Identification of novel driver risk genes in CNV loci associated with neurodevelopmental disorders.

Copy number variants (CNVs) are genome-wide structural variations involving the duplication or deletion of large nucleotide sequences. While these types of variations can be commonly found in humans, large and rare CNVs are known to contribute to the development of various neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD). Nevertheless, given that these NDD-risk CNVs cover broad regions of the genome, it is particularly challenging to pinpoint the critical gene(s) responsible for the manifestation of the phenotype. In this study, we performed a meta-analysis of CNV data from 11,614 patients with NDDs and 4,031 controls from SFARI database to identify 41 NDD-risk CNV loci, including 24 novel regions. We also found evidence for dosage-sensitive genes within these regions being significantly enriched for known NDD-risk genes and pathways. In addition, a significant proportion of these genes was found to i) converge in protein-protein interaction networks; ii) be among most expressed genes in the brain across all developmental stages; and iii) be hit by deletions that are significantly over-transmitted to individuals with ASD within multiplex ASD families from the iHART cohort. Finally, we conducted a burden analysis using 4,281 NDD cases from Decipher and iHART cohorts, and 2,504 neurotypical controls from 1,000 Genomes and iHART, that resulted in the validation of the association of 162 dosage sensitive genes driving risk for NDDs, including 22 novel NDD-risk genes. Importantly, most NDD-risk CNV loci entail multiple NDD-risk genes in agreement with a polygenic model associated with the majority of NDD cases.

Characterization of a Clinically and Biologically Defined Subgroup of Patients with Autism Spectrum Disorder and Identification of a Tailored Combination Treatment.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders (NDDs) with a high unmet medical need. The diagnosis of ASD is currently based on behavior criteria, which overlooks the diversity of genetic, neurophysiological, and clinical manifestations. Failure to acknowledge such heterogeneity has hindered the development of efficient drug treatments for ASD and other NDDs. DEPI® (Databased Endophenotyping Patient Identification) is a systems biology, multi-omics, and machine learning-driven platform enabling the identification of subgroups of patients with NDDs and the development of patient-tailored treatments. In this study, we provide evidence for the validation of a first clinically and biologically defined subgroup of patients with ASD identified by DEPI, ASD Phenotype 1 (ASD-Phen1). Among 313 screened patients with idiopathic ASD, the prevalence of ASD-Phen1 was observed to be ~24% in 84 patients who qualified to be enrolled in the study. Metabolic and transcriptomic alterations differentiating patients with ASD-Phen1 were consistent with an over-activation of NF-κB and NRF2 transcription factors, as predicted by DEPI. Finally, the suitability of STP1 combination treatment to revert such observed molecular alterations in patients with ASD-Phen1 was determined. Overall, our results support the development of precision medicine-based treatments for patients diagnosed with ASD.

Prognostic impact of the findings of the genetic test in left dominant arrhythmogenic cardiomyopathy.

Background: The diagnosis of left dominant arrhythmogenic cardiomyopathy (LDAC) is sometimes complex. The Padua group recently published a document with criteria to identify patients with LDAC, requiring a compatible genetic variant for diagnosis. Due to the gaps in the knowledge of the role of genetics in its pathogenesis, our objective is to describe the findings of the genetic test in patients with LDAC in our center and its prognostic impact. Methods: Single-center prospective cohort study, in which we recruited 77 patients diagnosed with LDAC or biventricular arrhythmogenic cardiomyopathy according to the criteria of Sen-Chowdhry et al. Results: We obtained a positive result in the genetic test in 53.2 %. The desmoplakin gene was the most affected (16.9 %). The mean value of left ventricular (LV) ejection fraction was 45.6 ± 13.1 %, with no significant differences in the severity of the dysfunction according to genetics (p = 0.187). Among the patients with positive genetics there was a greater number of segments in the LV affected by fibrosis (p = 0.043). Regarding fatty infiltration in the LV and number of affected segments, there were no significant differences between groups (p = 0.144). MACE was recorded in 23 patients (29.9 %). The positive result in the genetic test was not significantly associated with the occurrence of MACE (p = 0.902). Conclusion: In our study, we did not find mutations responsible for the disease in practically half of the cases. Despite the existence of a high proportion of MACE during follow-up, there were no prognostic differences according to the result of the genetic test.

A Novel Technique of Placing Desfibrillator Leads in Patients with Persistent Left Superior Vena Cava.

Persistent left superior vena cava is the most common thoracic venous anomaly. It is usually asymptomatic, but it can make implanting intracardiac devices difficult.We present a novel technique to facilitate desfibrillator lead implantation in patients with persistent left superior vena cava and the absence of the right superior vena cava. We used a fixed-curve Selectra 3D 65-42 cm sheath (Biotronik), orienting it toward the tricuspid valve (TV) by rotating it counter-clockwise. During follow-up, the electrodes remained stable.Our technique was safe, simple, and feasible for patients with this complex venous anatomy.

The contributions of rare inherited and polygenic risk to ASD in multiplex families.

Autism spectrum disorder (ASD) has a complex genetic architecture involving contributions from both de novo and inherited variation. Few studies have been designed to address the role of rare inherited variation or its interaction with common polygenic risk in ASD. Here, we performed whole-genome sequencing of the largest cohort of multiplex families to date, consisting of 4,551 individuals in 1,004 families having two or more autistic children. Using this study design, we identify seven previously unrecognized ASD risk genes supported by a majority of rare inherited variants, finding support for a total of 74 genes in our cohort and a total of 152 genes after combined analysis with other studies. Autistic children from multiplex families demonstrate an increased burden of rare inherited protein-truncating variants in known ASD risk genes. We also find that ASD polygenic score (PGS) is overtransmitted from nonautistic parents to autistic children who also harbor rare inherited variants, consistent with combinatorial effects in the offspring, which may explain the reduced penetrance of these rare variants in parents. We also observe that in addition to social dysfunction, language delay is associated with ASD PGS overtransmission. These results are consistent with an additive complex genetic risk architecture of ASD involving rare and common variation and further suggest that language delay is a core biological feature of ASD.

A case of recurring myocarditis: Magnetic resonance, key to diagnosis.

Arrhythmogenic cardiomyopathy is a hereditary disease characterized by the replacement of the myocardium by fibrofatty tissue. In recent years, three patterns have been described: the classic right variant, the biventricular variant, and the variant with predominant involvement of the left ventricle. Nowadays, cardiac magnetic resonance is a fundamental tool for diagnosis of arrhythmogenic left ventricular cardiomyopathy. Late gadolinium enhancement is a very sensitive indicator of early left-sided involvement, and is included as a marker in the current arrhythmogenic cardiomyopathy criteria. We report a case of arrhythmogenic left ventricular cardiomyopathy with atypical form of presentation as recurrent myocarditis. Clinical suspicion was important for the diagnosis, as the patient did not present data that would point to an infectious origin of the disease. However, the key to diagnosis was detecting a characteristic imaging pattern on cardiac magnetic resonance. Initially, a meso-subepicardial fibrosis located in lateral wall was observed, which progressively spread to other regions until it became practically global. In addition, irregularities were observed in the epicardial contour that were suggestive of fatty infiltration, all consistent with the diagnosis of arrhythmogenic left ventricular cardiomyopathy. Learning objective: Arrhythmogenic left ventricular cardiomyopathy has recently been recognized as part of the arrhythmogenic cardiomyopathy spectrum. Given the difficulties in its diagnosis, it is essential to have a high index of suspicion. We must pay attention to the clinical context and the cardiac magnetic resonance imaging findings, which has become an essential imaging tool for diagnosis.

A mesh microelectrode array for non-invasive electrophysiology within neural organoids.

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes and the culture of organoids on meshes for up to one year. Furthermore, we present proof-of-principle recordings of spontaneous electrical activity across the volume of an organoid. Our concept enables a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue.

Translating precision medicine for autism spectrum disorder: A pressing need.

Autism spectrum disorder (ASD) is a heterogenous group of neurodevelopmental disorders (NDDs) with a high unmet medical need. Currently, ASD is diagnosed according to behavior-based criteria that overlook clinical and genomic heterogeneity, thus repeatedly resulting in failed clinical trials. Here, we summarize the scientific evidence pointing to the pressing need to create a precision medicine framework for ASD and other NDDs. We discuss the role of omics and systems biology to characterize more homogeneous disease subtypes with different underlying pathophysiological mechanisms and to determine corresponding tailored treatments. Finally, we provide recent initiatives towards tackling the complexity in NDDs for precision medicine and cost-effective drug discovery.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro-derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9-mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Scipion3: A workflow engine for cryo-electron microscopy image processing and structural biology.

Image-processing pipelines require the design of complex workflows combining many different steps that bring the raw acquired data to a final result with biological meaning. In the image-processing domain of cryo-electron microscopy single-particle analysis (cryo-EM SPA), hundreds of steps must be performed to obtain the three-dimensional structure of a biological macromolecule by integrating data spread over thousands of micrographs containing millions of copies of allegedly the same macromolecule. The execution of such complicated workflows demands a specific tool to keep track of all these steps performed. Additionally, due to the extremely low signal-to-noise ratio (SNR), the estimation of any image parameter is heavily affected by noise resulting in a significant fraction of incorrect estimates. Although low SNR and processing millions of images by hundreds of sequential steps requiring substantial computational resources are specific to cryo-EM, these characteristics may be shared by other biological imaging domains. Here, we present Scipion, a Python generic open-source workflow engine specifically adapted for image processing. Its main characteristics are: (a) interoperability, (b) smart object model, (c) gluing operations, (d) comparison operations, (e) wide set of domain-specific operations, (f) execution in streaming, (g) smooth integration in high-performance computing environments, (h) execution with and without graphical capabilities, (i) flexible visualization, (j) user authentication and private access to private data, (k) scripting capabilities, (l) high performance, (m) traceability, (n) reproducibility, (o) self-reporting, (p) reusability, (q) extensibility, (r) software updates, and (s) non-restrictive software licensing.

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays.

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions. This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

Deterministic programming of human pluripotent stem cells into microglia facilitates studying their role in health and disease.

Microglia, the resident immune cells of the central nervous system (CNS), are derived from yolk-sac macrophages that populate the developing CNS during early embryonic development. Once established, the microglia population is self-maintained throughout life by local proliferation. As a scalable source of microglia-like cells (MGLs), we here present a forward programming protocol for their generation from human pluripotent stem cells (hPSCs). The transient overexpression of PU.1 and C/EBPß in hPSCs led to a homogenous population of mature microglia within 16 d. MGLs met microglia characteristics on a morphological, transcriptional, and functional level. MGLs facilitated the investigation of a human tauopathy model in cortical neuron-microglia cocultures, revealing a secondary dystrophic microglia phenotype. Single-cell RNA sequencing of microglia integrated into hPSC-derived cortical brain organoids demonstrated a shift of microglia signatures toward a more-developmental in vivo-like phenotype, inducing intercellular interactions promoting neurogenesis and arborization. Taken together, our microglia forward programming platform represents a tool for both reductionist studies in monocultures and complex coculture systems, including 3D brain organoids for the study of cellular interactions in healthy or diseased environments.

Predicting restriction of life-space mobility: a machine learning analysis of the IMIAS study.

BACKGROUND: Some studies have employed machine learning (ML) methods for mobility prediction modeling in older adults. ML methods could be a helpful tool for life-space mobility (LSM) data analysis. AIM: This study aimed to evaluate the predictive value of ML algorithms for the restriction of life-space mobility (LSM) among elderly people and to identify the most important risk factors for that prediction model. METHODS: A 2-year LSM reduction prediction model was developed using the ML-based algorithms decision tree, random forest, and eXtreme gradient boosting (XGBoost), and tested on an independent validation cohort. The data were collected from the International Mobility in Aging Study (IMIAS) from 2012 to 2014, comprising 372 older patients (≥ 65 years of age). LSM was measured by the Life-Space Assessment questionnaire (LSA) with five levels of living space during the month before assessment. RESULTS: According to the XGBoost algorithm, the best model reached a mean absolute error (MAE) of 10.28 and root-mean-square error (RMSE) of 12.91 in the testing portion. The variables frailty (39.4%), mobility disability (25.4%), depression (21.9%), and female sex (13.3%) had the highest importance. CONCLUSION: The model identified risk factors through ML algorithms that could be used to predict LSM restriction; these risk factors could be used by practitioners to identify older adults with an increased risk of LSM reduction in the future. The XGBoost model offers benefits as a complementary method of traditional statistical approaches to understand the complexity of mobility.

Evaluation of the Relationship between the Weight and Height Percentiles and the Sequence and Chronology of Eruption in Permanent Dentition.

The standard eruption of the permanent dentition in growing patients is influenced by multiple environmental factors. The objective of this research was to study the relationship between height and weight percentiles and the eruption of the permanent dentition. The design of the study was transversal based on the review of the clinical history, visual dental inspection, weight and height indicators, and their respective percentiles in patients in the mixed and definitive dentition stage. The descriptive and comparative analysis of the data was carried out with the statistical software R version 4.1.1. The sample size was 725 participants. The mean age of eruption of the first tooth was 8.0. The eruption sequence in the upper arch was first molar, central and lateral incisor, first premolar, canine, second premolar, and second molar. In the lower arch, the eruption sequence was: central incisor, first molar, lateral incisor, canine, first and second premolar, and second molar. The most frequent weight percentile was P50-97 (50.34%) and height P3-50 (53.38%). Weight (0.0129; 0.0426; 0.0495; 0.000166) and height (0.00768; 0.00473; 0.00927; 10-5) variables significantly influenced dental eruption. The factor that most influences the eruption of the permanent dentition is the height percentile.

Evaluation of the Relationship between the BMI and the Sequence and Chronology of Eruption in Permanent Dentition in Spanish Population.

The aim was to analyze the relationship between BMI (body mass index) and the sequence and chronology of the eruption of permanent teeth in a sample of Spanish children. Methods: The study design was descriptive, cross-sectional, observational, and epidemiological. Patients of pediatric age were included. Emerged teeth, and patient's age, race, and sex were recorded. The nutritional status of the child was assessed by calculating the BMI, according to the WHO parameters. Statistical analysis was carried out with a confidence interval of 95%. A prediction model with logistic regression models was obtained. Results: A total of 725 pediatric patients between 4 and 14 years old were analyzed. BMI acts as a predictor variable for eruption symmetry, as it was most frequent in overweight children (p < 0.001). The probability of symmetry in dental eruption increases for South American children, for an extra month of age, and each meter of height. BMI had an influence in the first tooth to appear only in the fourth quadrant. BMI did not seem to influence present teeth, and the sequence of permanent dental eruption was not influenced by the BMI category. Conclusions: Age, weight, height, and BMI act as significant predictors for eruption symmetry. BMI does not produce alterations in the eruption sequence of the permanent dentition.

Importance of family planning in patients with immune-mediated inflammatory diseases: A multidisciplinary approach.

BACKGROUND AND OBJECTIVES: Family planning in women with immune-mediated inflammatory diseases is a challenge for healthcare teams, highlighting the need for standardized available evidence to provide patients with objective and agreed information. This study reflects the work performed by a multidisciplinary team in reviewing available scientific evidence, and the strategy agreed for family planning, pregnancy, postpartum, and breastfeeding in patients with immune-mediated inflammatory diseases. METHODS: A literature search was conducted, information was structured across the different stages (preconception, pregnancy, postpartum and breastfeeding), and an on-site meeting was convened, in which patients and healthcare providers participated. RESULTS: Specific materials, which are included in this work, were developed to guide clinical decisions to be agreed upon by patients and healthcare providers. CONCLUSION: These materials meet the need for validated and updated information on the approach and use of indicated drugs for professionals responsible for the management of immune-mediated inflammatory diseases.

Cronología y secuencia de erupción de la dentición permanente. Actualización en la población infantil española / Chronology and sequence of the permanent dentition. A review in spanish children population

Estudiar la emergencia y secuencia deerupción es indispensable para establecer protocolos de prevención y tratamientos a nivel odontopediátrico, ortodóncicoy forense. En 1933 se estableció el iniciode la erupción definitiva a los 6 años y sutérmino a los 13 a excepción de los cordales. Sin embargo, posteriormente se hanobservado variaciones determinadas porfactores raciales, hormonales, mejoras enla alimentación y parámetros corporales.Con el objetivo de conocer posibles cambios en la cronología y secuencia de erupción en dentición permanente y poblaciónespañola se ha llevado a cabo una búsqueda bibliográfica en los buscadoresPubmed, Scielo, Dentistry y Oral Science.Empleando una estrategia avanzada conoperadores boleanos “AND” y “OR”. Losresultados obtenidos advierten de unaemergencia de la dentición permanentemás precoz en el sexo femenino. Los autores coinciden en que el primer diente enerupcionar es el incisivo central inferior yel último el segundo molar superior. Sinembargo, en cuanto a la edad media dela emergencia del primer diente no hayconsenso.Los dientes permanentes mandibulareserupcionaban antes que los maxilares.La secuencia de erupción más prevalente en la Arcada superior es: primer molar, incisivo central, incisivo lateral, primerpremolar, segundo premolar, canino ysegundo molar. Y en la inferior: incisivocentral, primer molar, incisivo lateral, canino, primer premolar, segundo premolary molar. Se concluyó que existía una correlación directa entre el tiempo de erupción de los dientes temporales y la de los permanentes canine, first premolar, second premolar and second molar.To conclude, we noticed an extremely direct relation between the eruption time of the temporal teeth and thepermanent teeth (AU)

In order, to develop prevention andtreatment protocols in pediatric,orthodontic and forensic dentistry, it isvitally needed to study the sequence andchronology of eruption of the permanentteeth. Logan and Kronfield establishedthat the eruption of permanent teethstarted at the age of six and finished atthe age of 13, except the third permanentmolars. Nevertheless, in later studies thisrange of age has been modified due toan early puberty, an improvement in thenutrition and due to other factors, suchas, race, weight, and heigh. Our objectiveis to get to know the current situationabout the chronology and sequence ofpermanent dentition.We did a bibliographic review in differentweb browsers, such as, PubMed, Scieloand Dentistry and Oral Science. Weapplied an advanced strategy searchingwith “AND” and “OR”. The results warn usthat the permanent teeth erupted earlieralways in the female sex. All the authorsagree that the first tooth to erupt is thelower central incisor and the last one isthe upper second molar. Despite this fact,regarding the media age of eruption therewas not agreement between the authors.Therefore, we can conclude thatpermanent teeth erupt before in themandible than in the maxilar. The mostprevalent sequence in the maxilar was:first molar, central incisor, lateral incisor,first premolar, second premolar, canineand second molar. In the mandible was:central incisor, first molar, lateral incisor(AU)

Importancia de la planificación familiar en pacientes con enfermedades inflamatorias inmunomediadas: un abordaje multidisciplinar / Importance of family planning in patients with immune-mediated inflammatory diseases: a multidisciplinary approach

Antecedentes y objetivo: El reto terapéutico que supone para los equipos asistenciales la planificación familiar en mujeres con enfermedades inflamatorias inmunomediadas remarca la necesidad de armonizar la evidencia disponible para proporcionar a las pacientes información objetiva y consensuada. Este artículo refleja el trabajo realizado por un equipo multidisciplinar de revisión de la evidencia científica disponible y la estrategia de actuación consensuada en la planificación familiar, embarazo, posparto y lactancia materna de pacientes con enfermedades inflamatorias inmunomediadas. Métodos: Se realizó una búsqueda bibliográfica, se estructuró la información a lo largo de las diferentes etapas (preconcepción, embarazo, posparto y lactancia materna) y se realizó una reunión presencial para consensuar dicha información en la que participaron tanto pacientes como profesionales de la salud. Resultados: Se desarrollaron materiales específicos incluidos en este trabajo y que pueden servir de guía en la toma de decisiones consensuada entre pacientes y profesionales de la salud. Conclusión: Estos materiales responden a la necesidad de que los profesionales responsables del manejo de pacientes con enfermedades inflamatorias inmunomediadas posean información validada y actualizada sobre las actuaciones y el uso de fármacos indicados para estas enfermedades.(AU)

Background and objectives: Family planning in women with immune-mediated inflammatory diseases is a challenge for healthcare teams, highlighting the need for standardized available evidence to provide patients with objective and agreed information. This study reflects the work performed by a multidisciplinary team in reviewing available scientific evidence, and the strategy agreed for family planning, pregnancy, postpartum, and breastfeeding in patients with immune-mediated inflammatory diseases. Methods: A literature search was conducted, information was structured across the different stages (preconception, pregnancy, postpartum and breastfeeding), and an on-site meeting was convened, in which patients and healthcare providers participated. Results: Specific materials, which are included in this work, were developed to guide clinical decisions to be agreed upon by patients and healthcare providers. Conclusion: These materials meet the need for validated and updated information on the approach and use of indicated drugs for professionals responsible for the management of immune-mediated inflammatory diseases.(AU)

Retrospective screening for SARS-CoV-2 among influenza-like illness hospitalizations: 2018-2019 and 2019-2020 seasons, Valencia region, Spain.

On 9 March 2020, the World Health Organization (WHO) Global Influenza Programme (GIP) asked participant sites on the Global Influenza Hospital Surveillance Network (GIHSN) to contribute to data collection concerning severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We re-analysed 5833 viral RNA archived samples collected prospectively from hospital admissions for influenza-like illness (ILI) in the Valencia Region of Spain by the Valencia Hospital Surveillance Network for the Study of Influenza and Other Respiratory Viruses (VAHNSI) network (four hospitals, catchment area population 1 118 732) during the pre-pandemic 2018/2019 (n = 4010) and pandemic 2019/2020 (n = 1823) influenza seasons for the presence of SARS-CoV-2. We did not find evidence for community-acquired SARS-CoV-2 infection in hospital admissions for ILI in our region before early March 2020.