Performance of the cobas EBV and cobas BKV assays: multi-site comparison of standardized quantitation.

Guidelines recommend monitoring of Epstein-Barr virus (EBV) and BK virus (BKV) in solid organ and hematopoietic stem cell transplant patients. The majority of quantitative DNA testing for EBV and BKV employs unstandardized individual laboratory-developed testing solutions (LDTs), with implications for accuracy, reproducibility, and comparability between laboratories. The performance of the cobas EBV and cobas BKV assays was assessed across five laboratories, using the World Health Organization International Standards (WHO IS) for EBV and BKV, and the National Institute of Standards and Technology Quantitative Standard for BKV, and results were compared with the LDTs in use at the time. Methods were also compared using locally sourced clinical specimens. Variation was high when laboratories reported EBV or BKV DNA values using LDTs, where quantitative values were observed to differ by up to 1.5 log10 unit/mL between sites. Conversely, results from the cobas EBV and cobas BKV assays were accurate and reproducible across sites and on different testing days. Adjustment of LDTs using the international standards led to closer alignment between the assays; however, day-to-day reproducibility of LDTs remained high. In addition, BKV continued to show bias, indicating challenges with the commutability of the BKV International Standard. The cobas EBV and cobas BKV assays are automated, aligned to the WHO IS, and have the potential to reduce the variability in viral load testing introduced by differences in LDTs. Standardization of reporting values may eventually allow different centers to compare data to allow clinical decision thresholds to be established supporting improvements in patient management.IMPORTANCEThe application of center-specific cut-offs for clinical decisions and the variability of LDTs often hinder interpretation; thus, the findings reported here support the need for standardization in the field of post-transplant monitoring of EBV and BKV to improve patient management. Alongside the choice of assay, it is also important to consider which standard to use when deciding upon a testing methodology. This is a call to action for standardization, as treatment for EBV and BKV is driven by viral load test results, and the more accurate and comparable the test results are across institutions, the more informed and better the treatment decisions can be.

Adenovirus-Associated Urethritis, a Not So Uncommon Entity. A Review of 91 Cases.

BACKGROUND: Human adenovirus (HAdV) is likely an underdiagnosed cause of urethritis, although it was already associated with urethritis in descriptions published more than 40 years ago. Differential clinical features of this entity, such as meatitis, conjunctivitis, and a predominance of mononuclear white blood cells in first-void urine and/or urethral smear, can be useful to increase diagnostic suspicion. METHODS: We retrospectively studied 91 episodes of HAdV-associated urethritis diagnosed for 9 years and 6 months after optimizing efforts to detect the pathogen mainly in patients with features suggestive of this condition. RESULTS: Dysuria was the main symptom (84%), whereas meatitis was observed in 34% of cases. Furthermore, 40% of patients had conjunctivitis. Human adenovirus type D was the most prevalent HAdV (56%), although HAdV-C6, a type not previously associated with urethritis, was observed in 12 patients (13%). CONCLUSIONS: Urethritis due to HAdV is not uncommon, and it is important to screen for it to avoid unnecessary treatments, contact tracing studies, and checkups. The use of multiplex polymerase chain reaction assays that include HAdV, for the diagnosis of urethritis, would raise awareness of its role in this entity.

Genetic Characterization of Non-Lymphogranuloma venereum Chlamydia trachomatis Indicates Distinct Infection Transmission Networks in Spain.

Chlamydia trachomatis infection is an important public health problem. Our objective was to assess the dynamics of the transmission of this infection, analysing the distribution of circulating ompA genotypes and multilocus sequence types of C. trachomatis in Spain as a function of clinical and epidemiological variables. During 2018 and 2019, we genetically characterized C. trachomatis in tertiary hospitals in six areas in Spain (Asturias, Barcelona, Gipuzkoa, Mallorca, Seville and Zaragoza), with a catchment population of 3.050 million people. Genotypes and sequence types were obtained using polymerase chain reaction techniques that amplify a fragment of the ompA gene, and five highly variable genes (hctB, CT058, CT144, CT172 and pbpB), respectively. Amplicons were sequenced and phylogenetic analysis was conducted. We obtained genotypes in 636/698 cases (91.1%). Overall and by area, genotype E was the most common (35%). Stratifying by sex, genotypes D and G were more common among men, and genotypes F and I among women (p < 0.05). Genotypes D, G and J were more common in men who have sex with men (MSM) than in men who have sex with women (MSW), in whom the most common genotypes were E and F. The diversity index was higher in sequence typing (0.981) than in genotyping (0.791), and the most common sequence types were ST52 and ST108 in MSM, and ST30, ST148, ST276 and ST327 in MSW. Differences in genotype distribution between geographical areas were attributable to differences in population characteristics. The transmission dynamics varied with sexual behaviour: the predominant genotypes and most frequent sequence types found in MSM were different to those detected in MSW and women.

Haemophilus influenzae Type a Sequence Type 23, Northern Spain.

Two consecutive cases of Haemophilus influenzae type a sequence type 23 invasive infection in 2 children attending the same daycare in 2019 triggered epidemiologic surveillance of H. influenzae infections in northern Spain. Despite the invasiveness potential of this virus strain, we detected no additional cases for 2013-2020.

Comparison of Four Methods for Streptococcus pneumoniae Capsular Typing.

BACKGROUND: Pneumococcal capsular-type identification is essential for monitoring the epidemiology of pneumococcal infections and for establishing the effectiveness of pneumococcal vaccines. The objective of this work was to compare the accuracy of four methods of Streptococcus pneumoniae capsular typing. METHODS: A prospective blind study was carried out at Donostia University Hospital (northern Spain) to determine the capsular types of 50 pneumococcal clinical isolates using four techniques: a) S. PneumoStripTM: a reverse-hybridization strip-based commercial assay that detects 76 pneumococcal serotypes: 42 individually and 34 in pairs. b) FAF-mPCR: a single-step multiplex-PCR (polymerase chain reaction) assay combined with fragment analysis using automated fluorescent capillary electrophoresis, which can differentiate 92 serotypes in a single tube: 31 individually, 28 in pairs, and 33 in groups of 3 to 5 serotypes. c) PCRSeqTyping: which enables the detection of 91 serotypes after sequencing the regions of the cpsB gene in two steps: 59 directly and the remaining 32 serotypes in a second step. d) The Quellung reaction. RESULTS: The S. PneumoStripTM, FAF-mPCR and PCRSeqTyping identified the serotypes of all the 50 clinical isolates. With the Quellung reaction 46/50 (92%) isolates were correctly serotyped. The quickest technique was the S. PneumoStripTM, followed by the single-step multiplex PCR assay and PCRSeqTyping. The Quellung reaction was the slowest technique. CONCLUSIONS: The S. PneumoStripTM, PCRSeqTyping, and FAF-mPCR were very accurate techniques for pneumococcal serotyping, with S. PneumoStripTM obtaining results more rapidly. The combination of any of these S. pneumoniae molecular typing techniques and the Quellung reaction as confirmation reference method is a highly precise and fast strategy for the serotyping of high number of pneumococcal clinical isolates.

Fatal Case of Nosocomial Legionella pneumophila Pneumonia, Spain, 2018.

A nosocomial case of Legionella pneumophila pneumonia likely caused by a serogroup 3 strain was detected by a urinary antigen test in Spain in 2018. Although Legionella bacteria could not be isolated from respiratory samples, molecular methods implicated the sink faucet of the patient's room as the probable infection source.

Immunosuppression for inflammatory bowel disease does not influence Epstein-Barr viral load in the short-term.

INTRODUCTION: Immunomodulators and biologics are two of the main drugs used for the treatment of inflammatory bowel disease (IBD). Some of these agents have been associated with certain infections and lymphoproliferative disorders, including Epstein-Barr virus (EBV) infection. Our aim was to determine the influence of immunosuppression in the EBV viral load in patients with IBD. MATERIALS AND METHODS: We prospectively included naïve patients with IBD who were starting immunosuppressive therapy in four IBD Units. All patients were assessed at baseline and four months after starting immunosuppression for clinical disease activity, biomarkers, EBV serology (IgM VCA, IgG VCA and IgG EBNA) and viral load. RESULTS: Thirty-two patients were included. At baseline, all patients showed positive results for IgG VCA or IgG EBNA with undetectable EBV viral load. No patient showed detectable EBV viral load after starting the immunosuppressive therapy. CONCLUSION: Immunosuppression did not influence on EBV viral load in the short-term in naïve IBD patients.

Surveillance of Helicobacter pylori resistance over 22 Years (2000-2021) in Northern Spain.

OBJECTIVES: Helicobacter pylori gastritis is considered an infectious disease, regardless of symptoms and stage of disease. Most consensus documents recommend empirical therapy based on local antimicrobial susceptibility patterns. We aimed to provide clinically useful information about primary and secondary antimicrobial resistance to antimicrobials commonly prescribed for H. pylori. METHODS: Overall, 31,406 gastroduodenal biopsies and 2,641 string tests from patients over 15 years of age were plated on selective media, isolating H. pylori in 36.7% of biopsies and 50.7% of string tests. Susceptibility testing could be performed in 96.6% (12,399/12,835) of H. pylori isolates. Polymerase chain reaction (PCR) was also used to detect H. pylori and its resistance to clarithromycin, providing susceptibility data for 112 patients with negative culture results. RESULTS: Resistance to amoxicillin and tetracycline was unusual (0.6% and 0.2%, respectively). Rates of primary resistance to clarithromycin and metronidazole remained steady over the 22-year study period, at around 14% for clarithromycin and 30% for metronidazole, while primary resistance to levofloxacin tripled (from 7.6% in 2000 to 21.7% in 2021, P < 0.001) and increased with patient age. Notably, 1.8% of isolates were multiresistant to clarithromycin, metronidazole, and levofloxacin. Overall, secondary resistance rates were higher (P < 0.0001) than primary resistance rates for clarithromycin (42.5% vs 14.1%), metronidazole (40.9% vs 32%), and levofloxacin (21.5% vs 17.1%). CONCLUSION: Determination of susceptibility for H. pylori by culture and/or PCR in patients undergoing endoscopy could facilitate the implementation of tailored therapy and guide the choice of empirical therapy when susceptibility testing cannot be performed, potentially helping limit the emergence of antimicrobial resistance.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain.

BACKGROUND: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. RESULTS: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. CONCLUSIONS: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Increases in the Macrolide Resistance of Mycoplasma genitalium and the Emergence of the A2058T Mutation in the 23S rRNA Gene: Clonal Spread?

The management of Mycoplasma genitalium sexually transmitted infection (STI) is hindered by increasing resistance to the recommended antibiotics, macrolides and quinolones, worldwide. In Gipuzkoa (Basque Country, Spain), macrolide and quinolone resistance rates in 2014−2018 were reported as <20% and <10%, respectively. The aims of this study were to compare these rates with those in 2019−2021 and analyse the genetic and epidemiological features of the strains and cases associated with striking changes in the resistance trends. Resistance to macrolides (n = 1019) and quinolones (n = 958) was studied, analysing mutations in 23S rRNA and parC/gyrA genes, respectively. The rate of macrolide resistance increased from 17.3% in 2014−2018 to 32.1% in 2019−2021, as much in the more prevalent A2058/2059G mutations (16.6−27.8%) as in the emergent A2058T mutations (0.5−4.1%) but with differences in the odds ratios and the relative risk increase between A2058T and A2058/2059G mutations. MG191 adhesin and MG309 lipoprotein of the 27 emergent strains detected with A2058T mutations were amplified, sequenced, and typed using phylogenetic and variable number tandem repeat analysis, respectively. Genetic clonal spread was ruled out, but most of the A2058T cases were men who had sex with men (24/27) with a history of STI and antibiotic treatments (19/27). No changes were observed in quinolone resistance trends, but the rate of resistance to both antibiotics rose from 2.9% to 8.3%, especially in cases with A2058T mutations. The genetic characterisation of strains and epidemiological surveillance of cases are needed to detect populations at increased risk of treatment failure in this infection.

Epidemiology and Clinical Features of Listeriosis in Gipuzkoa, Spain, 2010-2020.

Background: Listeriosis continues to be one of the most important notifiable foodborne diseases. Nonetheless, in Spain, there are few data on the molecular epidemiology of Listeria monocytogenes infections in recent years. Aim: To describe clinical features and the molecular epidemiology of human listeriosis over an 11-year period (2010-2020) in Gipuzkoa, Northern Spain. Methods: A total of 111 isolates, all but one from invasive disease, were studied. Serotyping (agglutination and multiplex polymerase chain reaction [PCR]) and multilocus sequence typing were performed for all isolates. Antibiotic susceptibility was assessed by the broth microdilution method. Results: The average annual incidence of listeriosis in non-pregnancy-associated cases was 1.55 per 100,000 population, with a 1-month mortality rate of 22.2%. In pregnant women, the average incidence was 0.45 cases per 1,000 pregnancies. Twenty-four sequence types were identified, serotype 4b ST1 (24.3%) being the most frequent followed by 1/2b ST87 (18.9%), which caused two long outbreaks in 2013-2014. A significant association was observed between ST219 and meningitis (p < 0.001). All isolates were susceptible to ampicillin as well as other antibiotics used in listeriosis treatment. Conclusion: Despite current control measures, listeriosis continues to be an important cause of mortality in the elderly, preterm birth, and miscarriages in pregnant women. Improvements in the control and diagnosis of listeriosis are needed to reduce the impact of this infection on vulnerable populations.

Identification of a New HIV-1 BC Intersubtype Circulating Recombinant Form (CRF108_BC) in Spain.

The extraordinary genetic variability of human immunodeficiency virus type 1 (HIV-1) group M has led to the identification of 10 subtypes, 102 circulating recombinant forms (CRFs) and numerous unique recombinant forms. Among CRFs, 11 derived from subtypes B and C have been identified in China, Brazil, and Italy. Here we identify a new HIV-1 CRF_BC in Northern Spain. Originally, a phylogenetic cluster of 15 viruses of subtype C in protease-reverse transcriptase was identified in an HIV-1 molecular surveillance study in Spain, most of them from individuals from the Basque Country and heterosexually transmitted. Analyses of near full-length genome sequences from six viruses from three cities revealed that they were BC recombinant with coincident mosaic structures different from known CRFs. This allowed the definition of a new HIV-1 CRF designated CRF108_BC, whose genome is predominantly of subtype C, with four short subtype B fragments. Phylogenetic analyses with database sequences supported a Brazilian ancestry of the parental subtype C strain. Coalescent Bayesian analyses estimated the most recent common ancestor of CRF108_BC in the city of Vitoria, Basque Country, around 2000. CRF108_BC is the first CRF_BC identified in Spain and the second in Europe, after CRF60_BC, both phylogenetically related to Brazilian subtype C strains.

Multicenter evaluation of the Panbio™ COVID-19 rapid antigen-detection test for the diagnosis of SARS-CoV-2 infection.

OBJECTIVES: The standard RT-PCR assay for coronavirus disease 2019 (COVID-19) is laborious and time-consuming, limiting testing availability. Rapid antigen-detection tests are faster and less expensive; however, the reliability of these tests must be validated before they can be used widely. The objective of this study was to determine the performance of the Panbio™ COVID-19 Ag Rapid Test Device (PanbioRT) (Abbott) in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swab specimens. METHODS: This prospective multicentre study was carried out in ten Spanish university hospitals and included individuals with clinical symptoms or epidemiological criteria of COVID-19. Only individuals with ≤7 days from the onset of symptoms or from exposure to a confirmed case of COVID-19 were included. Two nasopharyngeal samples were taken to perform the PanbioRT as a point-of-care test and a diagnostic RT-PCR test. RESULTS: Among the 958 patients studied, 325 (90.5%) had true-positive results. The overall sensitivity and specificity for the PanbioRT were 90.5% (95%CI 87.5-93.6) and 98.8% (95%CI 98-99.7), respectively. Sensitivity in participants who had a threshold cycle (CT) < 25 for the RT-PCR test was 99.5% (95%CI 98.4-100), and in participants with ≤5 days of the clinical course it was 91.8% (95%CI 88.8-94.8). Agreement between techniques was 95.7% (κ score 0.90; 95%CI 0.88-0.93). CONCLUSIONS: The PanbioRT performs well clinically, with even more reliable results for patients with a shorter clinical course of the disease or a higher viral load. The results must be interpreted based on the local epidemiological context.

Genomic Virulence Features of Two Novel Species Nocardia barduliensis sp. nov. and Nocardia gipuzkoensis sp. nov., Isolated from Patients with Chronic Pulmonary Diseases.

Strains 335427T and 234509T, isolated from two 76-year-old patients with chronic pulmonary diseases, were the subject of polyphasic taxonomic studies and comparative genomic analyses for virulence factors. The 16 rRNA gene sequence similarity between strains 335427T and 234509T and their closest phylogenetic neighbors Nocardia asiatica NBRC 100129T and Nocardia abscessus NBRC 100374T were 99.5% and 100%, respectively. Digital DNA-DNA hybridization values between the aforementioned studied strains were well below the 70% threshold for assigning prokaryotic strains to a novel species. Strains 335427T and 234509T have genome sizes of 8.49 Mpb and 8.07 Mpb, respectively, with G + C content of 68.5%. Isolate 335427T has C16:0, C18:1 ω9c, C18:0 and C18:0 10 methyl as major fatty acids (>15%) and mycolic acids formed of 52-54 carbon atoms. However, only C18:1 ω9c was detected for isolate 234509T, which had mycolic acids with 44-56 carbon. Based on phenotypic and genetic data, strains 335427T (DSM 109819T = CECT 9924T) and 234509T (DSM 111366T = CECT 30129T) merit recognition as novel species, which are named Nocardia barduliensis sp. nov. and Nocardia gipuzkoensis sp. nov., respectively. All the strains studied had homologous VF-associated genes to those described in M. tuberculosis, including experimentally verified virulence genes in humans related to tuberculosis. The narGHIJ (nitrate reduction pathway) and gvpAFGOJLMK (gas vesicles) genetic maps of strains 335427T, 234509T, NBRC 100129T and NBRC 100374T showed the same syntenic block and raise the question of whether their functions are interlinked during the infection of the human host. However, further research is required to decipher the role of the gas vesicle in the pathogenicity mechanism of Nocardia spp.

Molecular Typing of Mycoplasma genitalium-Positive Specimens Discriminates between Persistent and Recurrent Infections in Cases of Treatment Failure and Supports Contact Tracing.

Mycoplasma genitalium causes a sexually transmitted infection that sometimes persists or recurs despite adequate antibiotic treatment. Between 2014 and 2018, molecular typing was applied to 75 M. genitalium-positive samples from 48 patients with repeated infection and/or couples/groups of other infected sexual contacts. MG191 adhesin, MG309 lipoprotein, and the rRNA operon were amplified, sequenced, and typed using phylogenetic, variable number tandem repeat, and single-nucleotide polymorphism analysis, respectively. Amplicons were obtained in 74/75 samples, and the combination of locus patterns gave 44 different genetic profiles (discriminatory index of 0.987), with 43 considering only MG191 and MG309. Interestingly, 15/17 patients who presented a first sample sensitive and a second resistant to macrolides had the same genetic variant in the samples (persistence of the same strain). In 2/17 patients, discordant variants (one mixed infection and one recurrence due to incomplete contact tracing) were detected. In 31 additional not related and randomly distributed samples, MG191 typing obtained 23 different genotypes, with no appreciable clustering over time. The typing method allowed persistent and recurrent infections to be distinguished, indicating that macrolide resistance-associated mutations mostly developed during treatment. To detect these secondary resistant strains, prevent reinfections, and improve the control of M. genitalium infections, tests of cure and contact tracing of sexual partners should be mandatory.

Human metapneumovirus as cause of severe community-acquired pneumonia in adults: insights from a ten-year molecular and epidemiological analysis.

BACKGROUND: Information on the clinical, epidemiological and molecular characterization of human metapneumovirus in critically ill adult patients with severe community-acquired pneumonia (CAP) and the role of biomarkers identifying bacterial coinfection is scarce. METHODS: This is a retrospective epidemiological study of adult patients with hMPV severe CAP admitted to ICU during a ten-year period with admission PSI score ≥ 3. RESULTS: The 92.8% of the 28 patients with severe CAP due to human metapneumovirus were detected during the first half of the year. Median age was 62 years and 60.7% were male. The genotyping of isolated human metapneumovirus showed group B predominance (60.7%). All patients had acute respiratory failure. Median APACHE II and SOFA score were 13 and 6.55, respectively. The 25% were coinfected with Streptococcus pneumoniae. 60.7% of the patients had shock at admission and 50% underwent mechanical ventilation. Seven patients developed ARDS, three of them younger than 60 years and without comorbidities. Mortality in ICU was 14.3%. Among survivors, ICU and hospital stay were 6.5 and 14 days, respectively. Plasma levels of procalcitonin were higher in patients with bacterial coinfection (18.2 vs 0.54; p < 0.05). The levels of C-reactive protein, however, were similar. CONCLUSION: Human metapneumovirus was associated with severe CAP requiring ICU admission among elderly patients or patients with comorbidities, but also in healthy young subjects. These patients often underwent mechanical ventilation with elevated health resource consumption. While one out of four patients showed pneumococcal coinfection, plasma procalcitonin helped to implement antimicrobial stewardship.

Guided antibiotic therapy for Mycoplasma genitalium infections: Analysis of mutations associated with resistance to macrolides and fluoroquinolones. / Tratamiento antibiótico dirigido en infecciones por Mycoplasma genitalium: análisis de mutaciones asociadas con resistencia a macrólidos y fluoroquinolonas.

INTRODUCTION: The objective of this study was to analyse the susceptibility of Mycoplasma genitalium to macrolides and fluoroquinolones using molecular techniques. METHODS: Susceptibility to macrolides was tested (Gipuzkoa, 2014-2017) by a rapid probe-based real-time polymerase chain reaction assay (23S rRNA gene) and to fluoroquinolones by sequencing the parC and gyrA genes. RESULTS: Mutations associated with macrolide resistance were detected in 43/263 (16.3%) cases and potential fluoroquinolone resistance in 21/267 (7.9%). Macrolide resistance was more frequent in patients previously treated with azithromycin (76.5% vs 7.4%, P<.001) as well as in those treated with a single 1g dose (31.3%) vs the extended regimen (7%, P<.001). There were 5/245 (2%) cases with mutations probably associated with resistance to both antibiotics. CONCLUSIONS: The technique used for testing Mycoplasma genitalium susceptibility to azithromycin allowed the rapid implementation of resistance-guided antibiotic therapy. Moxifloxacin could be a good option in cases of macrolide resistance.

Enterovirus D68 Causing Acute Respiratory Infection: Clinical Characteristics and Differences With Acute Respiratory Infections Associated With Enterovirus Non-D68.

BACKGROUND: Enterovirus (EV) D68 is mainly associated with acute respiratory infection (ARI). Since 2014, when outbreaks in different countries were observed, this emerging virus was considered a potential threat to public health. METHODS: During 2015-2017, the presence of enterovirus RNA was investigated in all respiratory samples of children younger than 15 years of age with ARI, obtained for virologic studies in the Pediatric Emergency Care Units and wards of 2 hospitals in Gipuzkoa (Spain), using a commercial multiplex real-time polymerase chain reaction. When enterovirus was detected, a polymerase chain reaction to amplify a specific viral polyprotein (VP1) gene region of EV-D68 was performed. RESULTS: In 2016, EV-D68 circulation was associated to ARI, with the highest incidence in the spring months. EV-D68 was detected in 44 children, mean age 30.1 ± 31.7 months old, 23 (52.3%) of them females and 17 (38.6%) with underlying respiratory medical conditions. Thirty-two patients (72%) required hospital admission, receiving the discharge diagnosis of recurrent wheezing (37.5%), asthmatic crisis (37.5%) or bronchiolitis (12.5%). Seven children (15.9%) needed the support of the pediatric intensive care unit. When coinfections were excluded, children with EV-D68 infection presented with increased work of breathing, recurrent wheezing or asthmatic crisis, more frequently than those with ARI associated with EV non-D68. Moreover, clinical outcomes (hospitalization, respiratory support) were more severe. All 44 EV-D68 strains detected belonged to lineage B3. CONCLUSIONS: EV-D68 circulated widely in Gipuzkoa during 2016 and was associated with severe ARI. In children with severe ARI of unknown etiology, the presence of EV-D68 should be considered.

Molecular epidemiology of G12 rotavirus strains during eight consecutive epidemic seasons in the Basque Country (North of Spain), 2010-2018.

G12 rotaviruses were first detected in Spain (Gipuzkoa province) in December 2004. After four years with no detections, G12 strains re-emerged in the 2010-2011 epidemic season, when the first European epidemic circulation of this genotype was observed in Gipuzkoa. G12 rotaviruses were also the dominant strains in 2011-2012, 2014-2015 and 2015-2016 epidemic seasons and were sporadically detected in the remaining periods (2012-2014 and 2016-2018). The most frequently detected G-type between 2010 and 2018 was G12 (29.9%) rather than G1 rotavirus (17.8%), which historically had been the dominant genotype in our setting (1989-2009 period) and globally. Phylogenetic analysis of the VP4 and VP7 genome segments showed chronologically ordered clades, which spanned between two to four consecutive seasons. Overall, the circulating G12 rotavirus strains in Gipuzkoa between 2010 and 2018 belonged to four clades, which emerged in early 2009 potentially due to at least four importations from other regions followed by local evolution. Whole genome analysis of 16 G12 strains detected from 2010 to 2018 revealed a Wa-like genotype constellation, G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, and also showed that G12 strains from Gipuzkoa were similar to those identified in other countries. These findings suggest circulation of G12 rotavirus strains in different parts of the world leading to high genetic diversity.