Genomic evolution of human respiratory syncytial virus during a decade (2013-2023): bridging the path to monoclonal antibody surveillance.

OBJECTIVES: This study investigated the prevalence, genetic diversity, and evolution of human respiratory syncytial virus (HRSV) in Barcelona from 2013 to 2023. METHODS: Respiratory specimens from patients with RTI suspicion at Hospital Universitari Vall d'Hebron were collected from October 2013 to May 2023 for laboratory-confirmation of respiratory viruses. Next-generation sequencing was performed in randomly-selected samples with Illumina technology. Phylogenetic analyses of whole genome sequences were performed with BEAST v1.10.4. Signals of selection and evolutionary pressures were inferred by population dynamics and evolutionary analyses. Mutations in major surface proteins were genetic and structurally characterised, emphasizing those within antigenic epitopes. RESULTS: Analyzing 139,625 samples, 5.3% were HRSV-positive (3008 HRSV-A, 3882 HRSV-B, 56 HRSV-A and -B, and 495 unsubtyped HRSV), with a higher prevalence observed in the paediatric population. Pandemic-related shifts in seasonal patterns returned to normal in 2022-2023. A total of 198 whole-genome sequences were obtained for HRSV-A (6.6% of the HRSV-A positive samples) belonging to GA2.3.5 lineage. For HRSV-B, 167 samples were sequenced (4.3% of the HRSV-B positive samples), belonging to GB5.0.2, GB5.0.4a and GB5.0.5a. HRSV-B exhibited a higher evolution rate. Post-SARS-CoV-2 pandemic, both subtypes showed increased evolutionary rates and decreased effective population size initially, followed by a sharp increase. Analyses indicated negative selective pressure on HRSV. Mutations in antigenic epitopes, including S276N and M274I in palivizumab-targeted site II, and I206M, Q209R, and S211N in nirsevimab-targeted site Ø, were identified. DISCUSSION: Particularly in the context of the large-scale use in 2023-2024 season of nirsevimab, continuous epidemiological and genomic surveillance is crucial.

The emergence, impact, and evolution of human metapneumovirus variants from 2014 to 2021 in Spain.

BACKGROUND: Human metapneumovirus (HMPV) is an important aetiologic agent of respiratory tract infection (RTI). This study aimed to describe the prevalence, genetic diversity, and evolutionary dynamics of HMPV. METHODS: Laboratory-confirmed HMPV were characterised based on partial-coding G gene sequences with MEGA.v6.0. WGS was performed with Illumina, and evolutionary analyses with Datamonkey and Nextstrain. RESULTS: HMPV prevalence was 2.5%, peaking in February-April and with an alternation in the predominance of HMPV-A and -B until the emergence of SARS-CoV-2, not circulating until summer and autumn-winter 2021, with a higher prevalence and with the almost only circulation of A2c111dup. G and SH proteins were the most variable, and 70% of F protein was under negative selection. Mutation rate of HMPV genome was 6.95 × 10-4 substitutions/site/year. CONCLUSION: HMPV showed a significant morbidity until the emergence of SARS-CoV-2 pandemic in 2020, not circulating again until summer and autumn 2021, with a higher prevalence and with almost the only circulation of A2c111dup, probably due to a more efficient immune evasion mechanism. The F protein showed a very conserved nature, supporting the need for steric shielding. The tMRCA showed a recent emergence of the A2c variants carrying duplications, supporting the importance of virological surveillance.

Increased RSV-A Bronchiolitis Severity in RSV-Infected Children Admitted to a Reference Center in Catalonia (Spain) Between 2014 and 2018.

Between 2014 and 2018, we evaluated the severity of 687 cases of bronchiolitis caused by respiratory syncytial virus (RSV) in Catalonia, Spain. Compared to RSV-B, RSV-A cases required intensive care (adjusted relative risk (aRR) = 1.44, p < 0.01) and respiratory support (aRR = 1.07, p < 0.01) more often; hospital stay was one day longer (p < 0.01). Subgroup identification may aid clinical evaluation and seasonal healthcare planning.

Molecular characterization and clinical impact of human bocavirus at a tertiary hospital in Barcelona (Catalonia, Spain) during the 2014-2017 seasons.

PURPOSE: The aim was to describe the prevalence, molecular epidemiology and clinical manifestations of human bocavirus (HBoV) in patients attended at a tertiary hospital in Barcelona, Spain. METHODS: From October 2014 to May 2017, respiratory specimens from paediatric patients were collected for respiratory viruses' laboratory-confirmation. Phylogenetic analyses from partial VP1 sequences were performed from all HBoV laboratory-confirmed specimens. Clinical features were retrospectively studied. RESULTS: 178/10271 cases were HBoV laboratory-confirmed. The median age was 1.53 (IQR 1.0-2.3). Co-detection was highly reported (136; 76%). All viruses belonged into HBoV1 genotype but one into HBoV2. Non-reported mutations were observed and two sites were suggestive to be under negative selection. 61% (109/178) cases had lower RTI (LRTI), of whom 84 had co-detections (77%) and 76 had comorbidities (70%). LRTI was the cause of hospitalization in 85 out of 109 cases (78%), and no differences were found regarding severity factors during hospitalization between co- and single-detections, except for median length of respiratory support, which was longer in cases with co-detections. CONCLUSIONS: Close monitoring of predominant HBoV1 showed a high similarity between viruses. The presence of comorbidities might explain the high prevalence of LRTI. Symptomatology in HBoV single-detected cases suggest that HBoV is a true pathogen.

The burden of non-SARS-CoV2 viral lower respiratory tract infections in hospitalized children in Barcelona (Spain): A long-term, clinical, epidemiologic and economic study.

BACKGROUND: Viral lower respiratory tract infections (LRTI) are the leading cause of hospitalization in children. In Catalonia (Spain), information is scarce about the burden of viral LRTIs in paediatric hospitalizations. The aim of this study is to describe epidemiological, clinical, virological and economic features of paediatric hospitalizations due to viral LRTI. METHODS: From October 2012 to December 2020, children aged <16 years admitted to a tertiary paediatric hospital in Catalonia (Spain) with confirmed viral LRTI were included in the study. Virus seasonality, prevalence, age and sex distribution, clinical characteristics, hospital costs and bed occupancy rates were determined. RESULTS: A total of 3,325 children were included (57.17% male, 9.44% with comorbidities) accounting for 4056 hospitalizations (32.47% ≤ 12 months): 53.87% with wheezing/asthma, 37.85% with bronchiolitis and 8.28% with pneumonia. The most common virus was respiratory syncytial virus (RSV) (52.59%). Influenza A was associated with pneumonia (odds ratio [OR] 7.75) and caused longer hospitalizations (7 ± 31.58 days), while RSV was associated with bronchiolitis (OR 6.62) and was the most frequent reason for admission to the paediatric intensive care unit (PICU) (11.23%) and for respiratory support (78.76%). Male sex, age ≤12 months, chronic conditions and bronchiolitis significantly increased the odds of PICU admission. From October to May, viral LRTIs accounted for 12.36% of overall hospital bed days. The total hospitalization cost during the study period was €16,603,415. CONCLUSIONS: Viral LRTIs are an important cause of morbidity, hospitalization and PICU admission in children. The clinical burden is associated with significant bed occupancy and health-care costs, especially during seasonal periods.

Enterovirus D68 in Hospitalized Children, Barcelona, Spain, 2014-2021.

To determine molecular epidemiology and clinical features of enterovirus D68 (EV-D68) infections, we reviewed EV-D68-associated respiratory cases at a hospital in Barcelona, Spain, during 2014-2021. Respiratory samples were collected from hospitalized patients or outpatients with symptoms of acute respiratory tract infection or suggestive of enterovirus infection. Enterovirus detection was performed by real-time multiplex reverse transcription PCR and characterization by phylogenetic analysis of the partial viral protein 1 coding region sequences. From 184 patients with EV-D68 infection, circulating subclades were B3 (80%), D1 (17%), B2 (1%), and A (<1%); clade proportions shifted over time. EV-D68 was detected mostly in children (86%) and biennially (2016, 2018, 2021). In patients <16 years of age, the most common sign/symptom was lower respiratory tract infection, for which 11.8% required pediatric intensive care unit admission and 2.3% required invasive mechanical ventilation; neurologic complications developed in 1. The potential neurotropism indicates that enterovirus surveillance should be mandatory.

Diabetis insípida associada a holoprosencefàlia / Diabetes insípida asociada a holoprosencefalia / Diabetes insipidus associated with holoprosencephaly

Introducció. L’holoprosencefàlia és una malformació congènita greu que compromet estructures cerebrals situades ala línia mitjana. Per la seva localització, l’hipotàlem i lahipòfisi són estructures afectades sovint en aquesta entitat. La seva disfunció pot provocar trastorns de l’homeòstasii endocrinopaties com l’adípsia i la diabetis insípida.Cas clínic. Es presenta el cas d’una nena de 6 anys diagnosticada d’holoprosencefàlia als 9 mesos de vida. Consulta per somnolència i febre de 48 hores d’evolució i enl’exploració presenta signes de deshidratació. La mare refereix que no reclama ingesta hídrica tot i trobar-se deshidratada. Analíticament destaca deshidratació hipernatrèmica greu amb disfunció renal. A les 72 hores d’ingrés,després d’haver corregit les diselectrolitèmies i amb lapacient normohidratada, reapareix la hipernatrèmia ambpoliúria i osmolalitat urinària disminuïda. Es fa un test dedesmopressina que confirma la sospita diagnòstica de dèficit d’hormona antidiürètica associat a adípsia. S’instauratractament amb desmopressina i restricció hídrica amb elqual s’assoleix un bon control hidroelectrolític que esmanté en controls posteriors.Comentaris. Els pacients amb holoprosencefàlia tenen riscde presentar endocrinopaties per disfunció hipotalamohipofisiària. Per aquest motiu cal buscar-les activament encas de presentar-se la clínica clàssica de poliúria, hipernatrèmia i hipoosmolalitat urinària. (AU)

Introducción. La holoprosencefalia es una malformación congénitagrave que compromete estructuras cerebrales situadas en la líneamedia. Por su localización, el hipotálamo y la hipófisis son estructuras frecuentemente afectadas en esta entidad. Su disfunciónpuede generar trastornos de la homeostasis y endocrinopatíascomo la adipsia y la diabetes insípida.Caso clínico. Se presenta el caso de una niña de 6 años diagnosticada de holoprosencefalia a los 9 meses de vida. Consulta por somnolencia y fiebre de 48 horas de evolución y en la exploración físicadestacan signos de deshidratación. La madre refiere que no reclama ingesta hídrica pese a encontrarse deshidratada. Analíticamente destaca deshidratación hipernatrémica grave y disfunciónrenal. A las 72 horas de ingreso, corregidas las diselectrolitemias yencontrándose la paciente normohidratada, reaparece hipernatremia asociando poliuria e hipoosmolalidad urinaria. Se realiza untest de desmopresina que confirma la sospecha diagnóstica de déficit de hormona antidiurética asociado a adipsia. Se instaura tratamiento con desmopresina y restricción hídrica, alcanzando un buencontrol hidroelectrolítico que mantiene en controles posteriores.Comentarios. Los pacientes con holoprosencefalia tienen riesgo depresentar endocrinopatías por disfunción hipotálamo-hipofisaria.Por ese motivo es necesario buscarlas activamente en caso depresentarse la clínica clásica de poliuria, hipernatremia e hipoosmolalidad urinaria. (AU)

Introduction. Holoprosencephaly is a severe developmental defectthat compromise midline brain structures. Due to their location,the hypothalamus and the hypophysis are frequently involvedin this illness. Their dysfunction can lead to homeostatic andendocrine disorders such as thirst disturbances and diabetesinsipidus.Case report. Herein we report the case of a 6-year-old girl who wasdiagnosed with holoprosencephaly at 9 months old. She presentedwith a 48-hours history of somnolence and fever with signs of dehydration on examination. Her mother stated that despite beingdehydrated, she did not demand water intake. Laboratory evaluation revealed a severe hypernatremic dehydration with renal impairment. Seventy-two hours after admission, having successfullyattained fluid and electrolyte correction, she presented hypernatremia with polyuria and urine hypoosmolality. A desmopressin testwas performed confirming the suspected diagnosis of antidiuretichormone deficiency and adipsia. She responded well to desmopressin treatment and fluid restriction with restoration of serumelectrolytes, which remained within acceptable limits at follow-up.Discussion. Children with holoprosencephaly are at risk for endocrine disorders related to hypothalamic-pituitary dysfunction. It isimportant to rule out these disturbances if they present with theclassic triad of hypernatremia, polyuria and urine hypoosmolality. (AU)

Respiratory Syncytial Virus Infections in Young Children Presenting to Primary Care in Catalonia During the COVID-19 Pandemic.

We observed an unusual pattern of respiratory syncytial virus (RSV) in children under 5 years in Catalonia (Spain). We observed a near absence of RSV during winter months and a subsequent surge during the late spring. Primary care electronic health records combined with hospital RSV laboratory confirmation could be used to monitor trends of respiratory pathogens.

Management of Hospitalized Respiratory Syncytial Virus Bronchiolitis in the Pediatric Ward in Spain: Assessing the Impact of a New Clinical Practice Protocol.

BACKGROUND: Bronchiolitis caused by the respiratory syncytial virus (RSV) is the main reason for hospitalization in infants. Supportive care is the mainstay of treatment, and tests are restricted to a few indications. During 2015, our hospital bronchiolitis protocol (2015 HBP) was updated according to the latest practice guidelines. OBJECTIVE: The objective of this study was to assess implementation of the 2015 HBP and the clinical outcome of children aged ≤ 24 months with RSV bronchiolitis admitted to a pediatric ward. METHODS: We compared the use of treatments and tests, hospital length of stay (LOS), and oxygen requirements before implementation of the 2015 HBP (2014-2015 and 2015-2016 seasons) and after implementation (2016-2017 and 2017-2018 seasons). RESULTS: The study population comprised 251 children (44.90%) in the first period and 308 (55.10%) in the second (median age 99 days, interquartile range 44-233). After implementation of the 2015 HBP, a statistically significant reduction was found in the percentage of patients undergoing the following treatments or diagnostic tests: salbutamol, from 57.77 to 31.17% (p < 0.001); epinephrine, from 61.75 to 1.30% (p < 0.001); 3% hypertonic saline, from 70.12 to 6.82% (p < 0.001); antibiotics, from 33.07 to 23.05% (p = 0.008); and chest X-ray, from 43.82 to 31.17% (p = 0.001). No statistically significant reductions were observed in the use of corticosteroids and blood tests. Hospital LOS and oxygen requirements were similar in each period. CONCLUSIONS: Appropriate implementation of the 2015 HBP in the pediatric ward improves the use of medication and chest X-ray without modifying clinical outcomes. However, further efforts are needed to reduce the use of salbutamol, corticosteroids, and blood tests.

Liver, Renal, and Cardiovascular Failure After Unintentional Overdose of Tizanidine in a 2-Year-Old Child.

Tizanidine is a central alpha-2 adrenergic receptor agonist indicated for the treatment of spasticity in adults; however, its use in the pediatric population is considered off-label. In adults, the dose is gradually titrated until the desired reduction in muscle tone is achieved. Hypotension is a frequent adverse effect, but impaired liver function is not characteristic of alpha-2 adrenergic agonist overdose. We report a 2-year-old male affected with spastic quadriplegia (treated with clonazepam and tizanidine) and dysphagia (he was fed by nasogastric tube). Two days before admission caregivers ran out of clonazepam so they increased the tizanidine dose from 0.15 mg/kg/day to 1.6 mg/kg/day. Simultaneously his nasogastric tube fell out; therefore, he was unable to maintain proper oral nutrition and hydration. He presented to the emergency department hemodynamically unstable, with impaired consciousness and signs of severe dehydration. Blood tests revealed hepatic dysfunction without cholestasis and renal dysfunction. He was transferred to the pediatric intensive care unit. Treatment was mainly supportive, apart from tizanidine discontinuation. Metabolic and infectious diseases were ruled out so he was finally diagnosed as having liver, renal, and cardiovascular failure after tizanidine overdose, worsened by dehydration. His clinical status improved, and after 3 weeks he was discharged from the hospital, receiving clonidine instead of tizanidine to treat spasticity. Tizanidine overdose can result in serious complications that can be worsened because of patient comorbidities.

Insights into immune evasion of human metapneumovirus: novel 180- and 111-nucleotide duplications within viral G gene throughout 2014-2017 seasons in Barcelona, Spain.

BACKGROUND: Human metapneumovirus (HMPV) is an important aetiologic agent of respiratory tract infection (RTI). This study aimed to describe its genetic diversity and clinical impact in patients attended at a tertiary university hospital in Barcelona from the 2014-2015 to the 2016-2017 seasons, focusing on the emerging duplications in G gene and their structural properties. METHODS: Laboratory-confirmed HMPV were characterised based on partial-coding F and G gene sequences with MEGA.v6.0. Computational analysis of disorder propensity, aggregation propensity and glycosylation sites in viral G predicted protein sequence were carried out. Clinical data was retrospectively reviewed and further associated to virological features. RESULTS: HMPV prevalence was 3%. The 180- and 111-nucleotide duplications occurred in A2c lineage G protein increased in prevalence throughout the study, in addition to short genetic changes observed in other HMPV lineages. The A2c G protein without duplications was calculated to protrude over F protein in 23% of cases and increased to a 39% and a 46% with the 111- and 180-nucleotide duplications, respectively. Children did not seem to be more affected by these mutant viruses, but there was a strong association of these variants to LRTI in adults. DISCUSSION: HMPV presents a high genetic diversity in all lineages. Novel variants carrying duplications might present an evolutionary advantage due to an improved steric shielding, which would have been responsible for the reported increasing prevalence and the association to LRTI in adults.

The high genetic similarity between rhinoviruses and enteroviruses remains as a pitfall for molecular diagnostic tools: A three-year overview.

BACKGROUND: Enteroviruses (EVs) and rhinoviruses (RVs) belong to the Enterovirus genus within the Picornaviridae family, and show genetic similarities. These viruses are related to mild diseases, but EVs infections can sometimes lead to more severe complications. Current diagnostic molecular techniques should discriminate between the four EV and the three RV species that infect humans. The aim was to revise the EV and RV PCR-confirmed specimens by sequencing for genetic characterisation. MATERIAL AND METHODS: Respiratory tract specimens were collected from patients with suspicion of respiratory infection. Respiratory viruses' laboratory-confirmation was performed by commercial multiplex real-time RT-PCR assays. Genetic characterisation of all EV and in a selection of RV was performed based on the phylogenetic analyses of partial VP1 and VP4/2 sequences, respectively. RESULTS: From 19,957 tested specimens, 309 (1.5%) were EV-positive, 2546 (12%) were RV-positive, and 233 (1%) were EV/RV co-detections. The phylogenetic analyses revealed that: among single EV detections, 177/309 (57%) were characterised as EV, 2/309 (1%) as RV, and 130/309 (42%) could not be typed; among single 1771 RV detections (Ct < 35), 1651/1771 (93%) were characterised as RV, 3/1771 (0.3%) as EV and 117/1771 (6.7%) could not be typed. Among EV/RV co-detections, 62/233 (27%) were characterised as EV, 130/233 (56%) as RV and 41/233 (18%) could not be typed. CONCLUSIONS: A diagnostic method well considered for routine laboratory-confirmation of respiratory viruses should discriminate EV and RV targets. RVs are usually associated with mild respiratory disease, but the potential relatedness of EVs to neurological complications makes their monitoring mandatory. Therefore, an accurate detection and differentiation should be required in commercial diagnostic solutions.

Recombinant CV-A6 strains related to hand-foot-mouth disease and herpangina at primary care centers (Barcelona, Spain).

Aim: To describe the genetic diversity of enteroviruses (EV) causing hand, foot and mouth disease (HFMD) and herpangina, especially of coxsackievirus (CV)-A6, from patients attended at pediatric primary care centers during the 2017-2018 season. Methods: Phylogenetic analysis of partial VP1 region was performed for genetic characterization. The complete VP1 and 3Dpol proteins were sequenced for lineage determination and detection of recombination events. Results: An 80% of samples were EV laboratory-confirmed. CV-A6 was the most detected (70%) and associated with atypical HFMD (78%). The comparison of VP1 and 3Dpol phylogenies showed evidence of recombination in three strains, in which two shifted to CV-A16 3Dpol. Conclusion: The study provides recent information regarding the nonrecombinant and recombinant EVs related to HFMD at primary care centers.

Virological surveillance of human respiratory syncytial virus A and B at a tertiary hospital in Catalonia (Spain) during five consecutive seasons (2013-2018).

AIM: Human respiratory syncytial virus (HRSV) is the main cause of respiratory tract infections among infants. MATERIALS & METHODS: In the present study, the molecular epidemiology of HRSV detected from 2013 to 2017 has been described. RESULTS: A 10% of collected samples were laboratory confirmed for HRSV. Patients under 2 years of age were the main susceptible population to respiratory syncytial virus disease, but an increasingly number of confirmed patients over 65 years of age was reported. Epidemics usually started in autumn and ended in spring. Both HRSV groups co-circulated every season, but the HRSV-B was the most predominant. HRSV-A and HRSV-B strains mainly belonged to ON1 and BA9 genotypes, respectively. CONCLUSION: The present study reports recent data about the genetic diversity of circulating HRSV in Spain.

Surveillance of enteroviruses from paediatric patients attended at a tertiary hospital in Catalonia from 2014 to 2017.

BACKGROUND: Enterovirus (EV) infections are usually asymptomatic or mild, but symptomatic infections can evolve to severe complications. Outbreaks of EV-A71 and EV-D68 have been recently reported worldwide, sometimes related to severe clinical outcomes. OBJECTIVE: To describe EV genetic diversity and the clinical outcomes from paediatric patients attended at a tertiary university hospital in Barcelona (Catalonia, Spain) from 2014 to 2017. STUDY DESIGN: Specimens were collected from paediatric (<17 years old) cases with suspicion of respiratory tract infection or EV infection. EV laboratory-confirmation was performed by specific real-time multiplex RT-PCR assay. Partial viral VP1 protein was sequenced for genetic characterisation by phylogenetic analyses. RESULTS: A total of 376 (7%) from 5703 cases were EV laboratory-confirmed. Phylogenetic analyses of VP1 (210; 81%) sequences distinguished up to 27 different EV types distributed within EV-A (82; 40%), EV-B (90; 42%), EV-C (5; 2%), and EV-D (33; 15%), in addition to 50 (19%) rhinoviruses. The most predominant were EV-A71 (37; 45%) and EV-D68 (32; 99%). EV-A71 was highly related to neurological complications (25/39, 63%), of which 20/39 were rhombencephalitis, and most EV-D68 (28/32, 88%) were associated with lower respiratory tract infections (LRTI), and exceptionally one (3%) with acute flaccid paralysis. CONCLUSIONS: EV-A71 and EV-D68 were the most detected EV in respiratory specimens. EV-A71 was highly related to neurological disease and EV-D68 was often associated with LRTI. However, both potential relatedness to neurological diseases makes the monitoring of EV circulation obligatory.

Novel human metapneumovirus with a 180-nucleotide duplication in the G gene.

AIM: To describe the circulation, genetic diversity and clinical features of human metapneumovirus (HMPV) in pediatric patients that attended the Hospital Universitari Vall d'Hebron, Spain from 2014 to 2016. MATERIALS & METHODS: Partial G gene was sequenced from laboratory-confirmed HMPV respiratory specimens for subsequent phylogenetic analysis. RESULTS: A total of 121 different samples were HMPV laboratory-confirmed out of 6658 specimens received. The highest circulation was from February to April, with a prevalence of 3%. Different genetic groups within both genotypes were detected at variable levels. A 180-nucleotide duplication was first characterized within the G gene in nine cases, mostly related to lower respiratory-tract infection. CONCLUSION: This study reported on the circulation of a novel HMPV with a 180-nucleotide duplication in the G gene, but no clinical changes in related cases were observed. Their prevalence increased during the last season suggesting changes in viral features.