Hybrid capture shotgun sequencing detected unexpected viruses in the cerebrospinal fluid of children with acute meningitis and encephalitis.

PURPOSE: Investigation of undiagnosed cases of infectious neurological diseases, especially in the paediatric population, remains a challenge. This study aimed to enhance understanding of viruses in CSF from children with clinically diagnosed meningitis and/or encephalitis (M/ME) of unknown aetiology using shotgun sequencing enhanced by hybrid capture (HCSS). METHODS: A single-centre prospective study was conducted at Sant Joan de Déu University Hospital, Barcelona, involving 40 M/ME episodes of unknown aetiology, recruited from May 2021 to July 2022. All participants had previously tested negative with the FilmArray Meningitis/Encephalitis Panel. HCSS was used to detect viral nucleic acid in the patients' CSF. Sequencing was performed on Illumina NovaSeq platform. Raw sequence data were analysed using CZ ID metagenomics and PikaVirus bioinformatics pipelines. RESULTS: Forty episodes of M/ME of unknown aetiology in 39 children were analysed by HCSS. A significant viral detection in 30 CSF samples was obtained, including six parechovirus A, three enterovirus ACD, four polyomavirus 5, three HHV-7, two BKV, one HSV-1, one VZV, two CMV, one EBV, one influenza A virus, one rhinovirus, and 13 HERV-K113 detections. Of these, one sample with BKV, three with HHV-7, one with EBV, and all HERV-K113 were confirmed by specific PCR. The requirement for Intensive Care Unit admission was associated with HCSS detections. CONCLUSION: This study highlights HCSS as a powerful tool for the investigation of undiagnosed cases of M/ME. Data generated must be carefully analysed and reasonable precautions must be taken before establishing association of clinical features with unexpected or novel virus findings.

Factors Involved in the Immunological Protection against Rabies Virus in Dogs in Spain.

Rabies, a viral disease spread by infected animal bites that causes encephalitis in humans and other mammals, is a neglected infectious disease present on all continents except Antarctica. Spain has been free of terrestrial rabies since 1978. However, due to its geographical situation, it represents a bridge for imported cases from an endemic continent such as Africa to Europe. Rabies vaccination in dogs is an essential preventive tool against this zoonosis. The aim of this study was to determine the state of the immune response against rabies virus in dogs in Spain and to demonstrate whether several factors that have been previously related to the influence of the seroprevalence of this species are involved here. The seroconversion level of this zoonotic virus was assessed in a total of 1060 animals. Indirect ELISA was used to obtain data for statistical analysis to evaluate the studied variables. Working under the concept of One Health, this study provides relevant information to be taken into consideration not only to prevent re-emergence in countries free of this disease but also for prevention and control in endemic countries.

A novel high-resolution melting analysis strategy for detecting cystic fibrosis-causing variants.

Cystic fibrosis (CF), an autosomal recessive disease, is caused by variants in both alleles of the CF transmembrane conductance regulator (CFTR) gene. A new assay based on allele-specific polymerase chain reaction and high-resolution melting analysis was developed for the detection of 18 CF-causing CFTR variants previously identified in Cuba and Latin America. The assay is also useful for zygosity determination of mutated alleles and includes internal controls. The reaction mixtures were normalized and evaluated using blood samples collected on filter paper. The evaluation of analytical parameters demonstrated the specificity and sensitivity of the method to detect the included CFTR variants. Internal and external validations yielded a 100% agreement between the new assay and the used reference tests. This assay can complement CF newborn screening not only in Cuba but also in Latin America.

Patterns of Sexualized Drug Use among Gay, Bisexual, and Other Men Who Have Sex with Men Living with HIV: Insights from a Comprehensive Study-The U-SEX-2 GESIDA 9416 Study.

BACKGROUND: Sexualized drug use (SDU) has become a public health concern in recent years. This study aimed to estimate the prevalence of SDU in gay, bisexual, and other men who have sex with men living with HIV (HIV + GBMSM) in Madrid during 2019/2020 and compare it with data from 2016/2017 in order to detect changes in patterns. METHODS: We analyzed the frequency of SDU in a sample of HIV + GBMSM attending HIV clinics, who participated in an anonymous online survey regarding sexual behavior and recreational drug use. The association between SDU, sexual risk behaviors, and STIs was evaluated. RESULTS: This study included 424 HIV + GBMSM, with a mean age of 40 (10.43) years. Overall, 94% (396) reported being sexually active. Additionally, 33% (140) had been diagnosed with an STI within the previous year. Moreover, 54% (229) had used drugs in the last year, 25% (107) engaged in SDU, and 16% (17) reported engagement in slamsex. After adjusting for confounding factors, SDU was associated with STIs, fisting, unprotected anal intercourse, and having >24 sexual partners in the last year. According to the DUDIT test scores, 80% (81) probably had problematic drug use (≥6 points), and 8% (8) probable drug dependence (≥25 points). When comparing the U-SEX-1 (2016/2017) data with the U-SEX-2 (2019/2020) data, no significant differences were found in the proportion of participants practicing SDU or slamming. CONCLUSIONS: The prevalence of SDU among HIV + GBMSM has remained high in recent years and without significant changes. The risk of problematic drug use among those who practice SDU is high. We observed a clear association between SDU, high-risk sexual behaviors, and STIs.

Mutations in Coding and Non-Coding Regions in Varicella-Zoster Virus Causing Fatal Hemorrhagic Fever Without Rash in an Immunocompetent Patient: Case Report.

INTRODUCTION: We report the case of a fatal hemorrhagic varicella primary infection in an immunocompetent man and whole-genome characterization of the virus for the investigation of biomarkers of virulence. CASE: A 38-year-old patient born in Nigeria presented to the emergency department with abdominal pain and subsequently developed fatal hemorrhagic disease without skin rash. Extensive laboratory tests including serology and PCR for arenaviruses, bunyaviruses and ebolaviruses were negative. Varicella-zoster virus (VZV) PCR of sera, liver and spleen tissue samples from autopsy revealed the presence of VZV DNA. Primary infection by varicella-zoster virus with hemorrhagic manifestations was diagnosed after virological testing. The VZV genome was sequenced using a mWGS approach. Bioinformatic analysis showed 53 mutations across the genome, 33 of them producing non-synonymous variants affecting up to 14 genes. Some of them, such as ORF11 and ORF 62, encoded for essential functions related to skin or neurotropism. To our knowledge, the mutations reported here have never been described in a VZV causing such a devastating outcome. DISCUSSION: In immunocompetent patients, viral factors should be considered in patients with uncommon symptoms or severe diseases. Some relevant mutations revealed by using whole genome sequencing (WGS) directly from clinical samples may be involved in this case and deserves further investigation. CONCLUSION: Differential diagnosis of varicella-zoster virus in immunocompetent adults should be considered among patients with suspected VHF, even if the expected vesicular rash is not present at admission and does not arise thereafter. Whole genome sequencing of strains causing uncommon symptoms and/or mortality is needed for epidemiological surveillance and further characterization of putative markers of virulence. Additionally, this report highlights the recommendation for a VZV vaccination policy in non-immunized migrants from developing countries.

Suspensión de antibióticos a través de un programa de optimización de tratamiento antibiótico en pacientes atendidos en Urgencias con baja sospecha de infección / Antibiotic discontinuation through an antibiotic treatment optimization program in emergency department patients with low suspicion of infection

Introducción: Una de las estrategias más importantes de los PROA en los Servicios de Urgencias (SU) es el diagnóstico adecuado de infección para evitar la prescripción inadecuada. Nuestro objetivo es evaluar a los pacientes que reciben antibiótico a pesar de no tener datos objetivos de infección. Métodos: Realizamos un estudio transversal de los pacientes ingresados en el SU del Hospital Universitario Fundación Alcorcón durante 2 meses (mayo del 2019 y marzo del 2021) en los que se recomendó suspender el antibiótico a través del PROA. Se analizaron las características clínicas y epidemiológicas, y el seguimiento a 30 días para valorar los reingresos y la mortalidad. Resultados: Se analizaron 145 pacientes. Se recomendó suspender el antibiótico en 25. El 44% de ellos tenían diagnóstico de infección urinaria. La recomendación de suspensión se aceptó en el 88%. Ningún paciente falleció y uno reingresó. Conclusiones: Un porcentaje importante de pacientes tenían prescrito antibiótico a pesar de no tener criterios de infección, siendo la evolución clínica tras la de prescripción favorable.(AU)

Introduction: One of the most important strategies of PROA in the Emergency Department (ED) is the accurate diagnosis of infection to avoid inappropriate prescription. Our objective is to evaluate patients who receive antibiotics despite not having objective data of infection. Methods: We carried out a cross-sectional study of patients admitted to the ED of the Hospital Universitario Fundación Alcorcón in which it was recommended to suspend the antibiotic through the PROA. Clinical and epidemiological characteristics and 30-day follow-up were analyzed to assess readmissions and mortality. Results: 145 patients were analyzed. It was recommended to suspend the antibiotic in 25. 44% of them had a diagnosis of urinary infection. The suspension recommendation was accepted by 88%. No patient died and one was readmitted. Conclusions: An important percentage of patients are prescribed antibiotics despite not having infection criteria, the clinical evolution after suspension of antibiotics was favorable.(AU)

Vaccination trends in people with HIV infection participanting in the hospital-based survey of patients infected with HIV, 2006-2021.

OBJECTIVE: To assess the trend in hepatitis A, hepatitis B, pneumococcal, tetanus and seasonal influenza vaccination in people with HIV infection and to analyse associated factors. METHODS: The Hospital Survey of Patients with HIV, an annual cross-sectional study conducted on a fixed day (2006-2021), was used. Inpatients and outpatients were included. Trends in vaccination and associated factors were analysed using logistic regression. RESULTS: A total of 8643 participants were included. Vaccination rates increased to 65.3% for hepatitis A, 83.7% for hepatitis B, 49.3% for tetanus, 68.9% for pneumococcal and 74.5% for seasonal influenza in 2021. Factors positively associated with vaccination were older age for pneumococcal and influenza vaccination; higher educational level for hepatitis A and tetanus; living in a closed institutions or prison for tetanus, pneumococcal and influenza; and having acquired HIV through sex between men for hepatitis A, B and pneumococcal. In addition, being on antiretroviral treatment and having a high CD4 count were positively associated with vaccination for all these diseases. Factors inversely associated with vaccination were being older (hepatitis A, B and tetanus), being an immigrant (tetanus and seasonal influenza) and being an injection drug user/ex-user for hepatitis A and B. CONCLUSIONS: Vaccination in people with HIV has increased in the study period. The results are in line with the recommendations in this population, although there is still room to reach the established vaccination indicators.

First Report of Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst Nematode) Infecting Corn (Zea mays) in Spain.

Heterodera zeae Koshy, Swarup & Sethi, 1971 (corn cyst nematode) is an important disease of corn in several areas of the world, including India, Nepal, Pakistan, Egypt, USA, Greece and Portugal (Subbotin et al., 2010). It is a sedentary semi-endoparasite feeding on corn roots and other Poaceae plants and has been associated with significant yield losses in corn (Subbotin et al., 2010). During autumn 2022 a plant-parasitic nematode survey performed in corn at central-western area of Spain (Talavera de la Reina, Toledo), revealed a commercial field with stunted plants. Nematodes were extracted from soil by centrifugal-flotation method (Coolen, 1979). Corn roots inspection detected infections by immature and mature cysts, and soil revealed also mature live cysts and second-stage juveniles (J2s) with a population density of 1010 eggs and J2s/500 cm3 soil (including eggs from cysts). J2s and cysts were processed to pure glycerine using De Grisse's (1969) method. DNA was isolated from single live fresh J2s specimens for amplifying and sequencing of cytochrome c oxidase subunit II (COII) mitochondrial region using the primer pair species-specific H.Gly-COIIF_inFOR/P116F-1R (Riepsamen et al., 2011); D2 and D3 expansion domains of the 28S rRNA were amplified using the D2A/D3B primers (De Ley et al. 1999); internal transcribed spacer (ITS) region using primers TW81/AB28 (Subbotin et al., 2001); and cytochrome c oxidase subunit 1 (COI) gene was amplified using the primers JB3/JB5 (Bowles et al., 1992). Brown cysts were lemon-shaped with a protruding vulval cone with fenestra ambifenestrate, bullae prominent below underbridge and characteristically arranged in finger-like bullae (Fig. 1). J2 with slightly offset lip region (3-5 annuli), stylet strong with rounded stylet knobs, lateral field with four lines, and tail short and tapering conically. Measurements of cysts (n=10) included body length 559 (432-688) µm, body width 450 (340-522) µm, fenestral length 40 (36-43) µm, semifenestral width 19 (17-21) µm, and vulval slit 40 (35-44) µm. J2 measurements (n=10) included body length 477 (420-536) µm, stylet length 21 (20-22) µm, tail length 51 (47-56) µm, and tail hyaline region 23 (20-26) µm. Morphology and morphometrics of cysts and J2, fit with original description and others from several countries (Subbotin et al., 2010). Two J2s individuals were sequenced for COII region (OQ509010-OQ509011) showing 97.1-98.1% similarity with H. zeae from USA (HM462012). Six almost identical 28S rRNA sequences from J2s (OQ449649-OQ449654) were 99.2-99.4% similar to 28S rRNA sequences of H. zeae from Greece, Afghanistan and USA (GU145612, JN583885, DQ328695). Four identical ITS DNA fragments from J2s (OQ449655-OQ449658) were 97.0-97.8% similar to ITS sequences of H. zeae from Greece, and China (GU145616, MW785771, OP692770). Finally, six COI sequences of 400 bp obtained for J2s (OQ449699-OQ449704) were under 87% similarity to several COI sequences of Heterodera spp. in NCBI, being a new molecular barcoding for identifying this species. On the basis of these results, the cyst nematodes isolated from the corn plants from the central-western area of Spain (Talavera de la Reina, Toledo) were confirmed as H. zeae and up to our knowledge it is the first report in Spain. This is a well-known pest of corn, causing important losses in this crop (Subbotin et al., 2010) and it was previously regulated as a quarantine nematode in the Mediterranean region (EPPO).

An interregional measles outbreak in Spain with nosocomial transmission, November 2017 to July 2018.

Given sustained high vaccination coverage and enhanced surveillance for measles, Spain has been free of endemic measles transmission since 2014, achieving elimination certification from the World Health Organization in 2017. In November 2017, measles was introduced through an imported case travelling to the Valencian Community, causing an interregional outbreak. Here, we describe the outbreak using data reported to the national epidemiological surveillance network. The outbreak involved 154 cases (67 males, 87 females) notified in four regions; 148 were laboratory-confirmed and six epidemiologically linked. Most cases were adults aged 30-39 (n = 62, 40.3%) years. Sixty-two cases were hospitalised (40.3%) and 35 presented complications (22.7%). Two thirds of the cases (n = 102) were unvaccinated including 11 infants (≤ 1 year) not yet eligible for vaccination. The main route of transmission was nosocomial; at least six healthcare facilities and 41 healthcare workers and support personnel were affected. Sequencing of the viral nucleoprotein C-terminus (N450) identified genotype B3, belonging to the circulating MVs/Dublin.IRL/8.16-variant. Control measures were implemented, and the outbreak was contained in July 2018. The outbreak highlighted that raising awareness about measles and improving the vaccination coverage in under-vaccinated subgroups and personnel of healthcare facilities are key measures for prevention of future outbreaks.

A Blind-Identification Test on Criconema annuliferum (de Man, 1921) Micoletzky, 1925 Species Complex Corroborate the Hyper-Cryptic Species Diversity Using Integrative Taxonomy.

Ring nematodes are obligate ectoparasites on crops and natural herbaceous and woody plants, and some species are of economic importance and cause damage to roots of several crops. Recent integrative taxonomical analyses recognized the existence of two cryptic species within the Criconema annuliferum morphotype in Spain. In this study, we corroborated that morphometric, morphological and a multi-locus analysis (including the ribosomal markers D2-D3 expansion segments of 28S rRNA, ITS rRNA, 18S RNA, and the mitochondrial DNA cytochrome oxidase I gene) identified a new lineage clearly separated from C. annuliferum, C. paraannuliferum and C. plesioannuliferum. The new lineage was described herein as Criconema pseudoannuliferum sp. nov., confirming that C. annuliferum species complex species complex comprises a hyper-cryptic species complex. This research analysed soil samples from the rhizosphere of maritime pine (Pinus pinaster Ait.) forests in Bermeja-Crestellina Mountain, located at the western part of Málaga province, southern Spain. The integrative taxonomical analyses revealed the occurrence of a new cryptic species identified using females, males and juveniles with detailed morphology, morphometry and molecular markers, described herein as Criconema pseudoannuliferum sp. nov. All molecular markers (D2-D3, ITS, 18S and COI) were obtained from the same individual that was also used for morphological and morphometric analyses. This research demonstrated the hidden diversity within the C. annuliferum species complex species complex can reach to four lineages under ribosomal and mitochondrial gene markers for one morphospecies group, which includes four species, viz. C. annuliferum, C. paraannuliferum, C. plesioannuliferum, and C. pseudoannuliferum sp. nov. Criconema pseudoannuliferum sp. nov. was detected in moderate soil density in two maritime pine forests (5 and 25 nematodes/500 cm3 of soil) suggesting that does not cause damage to maritime pine.

Temporal Series Analysis of Population Cycle Threshold Counts as a Predictor of Surge in Cases and Hospitalizations during the SARS-CoV-2 Pandemic.

Tools to predict surges in cases and hospitalizations during the COVID-19 pandemic may help guide public health decisions. Low cycle threshold (CT) counts may indicate greater SARS-CoV-2 concentrations in the respiratory tract, and thereby may be used as a surrogate marker of enhanced viral transmission. Several population studies have found an association between the oscillations in the mean CT over time and the evolution of the pandemic. For the first time, we applied temporal series analysis (Granger-type causality) to validate the CT counts as an epidemiological marker of forthcoming pandemic waves using samples and analyzing cases and hospital admissions during the third pandemic wave (October 2020 to May 2021) in Madrid. A total of 22,906 SARS-CoV-2 RT-PCR-positive nasopharyngeal swabs were evaluated; the mean CT value was 27.4 (SD: 2.1) (22.2% below 20 cycles). During this period, 422,110 cases and 36,727 hospital admissions were also recorded. A temporal association was found between the CT counts and the cases of COVID-19 with a lag of 9-10 days (p ≤ 0.01) and hospital admissions by COVID-19 (p < 0.04) with a lag of 2-6 days. According to a validated method to prove associations between variables that change over time, the short-term evolution of average CT counts in the population may forecast the evolution of the COVID-19 pandemic.

Rare Recombinant GI.5[P4] Norovirus That Caused a Large Foodborne Outbreak of Gastroenteritis in a Hotel in Spain in 2021.

Noroviruses are among the most important causes of acute gastroenteritis (AGE). In summer 2021, a large outbreak of norovirus infections affecting 163 patients, including 15 norovirus-confirmed food handlers, occurred in a hotel in Murcia in southeast Spain. A rare GI.5[P4] norovirus strain was identified as the cause of the outbreak. The epidemiological investigation determined that norovirus transmission might have been initiated through an infected food handler. The food safety inspection found that some symptomatic food handlers continued working during illness. Molecular investigation with whole-genome and ORF1 sequencing provided enhanced genetic discrimination over ORF2 sequencing alone and enabled differentiation of the GI.5[P4] strains into separate subclusters, suggesting different chains of transmission. These recombinant viruses have been identified circulating globally over the last 5 years, warranting further global surveillance. IMPORTANCE Due to the large genetic diversity of noroviruses, it is important to enhance the discriminatory power of typing techniques to differentiate strains when investigating outbreaks and elucidating transmission chains. This study highlights the importance of (i) using whole-genome sequencing to ensure genetic differentiation of GI noroviruses to track chains of transmission during outbreak investigations and (ii) the adherence of symptomatic food handlers to work exclusion rules and strict hand hygiene practices. To our knowledge, this study provides the first full-length genome sequences of GI.5[P4] strains apart from the prototype strain.

Antibiotic discontinuation through an antibiotic treatment optimization program in emergency department patients with low suspicion of infection.

INTRODUCTION: One of the most important strategies of PROA in the Emergency Department (ED) is the accurate diagnosis of infection to avoid inappropriate prescription. Our objective is to evaluate patients who receive antibiotics despite not having objective data of infection. METHODS: We carried out a cross-sectional study of patients admitted to the ED of the Hospital Universitario Fundación Alcorcón in which it was recommended to suspend the antibiotic through the PROA. Clinical and epidemiological characteristics and 30-day follow-up were analyzed to assess readmissions and mortality. RESULTS: 145 patients were analyzed. It was recommended to suspend the antibiotic in 25. 44% of them had a diagnosis of urinary infection. The suspension recommendation was accepted by 88%. No patient died and one was readmitted. CONCLUSIONS: An important percentage of patients are prescribed antibiotics despite not having infection criteria, the clinical evolution after suspension of antibiotics was favorable.

ROD2 domain filamin C missense mutations exhibit a distinctive cardiac phenotype with restrictive/hypertrophic cardiomyopathy and saw-tooth myocardium.

INTRODUCTION AND OBJECTIVES: Missense mutations in the filamin C (FLNC) gene have been reported as cause of inherited cardiomyopathy. Knowledge of the pathogenicity and genotype-phenotype correlation remains scarce. Our aim was to describe a distinctive cardiac phenotype related to rare missense FLNC variants in the ROD2 domain. METHODS: We recruited 21 unrelated families genetically evaluated because of hypertrophic cardiomyopathy (HCM)/restrictive cardiomyopathy (RCM) phenotype carrying rare missense variants in the ROD2 domain of FLNC (FLNC-mRod2). Carriers underwent advanced cardiac imaging and genetic cascade screening. Myocardial tissue from 3 explanted hearts of a missense FLNC carrier was histologically analyzed and compared with an FLNC-truncating variant heart sample and a healthy control. Plasmids independently containing 3 FLNC missense variants were transfected and analyzed using confocal microscopy. RESULTS: Eleven families (52%) with 20 assessed individuals (37 [23.7-52.7]) years showed 15 cases with a cardiac phenotype consisting of an overlap of HCM-RCM and left ventricular hypertrabeculation (saw-tooth appearance). During a median follow-up of 6.49 years, they presented with advanced heart failure: 16 (80%) diastolic dysfunction, 3 heart transplants, 3 heart failure deaths) and absence of cardiac conduction disturbances or skeletal myopathy. A total of 6 families had moderate genotype-phenotype segregation, and the remaining were de novo variants. Differential extracellular matrix remodeling and FLNC distribution among cardiomyocytes were confirmed on histology. HT1080 and H9c2 cells did not reveal cytoplasmic aggregation of mutant FLNC. CONCLUSIONS: FLNC-mRod2 variants show a high prevalence of an overlapped phenotype comprising RCM, HCM and deep hypertrabeculation with saw-tooth appearance and distinctive cardiac histopathological remodeling.