A comparison of morphokinetic models and morphological selection for prioritizing euploid embryos: a multicentre cohort study.

STUDY QUESTION: Are morphokinetic models better at prioritizing a euploid embryo for transfer over morphological selection by an embryologist? SUMMARY ANSWER: Morphokinetic algorithms lead to an improved prioritization of euploid embryos when compared to embryologist selection. WHAT IS KNOWN ALREADY: PREFER (predicting euploidy for embryos in reproductive medicine) is a previously published morphokinetic model associated with live birth and miscarriage. The second model uses live birth as the target outcome (LB model). STUDY DESIGN, SIZE, DURATION: Data for this cohort study were obtained from 1958 biopsied blastocysts at nine IVF clinics across the UK from January 2021 to December 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ability of the PREFER and LB models to prioritize a euploid embryo was compared against arbitrary selection and the prediction of four embryologists using the timelapse video, blinded to the morphokinetic time stamp. The comparisons were made using calculated percentages and normalized discounted cumulative gain (NDCG), whereby an NDCG score of 1 would equate to all euploid embryos being ranked first. In arbitrary selection, the ploidy status was randomly assigned within each cycle and the NDGC calculated, and this was then repeated 100 times and the mean obtained. MAIN RESULTS AND THE ROLE OF CHANCE: Arbitrary embryo selection would rank a euploid embryo first 37% of the time, embryologist selection 39%, and the LB and PREFER ploidy morphokinetic models 46% and 47% of the time, respectively. The AUC for LB and PREFER model was 0.62 and 0.63, respectively. Morphological selection did not significantly improve the performance of both morphokinetic models when used in combination. There was a significant difference between the NDGC metric of the PREFER model versus embryologist selection at 0.96 and 0.87, respectively (t = 14.1, P < 0.001). Similarly, there was a significant difference between the LB model and embryologist selection with an NDGC metric of 0.95 and 0.87, respectively (t = 12.0, P < 0.001). All four embryologists ranked embryos similarly, with an intraclass coefficient of 0.91 (95% CI 0.82-0.95, P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Aside from the retrospective study design, limitations include allowing the embryologist to watch the time lapse video, potentially providing more information than a truly static morphological assessment. Furthermore, the embryologists at the participating centres were familiar with the significant variables in time lapse, which could bias the results. WIDER IMPLICATIONS OF THE FINDINGS: The present study shows that the use of morphokinetic models, namely PREFER and LB, translates into improved euploid embryo selection. STUDY FUNDING/COMPETING INTEREST(S): This study received no specific grant funding from any funding agency in the public, commercial or not-for-profit sectors. Dr Alison Campbell is minor share holder of Care Fertility. All other authors have no conflicts of interest to declare. Time lapse is a technology for which patients are charged extra at participating centres. TRIAL REGISTRATION NUMBER: N/A.

The CRYOSTAT2 trial: The rationale and study protocol for a multi-Centre, randomised, controlled trial evaluating the effects of early high-dose cryoprecipitate in adult patients with major trauma haemorrhage requiring major haemorrhage protocol activation.

OBJECTIVES: To describe the protocol for a multinational randomised, parallel, superiority trial, in which patients were randomised to receive early high-dose cryoprecipitate in addition to standard major haemorrhage protocol (MHP), or Standard MHP alone. BACKGROUND: Blood transfusion support for trauma-related major bleeding includes red cells, plasma and platelets. The role of concentrated sources of fibrinogen is less clear and has not been evaluated in large clinical trials. Fibrinogen is a key pro-coagulant factor that is essential for stable clot formation. A pilot trial had demonstrated that it was feasible to deliver cryoprecipitate as a source of fibrinogen within 90 min of admission. METHODS: Randomisation was via opaque sealed envelopes held securely in participating Emergency Departments or transfusion laboratories. Early cryoprecipitate, provided as 3 pools (equivalent to 15 single units of cryoprecipitate or 6 g fibrinogen supplementation), was transfused as rapidly as possible, and started within 90 min of admission. Participants in both arms received standard treatment defined in the receiving hospital MHP. The primary outcome measure was all-cause mortality at 28 days. Symptomatic thrombotic events including venous thromboembolism and arterial thrombotic events (myocardial infarction, stroke) were collected from randomisation up to day 28 or discharge from hospital. EQ5D-5Land Glasgow Outcome Score were completed at discharge and 6 months. All analyses will be performed on an intention to treat basis, with per protocol sensitivity analysis. RESULTS: The trial opened for recruitment in June 2017 and the final patient completed follow-up in May 2022. DISCUSSION: This trial will provide firmer evidence to evaluate the effectiveness and cost-effectiveness of early high-dose cryoprecipitate alongside the standard MHP in major traumatic haemorrhage.

Case Report: Congenital tuberculosis in a premature infant requiring extracorporeal membrane oxygenation.

Congenital tuberculosis is a rare infectious disease with less than 500 cases documented worldwide. Mortality is significant, ranging from 34 to 53%, and death without treatment is inevitable. Patients exhibit nonspecific symptoms such as fever, cough, respiratory distress, feeding intolerance, and irritability which can make appropriate diagnosis challenging in Peng et al. (2011) Pediatr Pulmonol 46(12), 1215-1224. Tuberculosis prevalence is particularly high in developing countries where access to resources can be limited in World Health Organization (2019) Global tuberculosis report 2019, Geneva. We present a 2.4-kg premature male infant with acute respiratory distress syndrome secondary to congenital tuberculosis caused by Mycobacterium bovis and tuberculosis-immune reconstitution inflammatory syndrome who was successfully supported with veno-arterial extracorporeal membrane oxygenation.

Early and Empirical High-Dose Cryoprecipitate for Hemorrhage After Traumatic Injury: The CRYOSTAT-2 Randomized Clinical Trial.

Importance: Critical bleeding is associated with a high mortality rate in patients with trauma. Hemorrhage is exacerbated by a complex derangement of coagulation, including an acute fibrinogen deficiency. Management is fibrinogen replacement with cryoprecipitate transfusions or fibrinogen concentrate, usually administered relatively late during hemorrhage. Objective: To assess whether survival could be improved by administering an early and empirical high dose of cryoprecipitate to all patients with trauma and bleeding that required activation of a major hemorrhage protocol. Design, Setting, and Participants: CRYOSTAT-2 was an interventional, randomized, open-label, parallel-group controlled, international, multicenter study. Patients were enrolled at 26 UK and US major trauma centers from August 2017 to November 2021. Eligible patients were injured adults requiring activation of the hospital's major hemorrhage protocol with evidence of active hemorrhage, systolic blood pressure less than 90 mm Hg at any time, and receiving at least 1 U of a blood component transfusion. Intervention: Patients were randomly assigned (in a 1:1 ratio) to receive standard care, which was the local major hemorrhage protocol (reviewed for guideline adherence), or cryoprecipitate, in which 3 pools of cryoprecipitate (6-g fibrinogen equivalent) were to be administered in addition to standard care within 90 minutes of randomization and 3 hours of injury. Main Outcomes and Measures: The primary outcome was all-cause mortality at 28 days in the intention-to-treat population. Results: Among 1604 eligible patients, 799 were randomized to the cryoprecipitate group and 805 to the standard care group. Missing primary outcome data occurred in 73 patients (principally due to withdrawal of consent) and 1531 (95%) were included in the primary analysis population. The median (IQR) age of participants was 39 (26-55) years, 1251 (79%) were men, median (IQR) Injury Severity Score was 29 (18-43), 36% had penetrating injury, and 33% had systolic blood pressure less than 90 mm Hg at hospital arrival. All-cause 28-day mortality in the intention-to-treat population was 26.1% in the standard care group vs 25.3% in the cryoprecipitate group (odds ratio, 0.96 [95% CI, 0.75-1.23]; P = .74). There was no difference in safety outcomes or incidence of thrombotic events in the standard care vs cryoprecipitate group (12.9% vs 12.7%). Conclusions and Relevance: Among patients with trauma and bleeding who required activation of a major hemorrhage protocol, the addition of early and empirical high-dose cryoprecipitate to standard care did not improve all cause 28-day mortality. Trial Registration: ClinicalTrials.gov Identifier: NCT04704869; ISRCTN Identifier: ISRCTN14998314.

Highly Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe PCR.

Tools to detect SARS-CoV-2 variants of concern and track the ongoing evolution of the virus are necessary to support public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. To this aim, an allele-specific probe PCR (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. The comparative advantage for ASP-PCR over NGS was most pronounced in samples with cycle threshold (CT) values between 26 and 30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. ASP-PCR is well suited to augment but not replace NGS. The method can differentiate SARS-CoV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer-target base mismatch through altered oligonucleotide chemistry or chemical additives.

Virological Characterization of Critically Ill Patients With COVID-19 in the United Kingdom: Interactions of Viral Load, Antibody Status, and B.1.1.7 Infection.

BACKGROUND: Convalescent plasma containing neutralizing antibody to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is under investigation for coronavirus disease 2019 (COVID-19) treatment. We report diverse virological characteristics of UK intensive care patients enrolled in the Immunoglobulin Domain of the REMAP-CAP randomized controlled trial that potentially influence treatment outcomes. METHODS: SARS-CoV-2 RNA in nasopharyngeal swabs collected pretreatment was quantified by PCR. Antibody status was determined by spike-protein ELISA. B.1.1.7 was differentiated from other SARS-CoV-2 strains using allele-specific probes or restriction site polymorphism (SfcI) targeting D1118H. RESULTS: Of 1274 subjects, 90% were PCR positive with viral loads 118-1.7 × 1011IU/mL. Median viral loads were 40-fold higher in those IgG seronegative (n = 354; 28%) compared to seropositives (n = 939; 72%). Frequencies of B.1.1.7 increased from <1% in November 2020 to 82% of subjects in January 2021. Seronegative individuals with wild-type SARS-CoV-2 had significantly higher viral loads than seropositives (medians 5.8 × 106 and 2.0 × 105 IU/mL, respectively; P = 2 × 10-15). CONCLUSIONS: High viral loads in seropositive B.1.1.7-infected subjects and resistance to seroconversion indicate less effective clearance by innate and adaptive immune responses. SARS-CoV-2 strain, viral loads, and antibody status define subgroups for analysis of treatment efficacy.

Effect of Convalescent Plasma on Organ Support-Free Days in Critically Ill Patients With COVID-19: A Randomized Clinical Trial.

IMPORTANCE: The evidence for benefit of convalescent plasma for critically ill patients with COVID-19 is inconclusive. OBJECTIVE: To determine whether convalescent plasma would improve outcomes for critically ill adults with COVID-19. DESIGN, SETTING, AND PARTICIPANTS: The ongoing Randomized, Embedded, Multifactorial, Adaptive Platform Trial for Community-Acquired Pneumonia (REMAP-CAP) enrolled and randomized 4763 adults with suspected or confirmed COVID-19 between March 9, 2020, and January 18, 2021, within at least 1 domain; 2011 critically ill adults were randomized to open-label interventions in the immunoglobulin domain at 129 sites in 4 countries. Follow-up ended on April 19, 2021. INTERVENTIONS: The immunoglobulin domain randomized participants to receive 2 units of high-titer, ABO-compatible convalescent plasma (total volume of 550 mL ± 150 mL) within 48 hours of randomization (n = 1084) or no convalescent plasma (n = 916). MAIN OUTCOMES AND MEASURES: The primary ordinal end point was organ support-free days (days alive and free of intensive care unit-based organ support) up to day 21 (range, -1 to 21 days; patients who died were assigned -1 day). The primary analysis was an adjusted bayesian cumulative logistic model. Superiority was defined as the posterior probability of an odds ratio (OR) greater than 1 (threshold for trial conclusion of superiority >99%). Futility was defined as the posterior probability of an OR less than 1.2 (threshold for trial conclusion of futility >95%). An OR greater than 1 represented improved survival, more organ support-free days, or both. The prespecified secondary outcomes included in-hospital survival; 28-day survival; 90-day survival; respiratory support-free days; cardiovascular support-free days; progression to invasive mechanical ventilation, extracorporeal mechanical oxygenation, or death; intensive care unit length of stay; hospital length of stay; World Health Organization ordinal scale score at day 14; venous thromboembolic events at 90 days; and serious adverse events. RESULTS: Among the 2011 participants who were randomized (median age, 61 [IQR, 52 to 70] years and 645/1998 [32.3%] women), 1990 (99%) completed the trial. The convalescent plasma intervention was stopped after the prespecified criterion for futility was met. The median number of organ support-free days was 0 (IQR, -1 to 16) in the convalescent plasma group and 3 (IQR, -1 to 16) in the no convalescent plasma group. The in-hospital mortality rate was 37.3% (401/1075) for the convalescent plasma group and 38.4% (347/904) for the no convalescent plasma group and the median number of days alive and free of organ support was 14 (IQR, 3 to 18) and 14 (IQR, 7 to 18), respectively. The median-adjusted OR was 0.97 (95% credible interval, 0.83 to 1.15) and the posterior probability of futility (OR <1.2) was 99.4% for the convalescent plasma group compared with the no convalescent plasma group. The treatment effects were consistent across the primary outcome and the 11 secondary outcomes. Serious adverse events were reported in 3.0% (32/1075) of participants in the convalescent plasma group and in 1.3% (12/905) of participants in the no convalescent plasma group. CONCLUSIONS AND RELEVANCE: Among critically ill adults with confirmed COVID-19, treatment with 2 units of high-titer, ABO-compatible convalescent plasma had a low likelihood of providing improvement in the number of organ support-free days. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02735707.

Sustained Total All-Region (STAR) Perfusion: An Optimized Perfusion Strategy for Norwood Reconstruction.

Early iterations of the Norwood procedure used aortic cross-clamping, myocardial arrest, and, sometimes, deep hypothermic circulatory arrest. The resulting hypothermia and prolonged ischemia caused frequent cardiac, neurologic, renal, and other end-organ dysfunctions. Our group describes a novel technique, sustained total all-region (STAR) perfusion, which circumvents these issues by providing continuous perfusion to the head, heart, and coronaries at temperatures of 32-34°C. A single DLP® straight venous cannula (Medtronic, Minneapolis, MN) is placed in the right atrium, and a DLP® pediatric arterial cannula, with a high-flow stopcock attached, is placed in the ascending aorta or innominate artery to provide flow to the head. A cardioplegia needle with walrus tubing is connected to the stopcock to provide flow to the coronary arteries. For lower body perfusion, an olive tip cannula is placed into the descending aorta lumen and attached to the 1/8″ line from the cardioplegia system which provides warm arterial blood flow. STAR perfusion allows the Norwood procedure to be completed with mild hypothermia and continuous perfusion to all vascular beds with reduced cardiopulmonary bypass as well as total operative times. This technique is successfully achieved with minimal changes to circuitry, minor modifications to heart-lung machine servoregulation and few additional cannulation disposables.

The Influence of Stigma on Suicide Bereavement: A Systematic Review.

Suicide loss affects a significant proportion of the population, and despite its prevalence there remains a stigma surrounding death by suicide. It is important for health professionals to understand the effects of suicide stigma on surviving friends and family members so that they can respond appropriately and provide effective support. A systematic review of the literature was conducted to evaluate the impact of public stigma on bereavement of suicide survivors. A total of 11 qualitative and quantitative studies were reviewed. Suicide survivors reported feeling shamed, blamed, and judged. They perceived a general discomfort and awkwardness surrounding the suicide, which contributed to avoidance and secrecy. Higher perceived stigma levels were associated with global psychological distress, depression, self-harm, and suicidality. Despite the recognized negative impact of suicide stigma on the bereaved, studies on grief interventions for suicide survivors are scarce. There is a critical need for research and evidence-based recommendations on how to best to support this vulnerable population. [Journal of Psychosocial Nursing and Mental Health Services, 58(4), 21-27.].

Evaluating the Impact of the Addition of Cladribine to Standard Acute Myeloid Leukemia Induction Therapy.

BACKGROUND: Treatment for acute myeloid leukemia (AML) has remained relatively unchanged over the past few decades. Although recent drug approvals have provided an increase in the number of treatment options in AML, further optimization of standard induction therapy is still necessary. The most commonly utilized induction options have been well studied, but there is a paucity of literature comparing the combination of idarubicin with cytarabine and cladribine. OBJECTIVE: To assess the clinical effectiveness of the addition of cladribine to idarubicin and cytarabine (7+3 IA) induction therapy in the treatment of AML. METHODS: This retrospective, propensity score-matched cohort study evaluated 37 patients with previously untreated AML who received either 7+3 IA or idarubicin, cytarabine, and cladribine (7+3+5 IAC) as induction therapy. The primary end point of this study was complete response (CR), with secondary end points including hospital length of stay (LOS), and adverse event rates. RESULTS: After propensity score matching, odds of reaching CR in the 7+3+5 IAC cohort were increased by 33% (95% CI = 1.09-1.55; P < 0.01) compared with the 7+3 IA cohort. Patients who received cladribine were also found to have a reduction in hospital LOS by 3.5 days (95% CI = 0.07-6.85; P = 0.045) without an increase in adverse event rates. CONCLUSION: The addition of cladribine to the 7+3 IA regimen may improve clinical outcomes when used as initial induction therapy, without increasing the incidence of adverse event rates.

A randomised controlled trial of the clinical and cost-effectiveness of ultrasound-guided intra-articular corticosteroid and local anaesthetic injections: the hip injection trial (HIT) protocol.

BACKGROUND: Evidence on the effectiveness of intra-articular corticosteroid injection for hip osteoarthritis is limited and conflicting. The primary objective of the Hip Injection Trial (HIT) is to compare pain intensity over 6 months, in people with hip OA between those receiving an ultrasound-guided intra-articular hip injection of corticosteroid with 1% lidocaine hydrochloride plus best current treatment with those receiving best current treatment alone. Secondary objectives are to determine specified comparative clinical and cost-effectiveness outcomes, and to explore, in a linked qualitative study, the lived experiences of patients with hip OA and experiences and impact of, ultrasound-guided intra-articular hip injection. METHODS: The HIT trial is a pragmatic, three-parallel group, single-blind, superiority, randomised controlled trial in patients with painful hip OA with a linked qualitative study. The current protocol is described, in addition to details and rationale for amendments since trial registration. 204 patients with moderate-to-severe hip OA will be recruited. Participants are randomised on an equal basis (1:1:1 ratio) to one of three interventions: (1) best current treatment, (2) best current treatment plus ultrasound-guided intra-articular hip injection of corticosteroid (triamcinolone acetonide 40 mg) with 1% lidocaine hydrochloride, or (3) best current treatment plus an ultrasound-guided intra-articular hip injection of 1% lidocaine hydrochloride alone. The primary endpoint is patient-reported hip pain intensity across 2 weeks, 2 months, 4 months and 6 months post-randomisation. Recruitment is over 29 months with a 6-month follow-up period. To address the primary objective, the analysis will compare participants' 'average' follow-up pain NRS scores, based on a random effects linear repeated-measures model. Data on adverse events are collected and reported in accordance with national guidance and reviewed by external monitoring committees. Individual semi-structured interviews are being conducted with up to 30 trial participants across all three arms of the trial. DISCUSSION: To ensure healthcare services improve outcomes for patients, we need to ensure there is a robust and appropriate evidence-base to support clinical decision making. The HIT trial will answer important questions regarding the clinical and cost-effectiveness of intra-articular corticosteroid injections. TRIAL REGISTRATION: ISRCTN: 50550256 , 28th July 2015.

Biosimilar perceptions among autoimmune prescribers and pharmacists in health system specialty pharmacy.

BACKGROUND: By the end of 2023, 10 self-administered biosimilars indicated for autoimmune conditions are expected to launch in the United States, resulting in alternative treatment options for patients and a potential for cost savings. However, studies about perception and knowledge of self-administered biosimilars among health system prescribers and health system specialty pharmacists are limited. OBJECTIVE: To assesses knowledge and perceptions of biosimilars among autoimmune prescribers and health system specialty pharmacists across the United States. METHODS: An anonymous, cross-sectional self-administered online questionnaire was conducted among prescribers and health system specialty pharmacists practicing in the specialties of rheumatology, dermatology, and gastroenterology across the United States. The survey was available from January 2023 to February 2023. RESULTS: 31 prescribers and 44 pharmacists completed the questionnaire. Only 16.0% of prescribers and 13.4% of pharmacists reported being "very prepared" to have conversations with patients about biosimilar options. 43% of prescribers indicated they would prescribe a biosimilar to biologic naive patients. However, 13.3% of prescribers would be willing to prescribe a biosimilar to patients successfully established on biologic therapy. Among pharmacists, 68.1% were comfortable recommending a biosimilar substitution to a biologic naive patient, but only 18.1% would recommend a biosimilar substitution to an existing patient successfully established on a biologic therapy. Less than half of prescribers (48.0%) and pharmacists (42.0%) understood regulations of interchangeability and substitution. CONCLUSIONS: Our study highlights several knowledge gaps and hesitancies that exist among health system specialty prescribers and pharmacists regarding biosimilar products. Education efforts are needed to overcome the lack of biosimilar adoption, which will increase affordability of therapy for patients and health care savings.

Survival after partial heart transplantation in a piglet model.

Partial heart transplantation (PHT) is a novel surgical approach that involves transplantation of only the part of the heart containing a valve. The rationale for this approach is to deliver growing heart valve implants that reduce the need for future re-operations in children. However, prior to clinical application of this approach, it was important to assess it in a preclinical model. To investigate PHT short-term outcomes and safety, we performed PHT in a piglet model. Yorkshire piglets (n = 14) were used for PHT of the pulmonary valve. Donor and recipient pairs were matched based on blood types. The piglets underwent PHT at an average age of 44 days (range 34-53). Post-operatively, the piglets were monitored for a period of two months. Of the 7 recipient piglets, one mortality occurred secondary to anesthesia complications while undergoing a routine echocardiogram on post-operative day 19. All piglets had appropriate weight gain and laboratory findings throughout the post-operative period indicating a general state of good health and rehabilitation after undergoing PHT. We conclude that PHT has good short-term survival in the swine model. PHT appears to be safe for clinical application.

Developing Guidance for Donor Intervention Randomized Controlled Trials: Initial Discussions From the Canada-United Kingdom 2022 Workshop.

BACKGROUND: Donor interventions, including medications, protocols, and medical devices administered to donors, can enhance transplantable organ quality and quantity and maximize transplantation success. However, there is paucity of high-quality evidence about their effectiveness, in part because of ethical, practical, and regulatory challenges, and lack of guidance about conduct of donor intervention randomized controlled trials (RCTs). METHODS: With the vision to develop authoritative guidance for conduct of donor intervention RCTs, we convened a workshop of Canadian-United Kingdom experts in organ donation and transplantation ethics, research, and policy to identify stakeholders, explore unique challenges, and develop research agenda to inform future work in this promising field. RESULTS: Donor intervention trials should consider perspectives of broad group of stakeholders including donors, transplant recipients, and their families; researchers in donation and transplantation; research ethics boards; and healthcare providers and administrators involved in donation and transplantation. Unique challenges include (1) research ethics (living versus deceased status of the donor at the time of intervention, intervention versus outcomes assessment in different individuals, harm-benefit analysis in donors versus recipients, consent, and impact on research bystanders); (2) outcome data standardization and linkage; and (3) regulatory and governance considerations. CONCLUSIONS: Donor intervention RCTs hold potential to benefit organ transplantation outcomes but face unique research ethics, outcome data, and regulatory challenges. By developing research agenda to address these challenges, our workshop was an important first step toward developing Canada-United Kingdom guidance for donor intervention RCTs that are poised to improve the quality and availability of transplantable organs.

Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae.

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.

A pilot PT scheme for external assessment of laboratory performance in testing synthetic opioid compounds in urine, plasma, and whole blood.

A proficiency testing (PT) scheme was prepared for laboratories engaged in bioanalytical testing for synthetic opioid compounds in urine, plasma, and whole blood. Samples were prepared using compounds included in the Opioid Certified Reference Material Kit (Opioid CRM Kit) developed by the U.S. Centers for Disease Control and Prevention. Laboratories received samples during a 2-year project with each year consisting of two PT events 6 months apart. In the first year (pilot test), participants included 10 public health laboratories throughout the United States. In the second year, the group of laboratories expanded to include clinical and forensic drug testing laboratories, and 12 additional participating labs joined the program. In Year 1, overall detection percentages for the compounds present in the PT samples were 95.5% in Event 1% and 97.2% in Event 2. There were 31 apparent false positives reported in Event 1 and four apparent false positives reported in Event 2. Carryover or contamination in laboratory analytical systems were found to be the most significant causes of the false positive results, and none of the laboratories that reported false positives in Event 1 did so in Event 2. In Year 2, overall detection percentages for the compounds present in the PT samples were 89.5% in Event 3% and 94.8% in Event 4. There was one apparent false positive reported in Event 3 and three apparent false positives reported in Event 4. Improvements in drug detection between the two PT events in each year demonstrated the benefit of PT schemes in identifying and addressing potential deficiencies in laboratory systems.

Performance of Hair Testing for Cocaine Use-Comparison of Five Laboratories Using Blind Reference Specimens.

The purpose of this study was to compare results from five commercial hair testing laboratories conducting workplace drug testing with regard to bias, precision, selectivity and decontamination efficiency. Nine blind hair specimens, including cocaine-positive drug user specimens (some contaminated with methamphetamine) and negative specimens contaminated with cocaine, were submitted in up to five replicates to five different laboratories. All laboratories correctly identified cocaine in all specimens from drug users. For an undamaged hair specimen from a cocaine user, within-laboratory Coefficients of Variation (CVs) of 5-22% (median 8%) were reported, showing that it is possible to produce a homogenous proficiency testing sample from drug user hair. Larger CVs were reported for specimens composed of blended hair (up to 29%) and curly/damaged hair (19-67%). Quantitative results appeared to be method-dependent, and the reported cocaine concentrations varied up to 5-fold between the laboratories, making interlaboratory comparisons difficult. All laboratories reported at least one positive result in specimens contaminated with cocaine powder, followed by sweat and shampoo treatments. Benzoylecgonine, norcocaine, cocaethylene and hydroxylated cocaine metabolites were all detected in cocaine powder-contaminated specimens. This indicates that current industry standards for analyzing and reporting positive cocaine results are not completely effective at identifying external contamination. Metabolite ratios between meta- or para-hydroxy-cocaine and cocaine were 6- and 10-fold lower in contaminated specimens compared to those observed in cocaine user specimens, supporting their potential use in distinguishing samples positive due to contamination and drug use.

British Association of Sexual Health and HIV (BASHH) United Kingdom national guideline for the management of sexually transmitted enteric infections 2023.

This is the first British Association of Sexual Health and HIV (BASHH) national guideline for the management of sexually transmitted enteric infections (STEI). This guideline is primarily aimed for level 3 sexual health clinics; however, it may also be applicable to other settings such as primary care or other hospital departments where individuals with STEI may present. This guideline makes recommendations on testing, management, partner notification and public health control of STEI.

Ex Vivo Gene Delivery to Porcine Cardiac Allografts Using a Myocardial-Enhanced Adeno-Associated Viral Vector.

Transplantation, the gold standard intervention for organ failure, is a clinical field that is ripe for applications of gene therapy. One of the major challenges in applying gene therapy to this field is the need for a method that achieves consistent and robust gene delivery to allografts. Normothermic ex vivo perfusion is a growing organ preservation method and a device for cardiac preservation was recently approved by the Food and Drug Administration (FDA) (Organ Care System, OCS™; TransMedics, Inc., Andover, MA); this device maintains donor hearts in a near physiologic state while they are transported from the donor to the recipient. This study describes the administration of recombinant adeno-associated viral vectors (rAAVs) during ex vivo normothermic perfusion for the delivery of transgenes to porcine cardiac allografts. We utilized a myocardial-enhanced AAV3b variant, SASTG, assessing its transduction efficiency in the OCS perfusate relative to other AAV serotypes. We describe the use of normothermic ex vivo perfusion to deliver SASTG carrying the Firefly Luciferase transgene to porcine donor hearts in four heterotopic transplant procedures. Durable and dose-dependent transgene expression was achieved in the allografts in 30 days, with no evidence of off-target transgene expression. This study demonstrates the feasibility and efficiency of delivering genes to a large animal allograft utilizing AAV vectors during ex vivo perfusion. These findings support the idea of gene therapy interventions to enhance transplantation outcomes.