregulator EfaR of E. faecalis controls the expression of several of its regulon members in a manganese-dependent way. We also show that efaR inactivation impairs the ability of E. faecalis to form biofilms, to survive inside macrophages, and to tolerate oxidative stress. Our results reveal that EfaR is an important modulator of E. faecalis virulence and link manganese homeostasis to enterococcal pathogenicity.

The effect of preimplantation genetic screening on neurological, cognitive and behavioural development in 4-year-old children: follow-up of a RCT.

STUDY QUESTION: Does embryo biopsy inherent to preimplantation genetic screening (PGS) affect neurological, cognitive and behavioural development of 4-year-old children? SUMMARY ANSWER: PGS does not seem to affect neurological, cognitive and behavioural development of 4-year-old singletons; however, our data suggest that it may be associated with altered neurodevelopment in twins. WHAT IS KNOWN ALREADY: Evidence concerning the safety of PGS on neurodevelopmental outcome in offspring is scarce. The present study provides information on neurodevelopmental, cognitive and behavioural outcome of 4-year-old PGS offspring. STUDY DESIGN, SIZE, DURATION: A prospective, assessor-blinded follow-up study of children born to women who participated in a multi-centre RCT on the effect of IVF with or without PGS. PARTICIPANTS/MATERIALS, SETTING, METHODS: At 4 years, 49 children (31 singletons, 9 sets of twins) born following IVF with PGS and 64 children (42 singletons, 11 sets of twins) born following IVF without PGS (controls) were assessed (post-natal attrition 18%). Neurological development was evaluated with the standardized, age-specific and sensitive neurological examination according to Hempel, resulting in a neurological optimality score (NOS), a fluency score and the rate of adverse neurological outcome. Primary outcome was the fluency score, as fluency of movements is easily reduced by subtle dysfunction of the brain. Cognitive development was evaluated with the Kaufman Assessment Battery for Children; behavioural development was evaluated with the Child Behavior Checklist. The effect of PGS was analysed with a mixed effects model. MAIN RESULTS AND THE ROLE OF CHANCE: Based on the intention to treat analysis, neurodevelopmental outcome of PGS children was similar to that of controls. However, additional analyses indicated that PGS affected neurodevelopmental outcome of twins in a different way than that of singletons. The fluency score of singletons born following PGS was similar to that of control singletons [mean values, 95% confidence intervals (CIs): 12.2 (11.5;12.8) and 12.2 (11.6;12.8)], respectively, P = 0.977) that was also true for the other neurodevelopmental parameters. The fluency score of PGS twins was significantly lower than that of control twins [mean values, 95% CIs: 10.6 (9.8;11.3) and 12.3 (11.5;13.1)], respectively, P = 0.001); the same was true for the NOS. In addition, PGS in twins was associated with a higher sequential intelligence quotient score. On the other hand, other neurodevelopmental parameters were similar for PGS twins and control twins. Post hoc sample size calculation for the primary outcome parameter, the fluency score, indicated that the study groups, including the subgroups of singletons and twins, were adequately powered. LIMITATIONS, REASONS FOR CAUTION: We assessed singletons and twins who contributed to the generalizability of the study. A limitation of our study is the relative small size of our study groups and the selective dropout in both groups (dropouts PGS group: higher gestational age; control group: less well-educated parents). These preclude the conclusion that PGS per se is not associated with neurodevelopmental, cognitive and behavioural problems in singletons and the conclusion that PGS is associated with altered neurodevelopmental outcome in twins. WIDER IMPLICATIONS OF THE FINDINGS: The need for careful long-term monitoring of children born following embryo biopsy remains, as it is still applied in the form of PGD and it is still unknown whether embryo biopsy affects long-term neurodevelopmental outcome.

Thermodynamic analysis of the thermal and exergetic performance of a mixed gas-steam aero derivative gas turbine engine for power generation.

A thermodynamic analysis is performed for an aero derivative gas turbine engine which utilizes steam injection to increase its efficiency. The target was to explore the performance of a high efficiency gas turbine unit for electric power generation without downstream Rankine cycle. A Rankine cycle for exhaust heat recovery is unattractive because of its large response time and cost of investment. The main purpose of this research was to develop a better understanding of how the optimal cycle efficiency is reached, when the steam for injection is generated by use of the turbine exhaust heat. The STIG cycle becomes attractive for grid stabilization because of its low CAPEX and small footprint and response time. A thermodynamic model has been developed to simulate the simple cycle gas turbine, steam generation and effects of steam injection. Reference input parameters for the model are taken for the GE LM6000 turbine as publicly available. The performance of the engine without steam injection as predicted by the model is compared with literature for validation and compares well. The performance of the STIG cycle as a function of operation parameter steam mass flow and design parameters pressure ratio and turbine inlet temperature is investigated and the optimal parameter settings determined. It is found that this type of cycle shows a very specific parameter setting for optimal efficiency. By using steam injection for the chosen turbine and its parameters an efficiency gain of around 11% points and an output power augmentation of 45% can be achieved.

Variations in the definition and perceived importance of positive resection margins in patients with colorectal cancer - an EYSAC international survey.

INTRODUCTION: Microscopically positive resection margins (R1) are associated with poorer outcomes in patients with colorectal cancer. However, different definitions of R1 margins exist. It is unclear to what extent the definitions used in everyday clinical practice differ within and between nations. This study sought to investigate variations in the definition of R1 margins in colorectal cancer and the importance of margin status in clinical decision-making. MATERIALS AND METHODS: A 14-point survey was developed by members of The European Society of Surgical Oncology (ESSO) Youngs Surgeons and Alumni Club (EYSAC) Research Academy targeting all members of the multidisciplinary team (MDT) treating patients with colorectal cancer. The survey was distributed on social media, in ESSO's monthly newsletter and via national societies. RESULTS: In total, 137 responses were received. Most respondents were from Europe (89.7%), with the majority from Denmark (56.9%). Less than 2/3 of respondents defined R1 margins as the presence of viable cancer cells ≤1 mm of the margin. Only 60% reported that subdivisions of R1 margins (primary tumour vs tumour deposit vs metastatic lymph node) are routinely available. More than 20% of respondents reported that pathology reports are not routinely reviewed at MDT meetings. Less than half of respondents considered margin status in decision-making for type and duration of adjuvant chemotherapy in Stage III colon cancer. CONCLUSION: The definitions and perceived clinical importance of microscopically positive margins in patients with colorectal cancer appear to vary. Adoption of an international dataset for pathology reporting may help to standardise current practices.

Reliability, sensitivity and responsiveness of the Infant Behavioral Assessment in very preterm infants.

AIM: The aim of this study is to investigate the reliability, sensitivity and responsiveness of the Infant Behavioral Assessment (IBA) to evaluate neurobehavioural organization in very preterm infants. METHODS: Videotaped assessments of very preterm infants participating in a recent trial served to evaluate a standardized IBA observation. Inter-rater reliability was based on 40 videos scored by two independent observers, using percentage agreement and weighted Kappa's. Sensitivity was evaluated by comparing the IBA results of 169 infants at 35-38 weeks postmenstrual age, dichotomized according to two developmental risk factors. For responsiveness, the effect size (ES) was calculated between 0 and 6 months corrected age in all intervention and control infants and in subgroups of high-risk intervention and control infants with oxygen dependency ≥28 days. RESULTS: Inter-rater agreement was 93% in the total assessment; Kappa agreement was moderate to good in the behavioural categories. Significant differences were found between groups with or without risk factors. Larger differences between ESs in the randomized groups with oxygen dependency ≥28 days than in the total randomized groups reflect the responsiveness of the IBA. CONCLUSION: In this study, we found satisfactory to good clinimetric characteristics of the IBA in very preterm born infants.

[Neurotoxicity in elderly patients being treated with lithium: is there a reason to adjust the dosage administered?]. / Neurotoxiciteit tijdens lithiumgebruik bij ouderen: reden om behandelbeleid aan te passen?

BACKGROUND: Neurotoxicity can occur in patients being treated with lithium. Features are cognitive dysfunctioning and motor symptoms. Some results of research performed on adults up to the age of 65 indicate that lithium can cause mild cognitive dysfunctioning. It is not clear, however, whether elderly patients are more susceptible than young adults to this form of neurotoxicity or, if this is the case, whether the dosage of lithium should be reduced. AIM: To determine whether elderly patients treated with lithium run an increased risk of neurotoxicity, and to discuss the pharmacodynamic aspects of lithium use in the elderly which may cause neurotoxicity. METHOD: Literature review in Cochrane (all EBM), Embase, PsycINFO, Medline and PubMed, with cross-checked references. RESULTS: Few studies described possible neurotoxic effects of lithium. We found no indications for cognitive dysfunctioning in elderly patients being treated with lithium. However, a lithiumassociated tremor was seen more often in elderly patients than in younger adults. Pharmacodynamic effects of aging, such as an increase in the lithium concentration in the brain with no change in the serum level, may give rise to side-effects. More research is needed into the relationship between the serum level and the neurotoxic effects of lithium in the elderly. CONCLUSION: There are no indications that lithium causes more neurotoxicity in the elderly than in younger adults. If the use of lithium is indicated, it can be safely prescribed for the elderly, provided age-related pharmacodynamics are taken into account.

Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

Gene cluster for biosynthesis of thermophilin 1277--a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide.

AIMS: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer-walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9-kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI-expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. CONCLUSIONS: Thermophilin 1277 biosynthesis genes were encoded by a 9·9-kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.

Outside of the intensive care unit: the safety and effectiveness of an insulin-dextrose infusion protocol.

A review of patients who were on an insulin-dextrose (I-D) infusion protocol in general hospital wards over a 6-month period was conducted to compare glycaemic control before, during and following I-D infusion. Among the 34 cases identified, blood glucose levels during the period of I-D infusion were significantly better than those before or after I-D infusion. This I-D infusion protocol was shown to be effective and safe in maintaining good glycaemic control for patients outside intensive care unit.

Parenting stress in mothers after very preterm birth and the effect of the Infant Behavioural Assessment and Intervention Program.

OBJECTIVE: Purpose of this study was to examine maternal parenting stress as a secondary outcome of the Infant Behavioural Assessment and Intervention Program (IBAIP). METHODS: In a randomized controlled trial 86 very preterm infants and their parents were assigned to the intervention group and 90 to the control group. Maternal parenting stress was assessed with the Dutch version of the Parenting Stress Index at 12 and 24 months post term. RESULTS: Mothers in the intervention group mothers assessed their infants as happier and less hyperactive/distractible compared with the control group mothers. However, mothers in the intervention group reported more feelings of social isolation. CONCLUSIONS: The IBAIP appears to have made mothers more satisfied about their infants' mood and distractibility, but also may have evoked more feelings of social isolation. Next to long-term evaluation of the development in very preterm born children, follow-up on functioning of their parents is important.

Neighborhood walkability, physical activity and changes in glycemic markers in people with type 2 diabetes: The Hoorn Diabetes Care System cohort.

Studies investigating neighborhood walkability and physical activity in people with type 2 diabetes (T2D) mainly used self-report measures, and only few studies assessed the association with glycemic control. This study assessed the associations between objectively measured (i.e. GIS based) and subjectively measured (i.e. questionnaire-based) neighborhood walkability and changes in glycemic markers in people with T2D, and whether this association was mediated by device-measured physical activity (PA), in the Diabetes Care System Cohort (n = 1230). Neither objective or subjectively measured walkability was associated with glycemic control. In mediation analyses we observed no overall mediation by PA.