Development and validation of a high throughput SARS-CoV-2 whole genome sequencing workflow in a clinical laboratory.

Monitoring new mutations in SARS-CoV-2 provides crucial information for identifying diagnostic and therapeutic targets and important insights to achieve a more effective COVID-19 control strategy. Next generation sequencing (NGS) technologies have been widely used for whole genome sequencing (WGS) of SARS-CoV-2. While various NGS methods have been reported, one chief limitation has been the complexity of the workflow, limiting the scalability. Here, we overcome this limitation by designing a laboratory workflow optimized for high-throughput studies. The workflow utilizes modified ARTIC network v3 primers for SARS-CoV-2 whole genome amplification. NGS libraries were prepared by a 2-step PCR method, similar to a previously reported tailed PCR method, with further optimizations to improve amplicon balance, to minimize amplicon dropout for viral genomes harboring primer-binding site mutation(s), and to integrate robotic liquid handlers. Validation studies demonstrated that the optimized workflow can process up to 2688 samples in a single sequencing run without compromising sensitivity and accuracy and with fewer amplicon dropout events compared to the standard ARTIC protocol. We additionally report results for over 65,000 SARS-CoV-2 whole genome sequences from clinical specimens collected in the United States between January and September of 2021, as part of an ongoing national genomics surveillance effort.

Successful alectinib desensitization in a patient with anaplastic lymphoma kinase-positive adenocarcinoma of the lung and alectinib-induced drug rash.

INTRODUCTION: Alectinib is an oral tyrosine kinase inhibitor currently recommended by the National Comprehensive Cancer Network (NCCN) as the preferred first-line treatment option for the treatment of metastatic anaplastic lymphoma kinase (ALK) gene rearrangement-positive non-small cell lung cancer (NSCLC). Skin toxicity is a known adverse effect of this medication, yet current recommendations are unclear regarding how to best manage patients who develop severe skin toxicity while taking alectinib. CASE REPORT: Here, we describe a case of successful rechallenge with alectinib by utilizing a desensitization procedure in a patient who had developed severe alectinib-induced skin toxicity about two weeks into treatment.Management and outcome: Upon resolution of the initial skin toxicity symptoms, the patient was rechallenged with alectinib using a modified version of a previously published desensitization procedure. The patient tolerated the rechallenge with no recurrence of skin toxicity or other adverse effects and was able to continue treatment with alectinib. DISCUSSION: Alectinib is currently recommended as the preferred first-line treatment option for the treatment of metastatic anaplastic lymphoma kinase gene rearrangement-positive NSCLC due to improved progression-free survival when compared to crizotinib. The development of skin toxicity can lead to early discontinuation of alectinib treatment, forcing providers and patients to select alternative, potentially less effective options. This case report provides evidence that patients who have experienced severe skin toxicity due to alectinib may be able to continue this first-line treatment option by rechallenging them using a desensitization procedure.

Neonatal Kawasaki syndrome presenting as out of hospital cardiac arrest rescued by extracorporeal cardiopulmonary resuscitation.

A 7-week-old girl presented in severe shock to a local emergency department. During transfer to the quaternary pediatric hospital, the child had a cardiac arrest and cardiopulmonary resuscitation was commenced en route. Upon arrival to the pediatric intensive care unit, extracorporeal life support was initiated via trans-sternal cannulation. Chest CT performed after extracorporeal life support cannulation, demonstrated widespread aneurysms and a diagnosis of Kawasaki disease was made. Immunomodulatory therapy with immunoglobulin and glucocorticoid medication was commenced and the child was separated from extracorporeal life support after 48 hours. Our case highlights both an unusual presentation of Kawasaki disease and the role extracorporeal cardiopulmonary resuscitation can play in the treatment of this disease. It describes the youngest reported patient in the literature with Kawasaki disease rescued by extracorporeal cardiopulmonary resuscitation and highlights how extracorporeal life support therapy can facilitate appropriate investigations to resolve diagnostic uncertainty and treat the underlying condition.

Responding to sustained poor outcomes in the management of non-communicable diseases (NCDs): an "incident control" approach is needed to improve and protect population health.

In 2017 Public Health England were asked to assist with investigating why 1-year cancer survival rates appeared lower than expected in a local area. We identified 50 premature deaths that surveillance data suggested we would not expect. These deaths highlighted a gap in recognising and responding to this kind of systematic non communicable disease (NCD) outcome variation. We hypothesise that the lack of a universally agreed systematic response to variations is not only counter-intuitive, but wholly unacceptable where non-communicable diseases (NCDs) rather than infectious diseases have become the leading causes of illness and death worldwide. In the United Kingdom (UK) alone over 89% of mortality in 2014 was attributable to NCDs. We argue that a new approach is urgently needed to turn the curve on NCD outcome variation to protect and improve the public's health. We set out a definition of an NCD "incident" and propose a phased approach that could be used to respond to local variation in NCD outcomes.Establishing parity of response for local variations in NCD outcomes and CD control is critically important. Although evidence shows that prevention and early intervention will make the biggest difference to NCD incidence, collective local whole health economy response, exploiting the wealth of surveillance data in real time, needs to be at the heart of responding to variations in NCD outcomes at a population level. We argue that local and national public health agencies should mandate a standardised 'incident' response to significant changes in outcomes from NCD to mitigate and reduce the loss of quality life.

Prenatal cell-free DNA screening for fetal aneuploidy in pregnant women at average or high risk: Results from a large US clinical laboratory.

BACKGROUND: We evaluated the performance of a cell-free DNA (cfDNA) prenatal screening assay for trisomies 21, 18, and 13, and sex chromosome aneuploidies (SCAs) among a population of pregnant women that included both those at average and high risk. METHODS: Specimen collection, cfDNA extraction, massively parallel sequencing, and bioinformatics analysis were conducted per laboratory protocol. Assay results, concordance with pregnancy outcomes, and performance characteristics were evaluated. RESULTS: A total 75,658 specimens from 72,176 individual pregnant women were received. Technical reasons accounted for 288 (0.4% of all received samples) tests not performed. In the final analysis cohort (N = 69,794), 13% of pregnancies were considered at average risk and 87% at high risk. Mean gestational age at specimen collection was 15.1 weeks. Of the 69,794 unique pregnancies, 1,359 (1.9%) had positive test results. Among the results with confirmed outcomes, PPV for trisomies 21, 18, and 13 was 98.1%, 88.2%, and 59.3%, respectively; the PPV was 69.0% for SCAs and 75.0% for microdeletions. Overall, PPV was 87.2%, sensitivity was 97.9%, and specificity was 99.9%. CONCLUSION: This cfDNA prenatal screening assay provides highly accurate discrimination between affected and unaffected pregnancies among a population of pregnant women at average and high risk for fetal genetic abnormalities.

Balloon Aortic Valvuloplasty for Congenital Aortic Stenosis: A 14-Year Single Centre Review.

BACKGROUND: The approach to intervention for congenital aortic valve stenosis (AS) differs depending upon centre bias toward a primary catheter or surgical approach. We therefore investigated associations with freedom from re-intervention (FFI) in the cohort of children who underwent primary balloon aortic valvuloplasty (BAV) for congenital AS in our centre. METHODS: All patients who underwent BAV as a primary procedure in the period between 2001 and 2015 in a single service were included. Echocardiographic parameters before and after catheterisation and procedural data was collected on all patients. RESULTS: Sixty-four (64) patients underwent BAV as the primary intervention during the study period. Follow-up data was available for 60 of these. Balloon aortic valvuloplasty was performed at a median age of 143 days (range 2 days-18.8 years). Freedom from re-intervention was observed in 75% of patients with a median follow-up of 6.8 years and a mean follow-up of 3 years. Catheter-based peak-to-peak aortic valve gradients decreased from 58±15.9mmHg to 22.9±13.1mmHg. There was no short- or long-term mortality. FFI was predicted by aortic valve morphology (p<0.01), post-BAV mean echo gradient (p=0.03) and post-BAV regurgitation (p<0.01). No patient had re-intervention for restenosis with post-BAV mean echo gradient <30mmHg. Catheter gradients before and after BAV approached significance for predicting FFI (p=0.06 and p=0.09 respectively). Fifteen (15) patients were neonates with significantly lower aortic valve (AoV) Z-scores (mean 0.63 vs 1.76, p=0.002) and no difference in FFI (p=0.19). Annulus size, balloon/annulus ratio (within the range utilised) and pre-BAV echo findings were not predictive for re-intervention. CONCLUSIONS: Balloon aortic valvuloplasty is an effective primary approach to congenital valvular AS with the potential of avoiding surgical intervention in the majority of patients at all ages. Freedom from re-intervention in our cohort was associated with valve morphology and the degree of stenosis and regurgitation immediately post BAV.

"Get to Know Me" Board.

For best outcomes, clinicians in the ICU need to attend not only to the immediate clinical needs of the critically ill patient, but also to higher human needs including psychologic, social, and spiritual. Understanding your patient as another human being with her or his fears, desires, preferences, and accomplishments is obviously important in order to provide compassionate care and achieve goal concordant outcomes. In an ever-evolving technological ICU environment, this may not be an easy task. All too often, we focus on monitors, devices, electronic records, and ignore the human being. Patients labeled with a disability are particularly vulnerable. Recently, I had the privilege to participate in the care of a Mayo Clinic patient with a history of cerebral palsy. In the midst of a life-threatening emergency, by paying attention to the human touch, the ICU team learned the story of a truly remarkable person. The essay below summarizes the patient's and physician's perspectives.

Coagulation monitoring correlation with heparin dose in pediatric extracorporeal life support.

OBJECTIVES: Extracorporeal Life Support (ECLS) risks thrombotic and hemorrhagic complications. Optimal anti-coagulation monitoring is controversial. We compared coagulation tests evaluating the heparin effect in pediatric ECLS. METHODS: A retrospective study of children (<18yrs) undergoing ECLS over 12 months in a tertiary pediatric intensive care unit (PICU). Variables included anti-Factor Xa activity (anti-Xa), activated partial thromboplastin time (aPTT), activated clotting time (ACT) and thromboelastogram (TEG®6s) parameters: ratio and delta reaction (R) times (the ratio and difference, respectively, between R times in kaolin assays with and without heparinase). Test results were correlated with unfractionated heparin infusion rate (IU/kg/hr) at the time of sampling. Mean test results of each ECLS run were evaluated according to the presence/absence of complications. RESULTS: Thirty-two ECLS runs (31 patients) generated 695 data-points for correlation. PICU mortality was 22% and the thrombotic complication rate was 66%. The proportion of variation in coagulation test results explained by heparin dose was 13.3% for anti-Xa, 11.9% for ratio R time, and 9.9% for delta R time, compared with <1% for ACT and aPTT. Incorporating individual variation, age and antithrombin activity in a model with heparin dose explained less than 50% of the variation in test results. Correlation varied according to age, day of ECLS run and between individuals, with parallel dose-response lines noted between patients. Significantly lower mean anti-Xa was observed in PICU non-survivors and runs with thrombosis. CONCLUSION: Lower anti-Xa was observed in ECLS runs with complications. Although absolute results from anti-Xa and TEG6®s showed the best correlation with heparin dose, a large proportion of variation in results was unexplained by heparin, while dose response was similar between individuals. Population pharmacokinetic/pharmacodynamic modelling is required, as well as prospective trials to delineate the superior means of adjusting heparin therapy to prevent adverse clinical outcomes.

Improving the Positive Predictive Value of Non-Invasive Prenatal Screening (NIPS).

We evaluated performance characteristics of a laboratory-developed, non-invasive prenatal screening (NIPS) assay for fetal aneuploidies. This assay employs massively parallel shotgun sequencing with full automation. GC sequencing bias correction and statistical smoothing were performed to enhance discrimination of affected and unaffected pregnancies. Maternal plasma samples from pregnancies with known aneuploidy status were used for assay development, verification, and validation. Assay verification studies using 2,085 known samples (1873 unaffected, 69 trisomy 21, 20 trisomy 18, 17 trisomy 13) demonstrated complete discrimination between autosomal trisomy (Z scores >8) and unaffected (Z scores <4) singleton pregnancies. A validation study using 552 known samples (21 trisomy 21, 10 trisomy 18, 1 trisomy 13) confirmed complete discrimination. Twin pregnancies showed similar results. Follow-up of abnormal results from the first 10,000 clinical samples demonstrated PPVs of 98% (41/42) for trisomy 21, 92% (23/25) for trisomy 18, and 69% (9/13) for trisomy 13. Adjustment for causes of false-positive results identified during clinical testing (eg, maternal duplications) improved PPVs to 100% for trisomy 21 and 96% for trisomy 18. This NIPS test demonstrates excellent discrimination between trisomic and unaffected pregnancies. The PPVs obtained in initial clinical testing are substantially higher than previously reported NIPS methods.

Impact of Viral Respiratory Pathogens on Outcomes After Pediatric Cardiac Surgery.

OBJECTIVES: Viral respiratory infection is commonly considered a relative contraindication to elective cardiac surgery. We aimed to determine the frequency and outcomes of symptomatic viral respiratory infection in pediatric cardiac surgical patients. DESIGN: Retrospective cohort study of children undergoing cardiac surgery. Symptomatic children were tested using a multiplex Polymerase Chain Reaction (respiratory virus polymerase chain reaction) panel capturing nine respiratory viruses. Tests performed between 72 prior to and 48 hours after PICU admission were included. Mortality, length of stay in PICU, and intubation duration were investigated as outcomes. SETTING: Tertiary PICU providing state-wide pediatric cardiac services. PATIENTS: Children less than 18 years admitted January 1, 2008 to November 29, 2014 for cardiac surgery. MEASUREMENTS AND MAIN RESULTS: Respiratory virus polymerase chain reaction was positive in 73 (4.2%) of 1,737 pediatric cardiac surgical admissions, including 13 children with multiple viruses detected. Commonly detected viruses included rhino/enterovirus (48%), adenovirus (32%), parainfluenza virus 3 (10%), and respiratory syncytial virus (3%). Pediatric Index of Mortality 2, Aristotle scores, and cardiopulmonary bypass times were similar between virus positive and negative/untested cohorts. Respiratory virus polymerase chain reaction positive patients had a median 2.0 days greater PICU length of stay (p < 0.001) and longer intubation duration (p < 0.001). Multivariate analysis adjusting for age, Aristotle score, cardiopulmonary bypass duration, and need for preoperative PICU admission confirmed that virus positive patients had significantly greater intubation duration and PICU length of stay (p < 0.001). Virus positive patients were more likely to require PICU admission greater than 4 days (odds ratio, 3.5; 95% CI, 1.9-6.2) and more likely to require intubation greater than 48 hours (odds ratio, 2.5; 95% CI, 1.4-4.7). There was no difference in mortality. No association was found between coinfection and outcomes. CONCLUSIONS: Pediatric cardiac surgical patients with a respiratory virus detected at PICU admission had prolonged postoperative recovery with increased length of stay and duration of intubation. Our results suggest that postponing cardiac surgery in children with symptomatic viral respiratory infection is appropriate, unless the benefits of early surgery outweigh the risk of prolonged ventilation and PICU stay.

A Simplified Technique for Interventional Extracardiac Fontan.

PURPOSE: We report a simple technique for an interventional extracardiac Fontan (ECF) procedure. DESCRIPTION: At the preparatory stage along with a bidirectional cavopulmonary connection (BCPC; cardiopulmonary bypass), a short piece of polytetrafluoroethylene (PTFE) tube graft is anastomosed to the inferior surface of the right pulmonary artery. Another longer PTFE graft is anastomosed to the transected inferior vena cava (IVC). A large medial opening in the lower PTFE graft is anastomosed to an atriotomy. These two PTFE tubes are anastomosed with a pericardial patch interposed between them. During the later interventional Fontan procedure, this pericardial patch is perforated using radiofrequency, and a covered stent is positioned entirely within the PTFE tubes, eliminating the window into the common atrium and leaving no intrapulmonary prosthetic material. EVALUATION: The hemodynamics after the preparatory stage is similar to those following a BCPC, with uninterrupted flow from the IVC to the right atrium. On completion, there is a nonfenestrated Fontan circuit. CONCLUSION: Our technique of interventional Fontan, anatomically and hemodynamically, mimics a standard ECF procedure.

Heat Vulnerability Index Mapping for Milwaukee and Wisconsin.

CONTEXT: Extreme heat waves elevate the population's risk for heat-related morbidity and mortality, specifically for vulnerable groups such as older adults and young children. In this context, we developed 2 Heat Vulnerability Indices (HVIs), one for the state of Wisconsin and one for the Milwaukee metropolitan area. OBJECTIVE: Through the creation of an HVI, state and local agencies will be able to use the indices as a planning tool for extreme heat events. DESIGN: Data used for the HVIs were grouped into 4 categories: (1) population density; (2) health factors; (3) demographic and socioeconomic factors; and (4) natural and built environment factors. These categories were mapped at the Census block group level. MAIN OUTCOME MEASURES: Unweighted z-score data were used to determine index scores, which were then mapped by quantiles ranging from "high" to "low" vulnerability. RESULTS: Statewide, Menominee County exhibited the highest vulnerability to extreme heat. Milwaukee HVI findings indicated high vulnerability in the city's inner core versus low vulnerability along the lakeshore. CONCLUSION: Visualization of vulnerability could help local public health agencies prepare for future extreme heat events.

Repair of congenital diaphragmatic hernia during extracorporeal life support: experience with six neonates.

BACKGROUND: The management of congenital diaphragmatic hernia (CDH) in neonates has evolved considerably over the last three decades. Initial stabilization followed by surgical repair is the current standard of care. A subset fails to achieve adequate oxygenation with medical management, including the use of high frequency oscillation and inhaled nitric oxide. The mortality in this group exceeds 80% without additional management strategies. Extracorporeal life support (ECLS) is a well-established modality for managing these neonates with CDH and has been shown to improve early survival in selected cases. METHODS: This is a retrospective analysis of six neonates with CDH who underwent repair during ECLS between September 2011 and November 2014. RESULTS: Of 24 admissions with CDH, there were six neonates (25%) who required ECLS. All the six had CDH repair during ECLS. There were no intra-operative bleeding complications. There were no clotting complications related to stopping heparin during CDH repair. There was one hospital death. Five neonates were weaned from ECLS and discharged home. CONCLUSIONS: Data from our small cohort of patients illustrate that early survival is possible in extremely compromised neonates who otherwise would have died without ECLS. Our experience demonstrates that CDH repair can safely be performed during ECLS. Use of ECLS, early repair during ECLS, lung protective ventilation strategies and aggressive management of pulmonary hypertension were associated with good early survival. ECLS should be considered as an integral part of therapeutic armamentarium for CDH in neonates.

Stenting of the left pulmonary artery after palliation of hypoplastic left heart syndrome.

BACKGROUND: Left pulmonary artery stenosis and hypoplasia is a well-recognized complication following surgical palliation of hypoplastic left heart syndrome. These lesions produce increased after load in a circulation in series so need to be effectively treated. METHODS: Between 2000 and 2011, 86 patients after surgical palliation for hypoplastic left heart syndrome had left pulmonary artery stents implanted. Median age at implantation was 4.7(1.3-15.2) years and median weight was 16.4(9.3-55.2) kg. Uncovered peripheral vascular stents were implanted (median diameter 10(8-15) mm). This is a retrospective review of the incidence of in-stent restenosis over the medium to long term. RESULTS: During primary stenting procedures, there were 2/88(2.3%) major complications of stent migration with no stroke or mortality. Follow-up was for a median period of 4.1(0.5-13.4) years. Follow-up catheter procedures were performed after a median time of 2.3(0.02-9.6) years in 59 patients (68.6%). 55/59(93.2%) had at most mild restenosis (≤10% loss of stent lumen) and 47/59(79.6%) had no evidence of any restenosis at all caused by neointimal in-growth. Freedom from reintervention was 77% at 5 years including stent dilation to compensate for somatic growth. Freedom from reintervention for restenosis was 93% at 5 years. Restenosis was successfully treated with standard balloon angioplasty or restenting. There was only 1/94 (1.1%) major complication in the follow-up catheterizations of stent embolization with successful transcatheter retrieval. CONCLUSIONS: Stenting of the left pulmonary artery after Norwood/Fontan palliation is safe and effective. Stents can be redilated to match somatic growth. The incidence of neointimal proliferation is extremely low and can be addressed by balloon dilation or stent implantation. © 2016 Wiley Periodicals, Inc.

In Vivo Evaluation of Active and Passive Physiological Control Systems for Rotary Left and Right Ventricular Assist Devices.

Preventing ventricular suction and venous congestion through balancing flow rates and circulatory volumes with dual rotary ventricular assist devices (VADs) configured for biventricular support is clinically challenging due to their low preload and high afterload sensitivities relative to the natural heart. This study presents the in vivo evaluation of several physiological control systems, which aim to prevent ventricular suction and venous congestion. The control systems included a sensor-based, master/slave (MS) controller that altered left and right VAD speed based on pressure and flow; a sensor-less compliant inflow cannula (IC), which altered inlet resistance and, therefore, pump flow based on preload; a sensor-less compliant outflow cannula (OC) on the right VAD, which altered outlet resistance and thus pump flow based on afterload; and a combined controller, which incorporated the MS controller, compliant IC, and compliant OC. Each control system was evaluated in vivo under step increases in systemic (SVR ∼1400-2400 dyne/s/cm(5) ) and pulmonary (PVR ∼200-1000 dyne/s/cm(5) ) vascular resistances in four sheep supported by dual rotary VADs in a biventricular assist configuration. Constant speed support was also evaluated for comparison and resulted in suction events during all resistance increases and pulmonary congestion during SVR increases. The MS controller reduced suction events and prevented congestion through an initial sharp reduction in pump flow followed by a gradual return to baseline (5.0 L/min). The compliant IC prevented suction events; however, reduced pump flows and pulmonary congestion were noted during the SVR increase. The compliant OC maintained pump flow close to baseline (5.0 L/min) and prevented suction and congestion during PVR increases. The combined controller responded similarly to the MS controller to prevent suction and congestion events in all cases while providing a backup system in the event of single controller failure.

Successful lysis of bilateral renal vein thrombosis following neonatal truncus repair.

Renal vein thrombosis (RVT) is the most common noncatheter-related thrombosis encountered in infancy, most of which occurs in neonates. The optimal management strategy for neonatal RVT is unclear. Fibrinolytic and heparin therapy may play a role in preventing chronic renal failure in neonates with bilateral RVT. However, the use of fibrinolytics early after any major surgery requires tremendous caution. In this report, we describe the successful use of fibrinolysis in a neonate with bilateral RVT after repair of truncus arteriosus in the early postoperative period.

Micro-concentrators for a microsystems-enabled photovoltaic system.

A 100X magnification, ± 2.5° field of view micro-concentrating optical system has been developed for a microsystems-enabled photovoltaic (MEPV) prototype module using 250 µm diameter multi-junction "stacked" PV cells.

Differential regulation of the Let-7 family of microRNAs in CD4+ T cells alters IL-10 expression.

MicroRNAs (miRNAs) are ∼22-nt small RNAs that are important regulators of mRNA turnover and translation. Recent studies have shown the importance of the miRNA pathway in HIV-1 infection, particularly in maintaining latency. Our initial in vitro studies demonstrated that HIV-1-infected HUT78 cells expressed significantly higher IL-10 levels compared with uninfected cultures. IL-10 plays an important role in the dysregulated cytotoxic T cell response to HIV-1, and in silico algorithms suggested that let-7 miRNAs target IL10 mRNA. In a time course experiment, we demonstrated that let-7 miRNAs fall rapidly following HIV-1 infection in HUT78 cells with concomitant rises in IL-10. To show a direct link between let-7 and IL-10, forced overexpression of let-7 miRNAs resulted in significantly reduced IL-10 levels, whereas inhibition of the function of these miRNAs increased IL-10. To demonstrate the relevance of these results, we focused our attention on CD4(+) T cells from uninfected healthy controls, chronic HIV-1-infected patients, and long-term nonprogressors. We characterized miRNA changes in CD4(+) T cells from these three groups and demonstrated that let-7 miRNAs were highly expressed in CD4(+) T cells from healthy controls and let-7 miRNAs were significantly decreased in chronic HIV-1 infected compared with both healthy controls and long-term nonprogressors. We describe a novel mechanism whereby IL-10 levels can be potentially modulated by changes to let-7 miRNAs. In HIV-1 infection, the decrease in let-7 miRNAs may result in an increase in IL-10 from CD4(+) T cells and provide the virus with an important survival advantage by manipulating the host immune response.