Detection of reassortant influenza B strains from 2004 to 2015 seasons in Barcelona (Catalonia, Spain) by whole genome sequencing.

BACKGROUND: Influenza B viruses (FLUBV) have segmented genomes which enables the virus to evolve by segment reassortment. Since the divergence of both FLUBV lineages, B/Victoria/2/87 (FLUBV/VIC) and B/Yamagata/16/88 (FLUBV/YAM), PB2, PB1 and HA have kept the same ancestor, while some reassortment events in the other segments have been reported worldwide. The aim of the present study was to find out reassortment episodes in FLUBV strains detected in cases attended at Hospital Universitari Vall d'Hebron and Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) from 2004 to 2015 seasons. METHODS: From October 2004 to May 2015, respiratory specimens were received from patients with respiratory tract infection suspicion. Influenza detection was carried out by either cell culture isolation, immunofluorescence or PCR-based assays. A RT-PCR was performed to distinguish both lineages by agarose gel electrophoresis. Whole genome amplification was performed using the universal primer set by Zhou et al. in 2012, and subsequently sequenced using Roche 454 GS Junior platform. Bioinformatic analysis was performed to characterise the sequences with B/Malaysia/2506/2007 and B/Florida/4/2006 corresponding sequences as reference of (B/VIC) and (B/YAM), respectively. RESULTS: A total of 118 FLUBV (75 FLUBV/VIC and 43 FLUBV/YAM), from 2004 to 2006, 2008-2011 and 2012-2015 seasons, were studied. The whole genome of 58 FLUBV/VIC and 42 FLUBV/YAM viruses was successfully amplified. Based on HA sequences, most FLUBV/VIC viruses (37; 64%) belonged to clade 1A (B/Brisbane/60/2008) except to 11 (19%), which fell within clade 1B (B/HongKong/514/2009) and 10 (17%) to B/Malaysia/2506/2004. Nine (20%) FLUBV/YAM viruses belonged to clade 2 (B/Massachusetts/02/2012), 18 (42%) to clade 3 (B/Phuket/3073/2013) and 15 (38%) fell within Florida/4/2006. Numerous intra-lineage reassortments in PB2, PB1, NA and NS were found in 2 2010-2011 viruses. An important inter-lineage reassortment event from 2008 to 2009 (11), 2010-2011 (26) and 2012-2013 (3) FLUBV/VIC (clade 1) strains to FLUBV/YAM (clade 3) was found, in addition to 1 reassortant NS in 2010-2011 B/VIC virus. CONCLUSIONS: Intra- and inter-lineage reassortment episodes were revealed by WGS. While PB2-PB1-HA remained in complex, NP and NS reassortant viruses were found in both lineages. Despite reassorment events are not often, the characterisation only by HA and NA sequences might be underestimating their detection.

Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma.

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

[Epidemiology of leptospirosis in the wetlands of Southern Spain]. / Epidemiología de la leptospirosis en los humedales del sur de España.

OBJECTIVE: To find out whether the leptospirosis incidence rate among red swamp crayfish collectors in the harvesting season is higher than in the general population, and to identify risk factors and assess the direct and indirect health costs associated with leptospirosis seroconversion. METHOD: This study was carried out between 1 July 2017 and 31 March 2018 in the municipality of Isla Mayor (Seville, Spain). It took the form of a prospective cohort study (exposed population: swamp crayfish collectors; non-exposed population: general population). The population was invited to take part in a prevalence study to be conducted using the ELISA qualitative technique, and informed consent was obtained from those who agreed. Negative serology cases were then included in the cohort study. Both cohorts were monitored clinically and symptomatic cases were serology tested. A second serum sample was taken from the swamp crayfish collectors at the end of the monitoring period to detect asymptomatic cases. Serovars were confirmed by microscopic agglutination testing. A bivariate descriptive analysis was carried out and cumulative incidence and relative risk were calculated, with positive serology being taken as the dependent variable. RESULTS: A total of 278 people were included in the study, of whom 92 made up the swamp crayfish collectors cohort and 186 the general population cohort. Women made up 46.8% of the sample, but only 29.3% of the collectors cohort. The mean age was 45.1 (±16.4) years. Nine cases of seroconversion were detected: eight among swamp crayfish collectors and one in the general population. Overall cumulative incidence was therefore 3.2%: 8.7% in the exposed group and 0.5% in the non-exposed group. Relative risk was 16.2% (95% confidence interval: 2.1-127.4). The total cost of medical assistance and illness-related losses associated with leptospirosis was 1568€/case. CONCLUSIONS: Leptospirosis in Isla Mayor is strongly associated with red swamp crayfish collecting. It's incidence here is much higher than that reported in studies published in other countries.

Epidemiología de la leptospirosis en los humedales del sur de España / Epidemiology of leptospirosis in the wetlands of Southern Spain

Objetivo: Conocer si la incidencia de leptospirosis en los capturadores de cangrejo rojo durante el periodo de captura es superior a la de la población general, así como identificar factores de riesgo y estimar los costes sanitarios directos e indirectos asociados a los casos de seroconversión de dicha enfermedad.Método: Este estudio se realizó desde el 1 de julio de 2017 hasta el 31 de marzo de 2018. Se llevó a cabo un estudio de cohortes prospectivo (población expuesta: capturadores de cangrejo rojo; población no expuesta: población general) en el municipio de Isla Mayor (Sevilla, España). Previo consentimiento informado, se invitó a la población al estudio de prevalencia mediante la técnica cualitativa ELISA. Los que tuvieron serología negativa fueron incluidos en el estudio de cohortes. Ambas cohortes se siguieron clínicamente y a los casos sintomáticos se les realizó serología. A los capturadores de cangrejo rojo se les tomó una segunda muestra de suero al final del seguimiento para detectar asintomáticos. La serovariedad se confirmó mediante aglutinación microscópica. Se realizó un análisis descriptivo bivariado y se calcularon la incidencia acumulada y el riesgo relativo. La serología positiva se tomó como variable dependiente.Resultados:Se incluyeron en el estudio 278 personas, de las que 92 constituían la cohorte de capturadores y 186 la de población general. El 46,8% de la muestra eran mujeres, aunque entre los capturadores de cangrejo rojo estas solo representaban el 29,3%. La edad media de la muestra fue de 45,1 (± 16,4) años. Se detectaron nueve seroconversiones: ocho en capturadores de cangrejo rojo y una en población general. Por lo tanto, la incidencia acumulada fue de 8,7% en capturadores de cangrejo rojo y de 0,5% en población general, siendo el riesgo relativo de 16,2 (intervalo de confianza del 95%: 2,1-127,4). El coste total de la asistencia sanitaria y de las pérdidas por enfermedad asociadas a la leptospirosis fue de 1568 € por caso(AU)

Objective: To find out whether the leptospirosis incidence rate among red swamp crayfish collectors in the harvesting season is higher than in the general population, and to identify risk factors and assess the direct and indirect health costs associated with leptospirosis seroconversion. Method: This study was carried out between 1 July 2017 and 31 March 2018 in the municipality of Isla Mayor (Seville, Spain). It took the form of a prospective cohort study (exposed population: swamp crayfish collectors; non-exposed population: general population). The population was invited to take part in a prevalence study to be conducted using the ELISA qualitative technique, and informed consent was obtained from those who agreed. Negative serology cases were then included in the cohort study. Both cohorts were monitored clinically and symptomatic cases were serology tested. A second serum sample was taken from the swamp crayfish collectors at the end of the monitoring period to detect asymptomatic cases. Serovars were confirmed by microscopic agglutination testing. A bivariate descriptive analysis was carried out and cumulative incidence and relative risk were calculated, with positive serology being taken as the dependent variable. Results: A total of 278 people were included in the study, of whom 92 made up the swamp crayfish collectors cohort and 186 the general population cohort. Women made up 46.8% of the sample, but only 29.3% of the collectors cohort. The mean age was 45.1 (±16.4) years. Nine cases of seroconversion were detected: eight among swamp crayfish collectors and one in the general population. Overall cumulative incidence was therefore 3.2%: 8.7% in the exposed group and 0.5% in the non-exposed group. Relative risk was 16.2% (95% confidence interval: 2.1-127.4). The total cost of medical assistance and illness-related losses associated with leptospirosis was 1568€/case. (AU)

Characteristics of 24 SARS-CoV-2-Sequenced Reinfection Cases in a Tertiary Hospital in Spain.

Background: Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the main concern is whether reinfections are possible, and which are the associated risk factors. This study aims to describe the clinical and molecular characteristics of 24 sequence-confirmed reinfection SARS-CoV-2 cases over 1 year in Barcelona (Catalonia, Spain). Methods: Patients with > 45 days between two positive PCR tests regardless of symptoms and negative tests between episodes were initially considered as suspected reinfection cases from November 2020 to May 2021. Whole-genome sequencing (WGS) was performed to confirm genetic differences between consensus sequences and for phylogenetic studies based on PANGOLIN nomenclature. Reinfections were confirmed by the number of mutations, change in lineage, or epidemiological criteria. Results: From 39 reported suspected reinfection cases, complete viral genomes could be sequenced from both episodes of 24 patients, all were confirmed as true reinfections. With a median age of 44 years (interquartile range [IQR] 32-65), 66% were women and 58% were healthcare workers (HCWs). The median days between episodes were 122 (IQR 72-199), occurring one-third within 3 months. Reinfection episodes were frequently asymptomatic and less severe than primary infections. The absence of seroconversion was associated with symptomatic reinfections. Only one case was reinfected with a variant of concern (VOC). Conclusion: Severe acute respiratory syndrome coronavirus 2 reinfections can occur in a shorter time than previously reported and are mainly found in immunocompetent patients. Surveillance through WGS is useful to identify viral mutations associated with immune evasion.

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.

A year living with SARS-CoV-2: an epidemiological overview of viral lineage circulation by whole-genome sequencing in Barcelona city (Catalonia, Spain).

Herein, we describe the genetic diversity of circulating SARS-CoV-2 viruses by whole-genome sequencing (WGS) in Barcelona city (Catalonia, Spain) throughout the first four pandemic waves. From weeks 11/2020-24/2021, SARS-CoV-2-positive respiratory samples were randomly selected per clinical setting (80% from primary care or 20% from the hospital), age group, and week. WGS was performed following the ARTICv3 protocol on MiSeq or NextSeq2000 Illumina platforms. Nearly complete consensus sequences were used for genetic characterization based on GISAID and PANGOLIN nomenclatures. From 2475 samples, 2166 (87%) were fully sequenced (78% from primary care and 22% from hospital settings). Multiple genetic lineages were co-circulating, but four were predominant at different periods. While B.1.5 (50.68%) and B.1.1 (32.88%) were the major lineages during the first pandemic wave, B.1.177 (66.85%) and B.1.1.7 (83.80%) were predominant during the second, third, and fourth waves, respectively. Almost all (96.4%) were carrying D614G mutation in the S protein, with additional mutations that define lineages or variants. But some mutations of concern, such as E484K from B.1.351 and P.1 lineages are currently under monitoring, together with those observed in the receptor-binding domain or N-terminal domain, such as L452R and T478K from B.1.617.2 lineage. The fact that a predominant lineage was observed in each pandemic wave suggests advantageous properties over other contemporary co-circulating variants. This genetic variability should be monitored, especially when a massive vaccination campaign is ongoing because the potential selection and emergence of novel antigenic SARS-CoV-2 strains related to immunological escapement events.

Functional gastrointestinal disorders: real-life results of a multidisciplinary non-pharmacological approach based on group-consultations.

INTRODUCTION: functional gastrointestinal disorders are prevalent and resource consuming. The use of group-consultations in these diseases is limited and no specific multidisciplinary programs have been developed. METHODS: a multidisciplinary approach was used in patients with diverse functional gastrointestinal disorders attending group-consultations (group A). Five two-hour sessions were scheduled over a four-month period. Sessions consisted of a theoretical introduction (Pathophysiology, Low fodmap diet, Over the counter medications, Mediterranean diet, and Laughter therapy workshop) followed by relaxation techniques. This group was compared to a similar group of patients who received written information covering the topics discussed during the group-consultations (group B). Severity of digestive and psychological symptoms, use of drugs and adherence to the diet were the main outcomes measured. RESULTS: the mean age of participants was 43 (± 1.38) years, 78 % were female and 73 % had at least two functional gastrointestinal disorders. Sixty-two patients were included in group A and 17 in group B. The severity of gastrointestinal and psychological symptoms at baseline was similar in both groups. Globally, there was an improvement in all symptoms in both groups. The proportion of participants with severe baseline gastrointestinal symptoms or pathologic anxiety scores that showed improvement was significantly higher in group A (74 % vs 23 %, p = 0.005; 47 % vs 8 %, p = 0.02, respectively). Symptoms were reassessed at six and 12 months after the intervention in participants from group A who attended ≥ 80 % sessions and a sustained response was observed. CONCLUSIONS: group-consultations are useful and efficient to alleviate gastrointestinal and psychological symptoms in patients with functional gastrointestinal disorders.

Naturally occurring SARS-CoV-2 gene deletions close to the spike S1/S2 cleavage site in the viral quasispecies of COVID19 patients.

The SARS-CoV-2 spike (S) protein, the viral mediator for binding and entry into the host cell, has sparked great interest as a target for vaccine development and treatments with neutralizing antibodies. Initial data suggest that the virus has low mutation rates, but its large genome could facilitate recombination, insertions, and deletions, as has been described in other coronaviruses. Here, we deep-sequenced the complete SARS-CoV-2 S gene from 18 patients (10 with mild and 8 with severe COVID-19), and found that the virus accumulates deletions upstream and very close to the S1/S2 cleavage site (PRRAR/S), generating a frameshift with appearance of a stop codon. These deletions were found in a small percentage of the viral quasispecies (2.2%) in samples from all the mild and only half the severe COVID-19 patients. Our results suggest that the virus may generate free S1 protein released to the circulation. We suggest that natural selection has favoured a "Don't burn down the house" strategy, in which free S1 protein may compete with viral particles for the ACE2 receptor, thus reducing the severity of the infection and tissue damage without losing transmission capability.

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.

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.

Fístula interesfinteriana sin orificio externo y con reentrada en el recto / Intersphincteric fistula without external opening and to re-enter the rectum

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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.

Variability in the expression of polycomb proteins in different normal and tumoral tissues. A pilot study using tissue microarrays.

In spite of the known function of polycomb group (PcG) genes in stem cell self-renewal, control of cellular proliferation and differentiation, its role in cancer pathogenesis is still poorly understood. We studied the expression by immunohistochemistry of several PcG-maintenance complex proteins (RING1, RNF2, BMI1, MEL18, HPH1 and RYBP) in nontumoral (154 samples) and tumoral (550 samples) human tissues using Tissue Microarrays. For selected genes (BMI1 and RING1) FISH analysis has been also carried out. PcG proteins had a tissue- and cell-type-specific expression pattern. Some of them were highly selectively expressed, such as HPH1, which was detected in germ cells in testis, pituitary and parathyroid glands and Langerhans islets, and RYBP, which was found in placenta, umbilical cord and thyroid gland. By contrast, RING1 was ubiquitously expressed in every normal tissue analyzed. Changes in expression associated with tumoral transformation have been found for BMI1 and RNF2, which exhibited increased expression in a large series of tumors, including gastrointestinal tumors, pituitary and parathyroid adenomas, and lymphomas, compared with their expression in normal-cell counterparts. The high level of expression of BMI1 protein observed in mantle-cell lymphomas and pituitary adenomas is associated in some cases with amplification of BMI1 locus. These findings imply that upregulation of BMI1 may constitute a malignancy marker in different types of cancer, mainly in lymphoid and endocrine tumors. RING1 was lost in a group of renal-cell carcinomas and testicular germ-cell tumors. Lastly, RYBP is anomalously expressed in Hodgkin's lymphomas and oligodendrogliomas, among others tumors. A significant finding of the study is the identification of unique PcG profiles for some tumors, such as testicular germ-cell tumors, which have high levels of HPH1 expression and loss of RING1 and/or BMI1; pituitary adenomas, which expressed every PcG protein analyzed; and clear-cell renal-cell carcinoma, which was the only tumor other than testicular germ-cell tumors that did not express RING1.