Serotypes and Clonal Composition of Streptococcus pneumoniae Isolates Causing IPD in Children and Adults in Catalonia before 2013 to 2015 and after 2017 to 2019 Systematic Introduction of PCV13.

The goal of this study was to investigate the distribution of serotypes and clonal composition of Streptococcus pneumoniae isolates causing invasive pneumococcal disease (IPD) in Catalonia, before and after systematic introduction of PCV13. Pneumococcal strains isolated from normally sterile sites obtained from patients of all ages with IPD received between 2013 and 2019 from 25 health centers of Catalonia were included. Two study periods were defined: presystematic vaccination period (2013 and 2015) and systematic vaccination period (SVP) (2017 to 2019). A total of 2,303 isolates were analyzed. In the SVP, there was a significant decrease in the incidence of IPD cases in children 5 to 17 years old (relative risk [RR] 0.61; 95% confidence interval [CI] 0.38 to 0.99), while there was a significant increase in the incidence of IPD cases in 18- to 64-year-old adults (RR 1.33; 95% CI 1.16 to 1.52) and adults over 65 years old (RR 1.23; 95% CI 1.09 to 1.38). Serotype 8 was the major emerging serotype in all age groups except in 5- to 17-year-old children. In children younger than 5 years old, the main serotypes in SVP were 24F, 15A, and 3, while in adults older than 65 years they were serotypes 3, 8, and 12F. A significant decrease in the proportions of clonal complexes CC156, CC191, and ST306 and an increase in those of CC180, CC53, and CC404 were observed. A steady decrease in the incidence of IPD caused by PCV13 serotypes indicates the importance and impact of systematic vaccination. The increase of non-PCV13 serotypes highlights the need to expand serotype coverage in future vaccines and rethink vaccination programs for older adults. IMPORTANCE We found that with the incorporation of the PCV13 vaccine, the numbers of IPD cases caused by serotypes included in this vaccine decreased in all of the age groups. Still, there was an unforeseen increase of the serotypes not included in this vaccine causing IPD, especially in the >65-year-old group. Moreover, a significant increase of serotype 3 included in the vaccine has been observed; this event has been reported by other researchers. These facts call for the incorporation of more serotypes in future vaccines and a more thorough surveillance of the dynamics of this microorganism.

Pneumococcal serotypes in children, clinical presentation and antimicrobial susceptibility in the PCV13 era.

The aim was to analyse invasive pneumococcal disease (IPD) serotypes in children aged ⩽17 years according to clinical presentation and antimicrobial susceptibility. We conducted a prospective study (January 2012-June 2016). IPD cases were diagnosed by culture and/or real-time polymerase chain reaction (PCR). Demographic, microbiological and clinical data were analysed. Associations were assessed using the odds ratio (OR) and 95% confidence intervals (CI). Of the 253 cases, 34.4% were aged <2 years, 38.7% 2-4 years and 26.9% 5-17 years. Over 64% were 13-valent pneumococcal conjugate vaccine (PCV13) serotypes. 48% of the cases were diagnosed only by real-time PCR. Serotypes 3 and 1 were associated with complicated pneumonia (P < 0.05) and non-PCV13 serotypes with meningitis (OR 7.32, 95% CI 2.33-22.99) and occult bacteraemia (OR 3.6, 95% CI 1.56-8.76). Serotype 19A was more frequent in children aged <2 years and serotypes 3 and 1 in children aged 2-4 years and 5-17 years, respectively. 36.1% of cases were not susceptible to penicillin and 16.4% were also non-susceptible to cefotaxime. Serotypes 14, 24F and 23B were associated with non-susceptibility to penicillin (P < 0.05) and serotypes 11, 14 and 19A to cefotaxime (P < 0.05). Serotype 19A showed resistance to penicillin (P = 0.002). In conclusion, PCV13 serotypes were most frequent in children aged ⩽17 years, mainly serotypes 3, 1 and 19A. Non-PCV13 serotypes were associated with meningitis and occult bacteraemia and PCV13 serotypes with pneumonia. Non-susceptibility to antibiotics of non-PCV13 serotypes should be monitored.

Rapid identification, capsular typing and molecular characterization of Streptococcus pneumoniae by using whole genome nanopore sequencing.

BACKGROUND: Whole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens. MinION (Oxford Nanopore) is a real-time third generation sequencer whose portability, affordability and speed in data production make of it an attractive device for whole genome sequencing. The objective of this study is to evaluate MinION sequencer for pathogen identification and molecular characterization of Streptococcus pneumoniae isolated at a children's Hospital. Whole genome sequencing of 32 Streptococcus pneumoniae invasive isolates, previously characterized by standard methods (Quellung reaction, Multiplex PCR and Sanger-MLST), were performed. DNA was extracted using ZymoBIOMICS DNA Microprep kit. Quantification and purity of DNA was assessed by Qubit and Nanodrop, respectively. Library preparation was performed using the Rapid Barcoding Kit. Real-time workflow EPI2ME platform "What's it in my pot" was used for species identification. Fast5 sequences were converted into FASTQ by Albacore software. Reads were assembled using CANU software. PathogenWatch, genomic epidemiology and pubmlst online tools were used for capsular typing and/or whole genome-MLST profile. RESULTS: Rapid identification of Streptococcus pneumoniae was achieved by "What's in my pot". Capsular typing was correctly assigned with PathogenWatch in all 32 isolates at serogroup level and 24 at serotype level. Whole genome-MLST results obtained by genomic epidemiology and pubmlst were consistent with double locus variant clonal complex obtained by Sanger-MLST in 31 isolates. CONCLUSION: MinION sequencer provides a rapid, cost-effective and promising pathway for performing WGS by a pocked-sized device for epidemiological purposes but improving its sequencing accuracy will make it more appealing to be used in clinical microbiology laboratories.

Enterovirus / No disponible

No disponible

Evaluation of the eazyplex MRSA assay for the rapid detection of Staphylococcus aureus in pleural and synovial fluid.

OBJECTIVE: Loop-mediated isothermal amplification (LAMP) is a simple and sensitive technique for rapid microbiological diagnosis. The aim of this study was to evaluate the analytical and diagnostic performance of the LAMP eazyplex MRSA test for the direct detection and differentiation of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) in synovial/pleural fluid. METHODS: Analytical validation included the determination of the limit of detection (LoD) and analytical specificity of the eazyplex MRSA test. A diagnostic evaluation of the eazyplex test against bacterial culture was performed on routine pleural/synovial samples collected prospectively from patients aged less than 18 years with complicated pneumonia with empyema or arthritis admitted to the Children's Hospital Sant Joan de Déu in Barcelona, Spain, between April 2015 and May 2016. RESULTS: The new system appropriately detected a quality control panel of clinical samples with DNA of MSSA, MRSA, and other pathogens. The LoD was established at 6.4×103 CFU/ml for S. aureus and 1.0×104 CFU/ml for MRSA. Diagnostic validation of the eazyplex MRSA assay was performed on 51 prospective clinical invasive samples, resulting in sensitivity and specificity values of 83.3% and 97.8%, respectively, for S. aureus detection. The mean turnaround time was 70min. CONCLUSIONS: The eazyplex MRSA assay was found to be a useful test for the rapid detection of S. aureus in invasive samples such as pleural/synovial fluid.

Outbreak of brainstem encephalitis associated with enterovirus-A71 in Catalonia, Spain (2016): a clinical observational study in a children's reference centre in Catalonia.

OBJECTIVES: To describe the characteristics of an outbreak of brainstem encephalitis and encephalomyelitis related to enterovirus (EV) infection in Catalonia (Spain), a setting in which these manifestations were uncommon. METHODS: Clinical and microbiological data were analysed from patients with neurological symptoms associated with EV detection admitted to a reference paediatric hospital between April and June 2016. RESULTS: Fifty-seven patients were included. Median age was 27.7 months (p25-p75 17.1-37.6). Forty-one (72%) were diagnosed with brainstem encephalitis, seven (12%) with aseptic meningitis, six (11%) with encephalitis, and three (5%) with encephalomyelitis (two out of three with cardiopulmonary failure). Fever, lethargy, and myoclonic jerks were the most common symptoms. Age younger than 12 months, higher white-blood-cell count, and higher procalcitonin levels were associated with cardiopulmonary failure. Using a PAN-EV real-time PCR, EV was detected in faeces and/or nasopharyngeal aspirate in all the patients, but it was found in cerebrospinal fluid only in patients with aseptic meningitis. EV was genotyped in 47 out of 57 and EV-A71 was identified in 40 out of 47, being the only EV type found in patients with brainstem symptoms. Most of the detected EV-A71 strains were subgenogroup C1. Intravenous immunoglobulins were used in 34 patients. Eight cases (14%) were admitted to the intensive care unit. All the patients but three, those with encephalomyelitis, showed a good clinical course and had no significant sequelae. No deaths occurred. CONCLUSIONS: The 2016 outbreak of brainstem encephalitis in Catalonia was associated with EV-A71 subgenogroup C1. Despite the clinical manifestations of serious disease, a favourable outcome was observed in the majority of patients.

Clinical characteristics and pertussis costs in cases reported to epidemiological services and cases detected in household contacts in Catalonia (Spain).

PURPOSE: To assess the clinical characteristics and direct health costs associated with pertussis cases reported to and confirmed by epidemiological services and cases detected among household contacts in Catalonia (Spain) in 2012-2013. METHODS: All pertussis cases confirmed by the epidemiological services (n = 641) and all cases detected among the household contacts (n = 422) were included in the study. The chi-square test and odds ratios were used to compare percentages and the t-test was used to compare mean pertussis costs, with p < 0.05 being considered statistically significant. RESULTS: Cases reported to epidemiological services had a higher percentage of hospitalizations (OR = 32.2, p < 0.001) and severe disease (OR = 27.7, p < 0.001) than cases detected among the household contacts. The total health costs associated with pertussis cases were €871,648, €799,704 (92 %) for cases reported to epidemiological services and €71,944 (8 %) for cases detected among the household contacts. Total treatment, detection, and quimiprophylaxis costs were € 809,702, € 44,312, and € 17,635, representing 92.5 %, 5.5 %, and 2 % of total pertussis costs respectively. The mean costs were significantly higher (p < 0.001) in cases reported to epidemiological services than in cases detected among the household contacts, for all cases (€1248 vs €170), and for severe (€4546 vs €1073), moderate (€204 vs €165), and mild (€153 vs €133) disease. CONCLUSIONS: The burden of pertussis in Catalonia was high in terms of health costs, especially in infants aged less than 1 year. Active epidemiological surveillance activities could prevent pertussis transmisison and reduce pertussis costs.

A comparison of human metapneumovirus and respiratory syncytial virus WHO-defined severe pneumonia in Moroccan children.

Acute respiratory infections remain the principal cause of morbidity and mortality in Moroccan children. Besides bacterial infections, respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are prominent among other viruses due to their high prevalence and association with severe clinical episodes. We aimed to describe and compare RSV- and hMPV-associated cases of WHO-defined severe pneumonia in a paediatric population admitted to Morocco's reference hospital. Children aged 2-59 months admitted to the Hôpital d'Enfants de Rabat, Morocco meeting WHO-defined severe pneumonia criteria were recruited during 14 months and thoroughly investigated to ascertain a definitive diagnosis. Viral prevalence of RSV, hMPV and other viruses causing respiratory symptoms was investigated in nasopharyngeal aspirate samples through the use of molecular methods. Of the 683 children recruited and included in the final analysis, 61/683 (8·9%) and 124/683 (18·2%) were infected with hMPV and RSV, respectively. Besides a borderline significant tendency for higher age in hMPV cases, patients infected with either of the viruses behaved similarly in terms of demographics, patient history, past morbidity and comorbidity, vaccination history, socioeconomic background and family environment. Clinical presentation on arrival was also similar for both viruses, but hMPV cases were associated with more severity than RSV cases, had a higher risk of intensive care need, and received antibiotic treatment more frequently. RSV and hMPV are common and potentially life-threatening causes of WHO-defined pneumonia in Moroccan children. Both viruses show indistinctive clinical symptomatology, but in Moroccan children, hMPV was associated with a more severe evolution.

Molecular epidemiology of severe respiratory disease by human rhinoviruses and enteroviruses at a tertiary paediatric hospital in Barcelona, Spain.

In order to describe the molecular epidemiology of human rhinovirus (HRV) and enterovirus (EV) infection in severely ill children, we studied all episodes of bronchospasm/bronchopneumonia in 6-month-old to 18-year-old patients from January 2010 to May 2012 who required mechanical ventilation. HRV/EVs were detected in 55 (57.3%) of 96 patients, of which 50 (91%) were HRV (HRV-A, 16; HRV-B, 1; HRV-C, 18) and 5 (9%) were EVs (EV-D68, 3). No significant differences in epidemiologic and clinical characteristics were found between different types. In six of the 13 patients who required invasive mechanical ventilation, HRV was the only pathogen detected.

Estimation of the invasive disease potential of Streptococcus pneumoniae in children by the use of direct capsular typing in clinical specimens.

Traditionally, invasiveness indexes have been based on culture methods. We aimed to establish a new classification of the invasive disease potential of pneumococcal serotypes causing invasive pediatric disease in the era of conjugate vaccines in Catalonia, Spain, by adding capsular typing of Streptococcus pneumoniae in direct sample. Two samples of children attended at the University Hospital Sant Joan de Déu (Barcelona, Spain) between 2007 and 2011 were compared: a first sample of 358 children with invasive pneumococcal disease and a second sample of 402 pneumococcal nasopharyngeal carriers selected from 714 healthy children admitted for minor surgical procedures. The most common invasive serotypes were 1 (20.1 %, n = 72), 19A (13.9 %, n = 50), 3 (12.3 %, n = 44), and 7FA (7.5 %, n = 27), whereas the most common serotypes in carriage were 19A (8.7 %, n = 38), 10FC33C (7.8 %, n = 34), 6C (6.9 %, n = 30), and 19FBC (5.5 %, n = 24). We detected a rate of cocolonization of 26.4 % (n = 89) among the 336 samples serotyped in the carriers population. Serotypes 1, 3, and 7FA were significantly associated with high invasiveness. Serotypes 6C, 10FC33C, 23A, 35B, 19FBC, 21, 11AD, 15BC, 23B, 34, and 6A were significantly associated with low invasiveness. Our results proved that the use of molecular techniques in direct sample for both the detection and the capsular identification of Streptococcus pneumoniae is very useful to obtain a more accurate calculation of the invasiveness of the different pneumococcal serotypes.

Pleuroneumonía y shock séptico por Streptococcus pneumoniae serotipo 19A multirresistente tratado mediante linezolid / Pleuropneumonia and septic shock due to multiresistant Streptococcus pneumoniae serotype 19A treated with linezolid

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Respiratory viruses, such as 2009 H1N1 influenza virus, could trigger temporal trends in serotypes causing pneumococcal disease.

In order to determine if the novel influenza A(H1N1)pdm09 was associated with temporal trends of main serotypes causing invasive pneumococcal disease (IPD), we studied 384 episodes of IPD in <18-year-old patients from 2007 to 2012. The number of IPD episodes diagnosed during the 2009 pandemic period meant almost one-third of all the episodes diagnosed in the five included influenza periods (51/156). The number of IPD episodes diagnosed during the 2009 pandemic period meant almost one-third of all the episodes diagnosed in the five included influenza periods. Most of them occurred in <5-year-old children. Serotype 1 was the main serotype detected over the period, except for the 2009 pandemic, when it practically disappeared. Seasonality and viral infections could trigger temporal trends of serotypes causing IPD.

Genotyping, local prevalence and international dissemination of ß-lactamase-producing Kingella kingae strains.

ß-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of ß-lactamase production among K. kingae isolates from different geographical origins and genetically characterized ß-lactamase-producing strains. Seven hundred and seventy-eight isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for ß-lactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, blaTEM and 16S rRNA sequencing. ß-lactamase was identified in invasive strains from Iceland (n=4/14, 28.6%), the USA (n=3/15, 20.0%) and Israel (n=2/190, 1.1%) and in carriage strains in the USA (n=5/17, 29.4%) and Israel (n=66/429, 15.4%). No French, Spanish or Canadian isolates were ß-lactamase producers. Among ß-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic ß-lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed from the four genetically unrelated Israeli ß-lactamase-producing clones. Representative strains of different genotypes produced the TEM-1 enzyme. K. kingae ß-lactamase producers exhibit a clear clonal distribution and have dissimilar invasive potential. The presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of ß-lactam resistance, and emphasizes the importance of routine testing of all K. kingae clinical isolates for ß-lactamase production.

High prevalence of genetically-determined mannose binding lectin deficiency in young children with invasive pneumococcal disease.

Susceptibility to invasive pneumococcal disease (IPD) correlates with age, younger children being the group with the highest burden of disease. The relevance of the innate immune response and particularly the role of mannose-binding lectin (MBL) in combating IPD is not well known. This is a 2-year prospective study (February 2011 to March 2013) including patients with IPD who attended two hospitals from Catalonia, Spain. Variables including attack rate of pneumococcal serotype (high or low invasive potential serotypes) and genotypes associated with low serum MBL levels were recorded. One hundred and forty-seven patients were included in the study. One hundred and two (69.4%) patients were children or adolescents <18 years and 45 (30.6%) were adults. Overall, low-MBL genotypes (O/O; XA/O) were detected in 23 (15.6%) patients. Children <2 years showed a higher frequency of low-MBL genotypes compared with other patients (31.0% vs. 11.9%; p = 0.031). Further sub-analysis revealed a higher proportion of low-MBL genotypes in children <2 years with IPD caused by opportunistic or low-attack-rate serotypes when compared with older patients (46.2% vs. 13.2%; p = 0.02). However, no statistically significant differences between the two groups were observed when including patients infected with invasive or high-attack-rate serotypes (18.8% vs. 10.0%; p = 0.59). Our data suggest that young children with a genetically determined low-MBL production are at a higher risk of developing IPD, particularly that caused by opportunistic or low-attack-rate pneumococcal serotypes.

High invasiveness of pneumococcal serotypes included in the new generation of conjugate vaccines.

The implementation of the seven-valent pneumococcal conjugate vaccine, PCV7, has resulted in significant changes in the pneumococcal population being carried and causing disease. We aimed to determine the invasive disease potential of serotypes causing invasive paediatric disease in the era of conjugate vaccines in Catalonia, Spain, and their potential coverage by the 13-valent pneumococcal conjugate vaccine, PCV13. As a secondary objective, we evaluated whether implementation of PCV7 had resulted in significant changes in the invasive disease potential of the most frequent serotypes circulating in the area. Two pneumococcal collections obtained from children admitted to the University Hospital Sant Joan de Déu (Barcelona, Spain) between 2007 and 2011 were compared: a first set of 159 invasive disease isolates, and a second set of 209 nasopharyngeal isolates recovered from healthy children admitted for minor surgery. The most common invasive serotypes were 1 (24.5%, n = 39), 19A (21.2%, n = 34), 5 (8.8%, n = 14), 7F (8.8%, n = 14) and 3 (5%, n = 8). The most common serotypes in carriage were 19A (10%, n = 21), 6C (9%, n = 19), 23B (8.1%, n = 17), 6A (7.6%, n = 16) and 19F (6.2%, n = 13). A significantly higher propensity to cause invasive disease was observed for serotypes 1, 3, 5, 7F and 19A, all of which are included in PCV13. After false-discovery-rate correction, the results were robust for serotypes 1, 5, 7F and 19A. Non-PCV13 serotypes had a low invasive disease potential. Our data reinforce the need for continuous surveillance and should encourage efforts to introduce universal vaccination with PCV13 in children in our region.

Molecular epidemiology of enterovirus 71, coxsackievirus A16 and A6 associated with hand, foot and mouth disease in Spain.

Hand, foot and mouth disease (HFMD) is a childhood illness frequently caused by genotypes belonging to the enterovirus A species, including coxsackievirus (CV)-A16 and enterovirus (EV)-71. Between 2010 and 2012, several outbreaks and sporadic cases of HFMD occurred in different regions of Spain. The objective of the present study was to describe the enterovirus epidemiology associated with HFMD in the country. A total of 80 patients with HFMD or atypical rash were included. Detection and typing of the enteroviruses were performed directly in clinical samples using molecular methods. Enteroviruses were detected in 53 of the patients (66%). CV-A6 was the most frequent genotype, followed by CV-A16 and EV-71, but other minority types were also identified. Interestingly, during almost all of 2010, CV-A16 was the only causative agent of HFMD but by the end of the year and during 2011, CV-A6 became predominant, while CV-A16 was not detected. In 2012, however, both CV-A6 and CV-A16 circulated. EV-71 was associated with HFMD symptoms only in three cases during 2012. All Spanish CV-A6 sequences segregated into one major genetic cluster together with other European and Asian strains isolated between 2008 and 2011, most forming a particular clade. Spanish EV-71 strains belonged to subgenogroup C2, as did most of the European sequences circulated. In conclusion, the recent increase of HFMD cases in Spain and other European countries has been due to a larger incidence of circulating species A enteroviruses, mainly CV-A6 and CV-A16, and the emergence of new genetic variants of these viruses.

Lactante sano con reactivación de citomegalovirus en contexto de bronquiolitis por metapneumovirus / Healthy infant with cytomegalovirus reactivation in the setting of metapneumovirus bronchiolitis

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Multiplex real-time PCR for prompt diagnosis of an outbreak of human parainfluenza 3 virus in children with acute leukemia.

INTRODUCTION: Human parainfluenza virus type 3 (HPIV-3) causes significant morbimortality in immunocompromised patients. Outbreaks of severe pneumonitis have been previously described in this setting. MATERIALS AND METHODS: Retrospective observational study in children diagnosed with acute leukemia and a documented HPIV-3 infection in the context of a nosocomial outbreak occurred in a single center. RESULT: During summer 2012, an HPIV-3 infection was detected in six hospitalized children with acute leukemia. All the patients had respiratory symptoms and one of them suffered from parotitis. CONCLUSION: Early diagnoses using multiplex real-time polymerase chain reaction (PCR) let us control this outbreak. A phylogenetic analysis confirmed person-to-person transmission of a single HPIV-3 variant.