The 8th International RASopathies Symposium: Expanding research and care practice through global collaboration and advocacy.

Germline pathogenic variants in the RAS/mitogen-activated protein kinase (MAPK) signaling pathway are the molecular cause of RASopathies, a group of clinically overlapping genetic syndromes. RASopathies constitute a wide clinical spectrum characterized by distinct facial features, short stature, predisposition to cancer, and variable anomalies in nearly all the major body systems. With increasing global recognition of these conditions, the 8th International RASopathies Symposium spotlighted global perspectives on clinical care and research, including strategies for building international collaborations and developing diverse patient cohorts in anticipation of interventional trials. This biannual meeting, organized by RASopathies Network, was held in a hybrid virtual/in-person format. The agenda featured emerging discoveries and case findings as well as progress in preclinical and therapeutic pipelines. Stakeholders including basic scientists, clinician-scientists, practitioners, industry representatives, patients, and family advocates gathered to discuss cutting edge science, recognize current gaps in knowledge, and hear from people with RASopathies about the experience of daily living. Presentations by RASopathy self-advocates and early-stage investigators were featured throughout the program to encourage a sustainable, diverse, long-term research and advocacy partnership focused on improving health and bringing treatments to people with RASopathies.

Novel Approach to Improve the Identification of the Bleeding Phenotype in Noonan Syndrome and Related RASopathies.

OBJECTIVES: To characterize the bleeding phenotype in Noonan syndrome (NS), to test the utility of following national guidelines in detecting this phenotype, to evaluate thromboelastography (TEG) as a diagnostic tool, and to evaluate the cohort for genotype-phenotype correlations. STUDY DESIGN: Participants with a clinical diagnosis of NS or related RASopathies were enrolled in a cohort study. Study procedures included clinical bleeding assessment, coagulation testing per guidelines, and hematology consultation. TEG was completed in a subset, and genetic testing was conducted for those without a molecular diagnosis. International Society of Haemostasis and Thrombosis Bleeding Assessment Tool scores were calculated with hematology consultation. Bleeding phenotype was defined as abnormal bleeding score. RESULTS: Twenty participants were enrolled; 12 completed clinical and laboratory evaluation, and five of whom met the definition for bleeding phenotype. Four of the five participants with a bleeding phenotype had platelet aggregation defects and at least one additional coagulation defect. TEG was performed in nine participants, four with bleeding phenotype and five without, and results were normal in all cases. No genotype-phenotype correlation was found. CONCLUSION: Five of the 20 participants had a bleeding phenotype identified. Based on available data, we do not recommend incorporating TEG into clinical practice for patients with NS. Platelet aggregation defects were the most common abnormalities, which would not be detected on tier 1 testing of current guidelines; therefore, we propose a new algorithm.

Clinical Metagenomic Sequencing for Diagnosis of Meningitis and Encephalitis.

BACKGROUND: Metagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test. METHODS: In a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review. RESULTS: We enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment. CONCLUSIONS: Routine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, NCT02910037.).

The Evaluation of Hematologic Screening and Perioperative Management in Patients with Noonan Syndrome: A Retrospective Chart Review.

OBJECTIVES: To assess the potential impact of using screening recommendations for bleeding disorders in patients with Noonan syndrome on perioperative bleeding complications. STUDY DESIGN: We performed a retrospective, single-site cohort study; patients were identified by query of the electronic medical record. All patients with a clinical diagnosis of Noonan syndrome over a 10-year period were included. Data on surgeries, hematologic evaluation, bleeding symptoms, and bleeding complications were extracted. Surgeries were graded as major or minor. RESULTS: We identified 101 patients with Noonan syndrome, 70 of whom required surgery for a total of 164 procedures. Nine patients (9/70; 12.8%) had bleeding complications, occurring in those without comprehensive testing or perioperative intervention and undergoing major or dental surgery. Based on these findings, the risk of a bleeding complication for patients with Noonan syndrome who did not have comprehensive testing or perioperative intervention was 6.2% (95% CI 2.3%-10.1%), indicating the number needed to treat or screen would be 16 to prevent 1 bleeding complication (95% CI 9.9-43.9). The majority of patients had either no or incomplete evaluation (59 of 101; 58.4%). CONCLUSIONS: With proper evaluation and management, the bleeding risk in patients with Noonan syndrome can be minimized. Efforts are needed to address the knowledge and implementation gap in this evaluation.

Rapid Whole Genome Sequencing Has Clinical Utility in Children in the PICU.

OBJECTIVES: Genetic disorders are a leading contributor to mortality in the neonatal ICU and PICU in the United States. Although individually rare, there are over 6,200 single-gene diseases, which may preclude a genetic diagnosis prior to ICU admission. Rapid whole genome sequencing is an emerging method of diagnosing genetic conditions in time to affect ICU management of neonates; however, its clinical utility has yet to be adequately demonstrated in critically ill children. This study evaluates next-generation sequencing in pediatric critical care. DESIGN: Retrospective cohort study. SETTING: Single-center PICU in a tertiary children's hospital. PATIENTS: Children 4 months to 18 years admitted to the PICU who were nominated between July 2016 and May 2018. INTERVENTIONS: Rapid whole genome sequencing with targeted phenotype-driven analysis was performed on patients and their parents, when parental samples were available. MEASUREMENTS AND MAIN RESULTS: A molecular diagnosis was made by rapid whole genome sequencing in 17 of 38 children (45%). In four of the 17 patients (24%), the genetic diagnoses led to a change in management while in the PICU, including genome-informed changes in pharmacotherapy and transition to palliative care. Nine of the 17 diagnosed children (53%) had no dysmorphic features or developmental delay. Eighty-two percent of diagnoses affected the clinical management of the patient and/or family after PICU discharge, including avoidance of biopsy, administration of factor replacement, and surveillance for disorder-related sequelae. CONCLUSIONS: This study demonstrates a retrospective evaluation for undiagnosed genetic disease in the PICU and clinical utility of rapid whole genome sequencing in a portion of critically ill children. Further studies are needed to identify PICU patients who will benefit from rapid whole genome sequencing early in PICU admission when the underlying etiology is unclear.

Tularemia-induced erythema multiforme minor in an 11-year-old girl.

Tularemia is a rare and potentially life-threatening infection caused by the highly infectious gram-negative coccobacillus Francisella tularensis. We present the case of an 11-year old girl who presented with erythema multiforme minor in the setting of an indolent but progressive soft tissue infection and was found to have tularemia. We review the role of dermatologists in identifying the features of and complications associated with this rare zoonosis and discuss the potential effect of climate change on its incidence.

The burden of RSV, hMPV, and PIV amongst hospitalized adults in the United States from 2016 to 2019.

BACKGROUND: Respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and parainfluenza virus (PIV) hospitalize many people yearly. Though severe lower respiratory tract disease has been described in children, the elderly, and the immunocompromised, there is a gap in our understanding of RSV, hMPV, and PIV in hospitalized adults. We sought to evaluate the association of RSV, hMPV, and PIV with severe respiratory disease requiring noninvasive or mechanical ventilation and death in hospitalized adults in the United States. METHODS: We conducted a retrospective, pooled, cross-sectional study of general medicine hospitalizations in the United States from 2016 to 2019 using the National Inpatient Sample published by the Agency for Healthcare Quality and Research. We used multivariable Poisson regression to estimate the likelihood of severe respiratory disease or death. We used linear regression to estimate the mean difference in length of stay for those hospitalized with and without a respiratory virus. RESULTS: We found that RSV (incidence rate ratio [IRR]: 1.68, 95% confidence interval [CI]: 1.61-1.74, p < .001), hMPV (IRR: 1.82, 95% CI: 1.71-1.93, p < .001), and PIV (IRR: 1.81, 95% CI: 1.68-1.94, p < .001) were independently associated with severe respiratory disease, even after adjustment. Additionally, we found the presence of a respiratory virus prolonged hospitalizations by (0.79 ± 0.27 days, p < .003) for RSV, (0.88 ± 0.28 days, p < .002) for hMPV, and (1.43 ± 0.30 days, p < .001) for PIV. CONCLUSIONS: RSV, hMPV, and PIV have a significant burden on hospitalized adults, even without classic risk factors.

A Prospective Clinical Study to EvaluAte the AbiliTy of the CloudCath System to Detect Peritonitis During In-Home Peritoneal Dialysis (CATCH).

Introduction: Peritonitis is the leading complication of peritoneal dialysis (PD). Patients are instructed to seek care promptly for signs (cloudy effluent) or symptoms (abdominal pain), and earlier treatment improves outcomes. The CloudCath Peritoneal Dialysis Drain Set Monitoring (CloudCath) system monitors turbidity in dialysis effluent and sends notifications of changes signaling possible peritonitis. Methods: We conducted this single-arm, open-label, multicenter study of CloudCath system use during PD. We deactivated system notifications to participants and investigators, who followed standard-of-care for peritonitis signs and symptoms. Effectiveness endpoints measured time between CloudCath system notifications and peritonitis events using International Society of Peritoneal Dialysis (ISPD) criteria. Results: Two hundred forty-three participants used the CloudCath system for 178.8 patient-years. Of 71 potential peritonitis events, 51 events (0.29 per patient-year) met ISPD white blood cell (WBC) count criteria. The system triggered notifications for 41 of 51 events (80.4%), with a median lead time of 2.6 days (10%-90% range, -1.0 to 15.7; P < 0.0001). Excluding 6 peritonitis events that occurred when the system was not in use, the system triggered notifications for 41 of 45 events (91.1%), with a median lead time of 3.0 days (10%-90% range, -0.5 to 18.8; P < 0.0001). Of the 0.78 notifications per patient-year, the majority were peritonitis events or nonperitonitis events such as exit site and tunnel infections or catheter/cycler issues. Conclusion: The CloudCath system detected peritonitis events during PD several days earlier than the current standard-of-care and has the capacity to send notifications that could expedite peritonitis diagnosis and treatment.

Performance characteristics of a prototype dialysate turbidity monitoring system to detect peritonitis in patients receiving peritoneal dialysis.

BACKGROUND: The risk of peritonitis has limited wider adoption of peritoneal dialysis (PD) in the United States. We developed a prototype bedside dialysate turbidity monitoring system, aiming to improve diagnostic accuracy relative to conventional approaches which depend on visual inspection and reporting of insensitive and non-specific symptoms. METHODS: The prototype system was tested in a single-centre, proof-of-principle clinical study in patients receiving intermittent PD. We obtained multiple effluent dialysate samples from each consenting participant. We compared turbidity measurements with diagnostic criteria endorsed by the International Society of Peritoneal Dialysis (ISPD). RESULTS: Overall, we analysed 983 specimens from 65 patients, including 105 samples from patients with peritonitis and 878 samples from patients without peritonitis. An operating point derived from a previous in vitro study yielded an unadjusted sensitivity and specificity of 95.2% and 91.5%, respectively. The majority of samples that did not meet ISPD diagnostic criteria were either cases detected before criteria were met or were related to active peritonitis treatment and resolution. CONCLUSION: This proof-of-principle study demonstrates the feasibility and diagnostic accuracy of a prototype dialysate turbidity monitoring system for peritonitis surveillance.

Effectiveness of Self-Collected, Ambient Temperature-Preserved Nasal Swabs Compared to Samples Collected by Trained Staff for Genotyping of Respiratory Viruses by Shotgun RNA Sequencing: Comparative Study.

BACKGROUND: The SARS-CoV-2 pandemic has underscored the need for field specimen collection and transport to diagnostic and public health laboratories. Self-collected nasal swabs transported without dependency on a cold chain have the potential to remove critical barriers to testing, expand testing capacity, and reduce opportunities for exposure of health professionals in the context of a pandemic. OBJECTIVE: We compared nasal swab collection by study participants from themselves and their children at home to collection by trained research staff. METHODS: Each adult participant collected 1 nasal swab, sampling both nares with the single swab, after which they collected 1 nasal swab from 1 child. After all the participant samples were collected for the household, the research staff member collected a separate single duplicate sample from each individual. Immediately after the sample collection, the adult participants completed a questionnaire about the acceptability of the sampling procedures. Swabs were placed in temperature-stable preservative and respiratory viruses were detected by shotgun RNA sequencing, enabling viral genome analysis. RESULTS: In total, 21 households participated in the study, each with 1 adult and 1 child, yielding 42 individuals with paired samples. Study participants reported that self-collection was acceptable. Agreement between identified respiratory viruses in both swabs by RNA sequencing demonstrated that adequate collection technique was achieved by brief instructions. CONCLUSIONS: Our results support the feasibility of a scalable and convenient means for the identification of respiratory viruses and implementation in pandemic preparedness for novel respiratory pathogens.

Bleeding disorders in Noonan syndrome.

Noonan Syndrome (NS) is a common genetic disease with multiple organ defects including bleeding disorders, which was last reviewed in 1997. Since then significant information has been acquired regarding bleeding problems in NS, specifically on the underlying genetics. Associations between mutated genes and bleeding disorders are reviewed along with prevalence and underlying etiologies. Between 50-89% of NS patients will have a bleeding disorder and since a significant number will require surgery it is important to identify which ones are at risk prior to their procedure. Recommendations regarding screening for bleeding disorders and their treatment are discussed.

Tick-borne encephalitis virus, Kyrgyzstan.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and Asia. We investigated TBEV in Kyrgyzstan by collecting small mammals and ticks from diverse localities and analyzing them for evidence of TBEV infection. We found TBEV circulating in Kyrgyzstan much farther south and at higher altitudes than previously reported.

Use of Metagenomic Next-Generation Sequencing to Identify Pathogens in Pediatric Osteoarticular Infections.

BACKGROUND: Osteoarticular infections (OAIs) are frequently encountered in children. Treatment may be guided by isolation of a pathogen; however, operative cultures are often negative. Metagenomic next-generation sequencing (mNGS) allows for broad and sensitive pathogen detection that is culture-independent. We sought to evaluate the diagnostic utility of mNGS in comparison to culture and usual care testing to detect pathogens in acute osteomyelitis and/or septic arthritis in children. METHODS: This was a single-site study to evaluate the use of mNGS in comparison to culture to detect pathogens in acute pediatric osteomyelitis and/or septic arthritis. Subjects admitted to a tertiary children's hospital with suspected OAI were eligible for enrollment. We excluded subjects with bone or joint surgery within 30 days of admission or with chronic osteomyelitis. Operative samples were obtained at the surgeon's discretion per standard care (fluid or tissue) and based on imaging and operative findings. We compared mNGS to culture and usual care testing (culture and polymerase chain reaction [PCR]) from the same site. RESULTS: We recruited 42 subjects over the enrollment period. mNGS of the operative samples identified a pathogen in 26 subjects compared to 19 subjects in whom culture identified a pathogen. In 4 subjects, mNGS identified a pathogen where combined usual care testing (culture and PCR) was negative. Positive predictive agreement and negative predictive agreement both were 93.0% for mNGS. CONCLUSIONS: In this single-site prospective study of pediatric OAI, we demonstrated the diagnostic utility of mNGS testing in comparison to culture and usual care (culture and PCR) from operative specimens.

Metagenomic Next-Generation Sequencing for Pathogen Detection and Transcriptomic Analysis in Pediatric Central Nervous System Infections.

BACKGROUND: Pediatric central nervous system (CNS) infections are potentially life-threatening and may incur significant morbidity. Identifying a pathogen is important, both in terms of guiding therapeutic management and in characterizing prognosis. Usual care testing by culture and polymerase chain reaction is often unable to identify a pathogen. We examined the systematic application of metagenomic next-generation sequencing (mNGS) for detecting organisms and transcriptomic analysis of cerebrospinal fluid (CSF) in children with central nervous system (CNS) infections. METHODS: We conducted a prospective multisite study that aimed to enroll all children with a CSF pleocytosis and suspected CNS infection admitted to 1 of 3 tertiary pediatric hospitals during the study timeframe. After usual care testing had been performed, the remaining CSF was sent for mNGS and transcriptomic analysis. RESULTS: We screened 221 and enrolled 70 subjects over a 12-month recruitment period. A putative organism was isolated from CSF in 25 (35.7%) subjects by any diagnostic modality. Metagenomic next-generation sequencing of the CSF samples identified a pathogen in 20 (28.6%) subjects, which were also all identified by usual care testing. The median time to result was 38 hours. CONCLUSIONS: Metagenomic sequencing of CSF has the potential to rapidly identify pathogens in children with CNS infections.

Cost Efficacy of Rapid Whole Genome Sequencing in the Pediatric Intensive Care Unit.

The diagnostic and clinical utility of rapid whole genome sequencing (rWGS) for critically ill children in the intensive care unit (ICU) has been substantiated by multiple studies, but comprehensive cost-effectiveness evaluation of rWGS in the ICU outside of the neonatal age group is lacking. In this study, we examined cost data retrospectively for a cohort of 38 children in a regional pediatric ICU (PICU) who received rWGS. We identified seven of 17 patients who received molecular diagnoses by rWGS and had resultant changes in clinical management with sufficient clarity to permit cost and quality adjusted life years (QALY) modeling. Cost of PICU care was estimated to be reduced by $184,846 and a total of 12.1 QALYs were gained among these seven patients. The total cost of rWGS for patients and families for the entire cohort (38 probands) was $239,400. Thus, the net cost of rWGS was $54,554, representing $4,509 per QALY gained. This quantitative, retrospective examination of healthcare utilization associated with rWGS-informed medicine interventions in the PICU revealed approximately one-third of a QALY gained per patient tested at a cost per QALY that was approximately one-tenth of that typically sought for cost-effective new medical interventions. This evidence suggests that performance of rWGS as a first-tier test in selected PICU children with diseases of unknown etiology is associated with acceptable cost-per-QALY gained.

Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids.

We developed a metagenomic next-generation sequencing (mNGS) test using cell-free DNA from body fluids to identify pathogens. The performance of mNGS testing of 182 body fluids from 160 patients with acute illness was evaluated using two sequencing platforms in comparison to microbiological testing using culture, 16S bacterial PCR and/or 28S-internal transcribed ribosomal gene spacer (28S-ITS) fungal PCR. Test sensitivity and specificity of detection were 79 and 91% for bacteria and 91 and 89% for fungi, respectively, by Illumina sequencing; and 75 and 81% for bacteria and 91 and 100% for fungi, respectively, by nanopore sequencing. In a case series of 12 patients with culture/PCR-negative body fluids but for whom an infectious diagnosis was ultimately established, seven (58%) were mNGS positive. Real-time computational analysis enabled pathogen identification by nanopore sequencing in a median 50-min sequencing and 6-h sample-to-answer time. Rapid mNGS testing is a promising tool for diagnosis of unknown infections from body fluids.

Tick-borne encephalitis and golden agers: position paper of the International Scientific Working Group on Tick-borne encephalitis (ISW-TBE).

One of the primary goals of the 11th Annual Meeting of the International Scientific Working Group on Tick-borne encephalitis (ISW-TBE) held in 2009 was to develop the first update of the Position Paper on TBE in Golden Agers, summarizing the most essential aspects of the disease in this age group. TBE morbidity has continued to increase in recent years, which is thought to be due to an interplay of social, political, ecological, economic and demographic factors combined with climate changes. Today's golden agers i.e. individuals aged 50 years or above, are healthier and more mobile, lead more active lifestyles and spend more time travelling and performing outdoor leisure activities. This places them at an increased risk of infection. At the same time, increasing age is associated with a quantitative and qualitative decline in innate and adaptive immunity, which is why elderly individuals are more susceptible to infection and severe disease than younger people. Also, their response to vaccination tends to be slower, antibody titres generally reach lower levels and titres tend to decrease earlier than in younger individuals. Evidence is accumulating that this is also the case with TBE vaccination, emphasizing the importance of administering the first TBE booster vaccination no later than 3 years after the completion of primary immunization or at an even shorter interval. Encouragingly, recent data have shown that the field effectiveness of TBE vaccination exceeds 97%, with no significant differences between age groups.

Partially automated whole-genome sequencing reanalysis of previously undiagnosed pediatric patients can efficiently yield new diagnoses.

To investigate the diagnostic and clinical utility of a partially automated reanalysis pipeline, forty-eight cases of seriously ill children with suspected genetic disease who did not receive a diagnosis upon initial manual analysis of whole-genome sequencing (WGS) were reanalyzed at least 1 year later. Clinical natural language processing (CNLP) of medical records provided automated, updated patient phenotypes, and an automated analysis system delivered limited lists of possible diagnostic variants for each case. CNLP identified a median of 79 new clinical features per patient at least 1 year later. Compared to a standard manual reanalysis pipeline, the partially automated pipeline reduced the number of variants to be analyzed by 90% (range: 74%-96%). In 2 cases, diagnoses were made upon reinterpretation, representing an incremental diagnostic yield of 4.2% (2/48, 95% CI: 0.5-14.3%). Four additional cases were flagged with a possible diagnosis to be considered during subsequent reanalysis. Separately, copy number analysis led to diagnoses in two cases. Ongoing discovery of new disease genes and refined variant classification necessitate periodic reanalysis of negative WGS cases. The clinical features of patients sequenced as infants evolve rapidly with age. Partially automated reanalysis, including automated re-phenotyping through CNLP, has the potential to identify molecular diagnoses with reduced expert labor intensity.

Bioinformatic and comparative localization of Rab proteins reveals functional insights into the uncharacterized GTPases Ypt10p and Ypt11p.

A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance.

Biallelic loss of GNAS in a patient with pediatric medulloblastoma.

Genome sequencing was performed on matched normal and tumor tissue from a 6.5-yr-old boy with a diagnosis of recurrent medulloblastoma. A pathogenic heterozygous c.432+1G>A canonical splice donor site variant in GNAS was detected on analysis of blood DNA. Analysis of tumor DNA showed the same splice variant along with copy-neutral loss of heterozygosity on Chromosome 20 encompassing GNAS, consistent with predicted biallelic loss of GNAS in the tumor specimen. This case strengthens the evidence implicating GNAS as a tumor-suppressor gene in medulloblastoma and highlights a scenario in which therapeutics targeting the cAMP pathway may be of great utility.