Validation and implementation of a diagnostic algorithm for DNA Detection of Bordetella pertussis, B. parapertussis, and B-holmesii in a Pediatric Referral Hospital in Barcelona, Spain

This study aimed to validate a comprehensive diagnostic protocolbased on real-time PCR for the rapid detection and identification ofBordetella per-tussis,Bordetella parapertussis, andBordetella holmesii, as well as its implementationin the diagnostic routine of a reference children’s hospital. The new algorithm in-cluded a triplex quantitative PCR (qPCR) targeting IS481gene (inB. pertussis,B. hol-mesii, and someBordetella bronchisepticastrains), pIS1001(B. parapertussis-specific)andrnaseP as the human internal control. Two confirmatory singleplex tests forB.pertussis(ptxA-Pr) andB. holmesii(hIS1001) were performed if IS481was positive. An-alytical validation included determination of linear range, linearity, efficiency, preci-sion, sensitivity, and a reference panel with clinical samples. Once validated, the newalgorithm was prospectively implemented in children with clinical suspicion ofwhooping cough presenting to Hospital Sant Joan de Deu (Barcelona, Spain) over12 months. Lower limits of detection obtained were 4.4, 13.9, and 27.3 genomicequivalents/ml of sample for IS481(onB. pertussis), pIS1001and hIS1001, and 777.9forptxA-Pr. qPCR efficiencies ranged from 86.0% to 96.9%. Intra- and interassay vari-abilities were 3% and 5%, respectively. Among 566 samples analyzed,B. pertus-sis,B. holmesii, andB. parapertussiswere detected in 11.1%, 0.9% (only in females 4 years old), and 0.2% of samples, respectively. The new algorithm proved to be auseful microbiological diagnostic tool for whooping cough, demonstrating a low rateof other non-pertussis Bordetellaspecies in our surveilled areaPeer ReviewedPostprint (author's final draft

Recurrent invasive pneumococcal disease in children: Underlying clinical conditions, and immunological and microbiological characteristics.

Purpose Clinical, immunological and microbiological characteristics of recurrent invasive pneumo-coccal disease (IPD) in children were evaluated, differentiating relapse from reinfection, in order to identify specific risk factors for both conditions. Methods All patients<18 years-old with recurrent IPD admitted to a tertiary-care pediatric center from January 2004 to December 2011 were evaluated. An episode of IPD was defined as the presence of clinical findings of infection together with isolation and/or pneumococcal DNA detection by Real-Time PCR in any sterile body fluid. Recurrent IPD was defined as 2 or more episodes in the same individual at least 1 month apart. Among recurrent IPD, we differentiated relapse (same pneumococcal isolate) from reinfection. Results 593 patients were diagnosed with IPD and 10 patients died. Among survivors, 23 episodes of recurrent IPD were identified in 10 patients (1.7%). Meningitis was the most frequent form of recurrent IPD (10 episodes/4 children) followed by recurrent empyema (8 episodes/4 children). Three patients with recurrent empyema caused by the same pneumococcal clone ST306 were considered relapses and showed high bacterial load in their first episode. In contrast, all other episodes of recurrent IPD were considered reinfections. Overall, the rate of relapse of IPD was 0.5% and the rate of reinfection 1.2%. Five out of 7 patients with rein- fection had an underlying risk factor: cerebrospinal fluid leak (n = 3), chemotherapy treatment (n = 1) and a homozygous mutation in MyD88 gene (n = 1). No predisposing risk factors were found in the remainder. Conclusions recurrent IPD in children is a rare condition associated with an identifiable risk factor in case of reinfection in almost 80% of cases. In contrast, recurrent IPD with pleuropneumonia is usually a relapse of infection

Changes in the Serotype Distribution of Streptococcus Pneumoniae Causing Otitis Media After PCV13 Introduction in Spain

One of the beneficial effects of pneumococcal conjugate vaccines (PCVs) has been a decrease in the incidence of non-invasive infections, such as otitis media (OM) caused by vaccine serotypes. In this study, we analyzed the epidemiology of pneumococcal OM before and after PCV13 introduction in 2010. Between 2008 and 2016, the middle ear exudates from 2653 children under 14 years of age with OM were studied in two Spanish provinces (Gipuzkoa and Barcelona), and S. pneumoniae was isolated in 235 (8.9%) of cases. The 204 available isolates were serotyped and distributed in three 3-year periods: one before and two after PCV13 introduction (early and late post-PCV13). A significant decrease in the rate of OM caused by S. pneumoniae was observed mainly due to a decrease in infections caused by all PCV13 serotypes, although exceptions were observed including the persistence of serotype 3 in Gipuzkoa and a weak re-emergence of serotype 19F in both regions. The rate and diversity of non-PCV13 serotypes increased in both regions and an emerging clone causing OM was detected in each region: serotype 23B ST2372 in Gipuzkoa and serotype 11A ST838/ST6521 in Barcelona. The introduction of PCV13 has been followed by a change in the epidemiology of pneumococcal OM, with a decrease in the rate of vaccine serotypes accompanied by an increase in the diversity of non-vaccine serotype and the clonal spreading of different single clones in each region.JMM was funded in part by the Centro de Investigacion Biomedica en Red de Enfermedades Respiratorias (CIBERES-26). CMA was funded in part by the Centro de Investigacion Biomedica en Red de de Epidemiologia y Salud Publica (CIBERESP-57). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was funded in part by the Centro de Investigacion Biomedica en Red de Enfermedades Respiratorias (CIBERES-26) and de Epidemiologia y Salud Publica (CIBERESP-57)

Characteristics of hospitalized cases of Pertussis in Catalonia and Navarra, two regions in the north of Spain

Pertussis causes a large number of cases and hospitalizations in Catalonia and Navarra. We made a study of household cases of pertussis during 2012 and 2013 in order to identify risk factors for hospitalization in pertussis cases. Each primary case reported triggered the study of their contacts. Close contacts at home and people who were in contact for >2 hours during the transmission period of cases were included. The adjusted OR and 95% confidence intervals (CI) was calculated using logistic regression. A total of 1124 pertussis cases were detected, of which 14.9% were hospitalized. Inspiratory whoop (aOR: 1.64; CI: 1.02–2.65), apnoea (aOR: 2.47; CI: 1.51–4.03) and cyanosis (aOR: 15.51; CI: 1.87–128.09) were more common in hospitalized than in outpatient cases. Hospitalization occurred in 8.7% of correctly-vaccinated cases, 41.1% of non-vaccinated cases and 9.4% of partially-vaccinated cases. In conclusion, inspiratory whoop, apnoea and cyanosis were associated factors to hospitalization while vaccination reduced hospitalizations due to pertussis.Ministry of Science and Innovation, Institute of Health Carlos III (Project PI11/02557) and Fondo Europeo de Desarrollo Regional (FEDER-Una manera de hacer Europa); the Catalan Agency for the Management of Grants for University Research (AGAUR Grant number 2014/ SGR 1403)

Congenital cytomegalovirus, parvovirus and enterovirus infection in Mozambican newborns at birth: A cross-sectional survey

BACKGROUND: Congenital cytomegalovirus (cCMV) infection is the most prevalent congenital infection acquired worldwide, with higher incidence in developing countries and among HIV-exposed children. Less is known regarding vertical transmission of parvovirus B19 (B19V) and enterovirus (EV). We aimed to assess the prevalence of CMV, B19V and EV vertical transmission and compare results of screening of congenital CMV obtained from two different specimens in a semirural Mozambican maternity. METHODS: A cross sectional study was conducted among pregnant mothers attending Manhica District Hospital upon delivery. Information on maternal risk factors was ascertained. Dried umbilical cord (DUC) samples were collected in filter paper for CMV, B19V and EV detection by real-time polymerase chain reaction (RT-PCR), and nasopharyngeal aspirates (NPA) to test for CMV by RT-PCR. Maternal blood samples and placental biopsy samples were also obtained to investigate CMV maternal serology, HIV status and immunopathology. RESULTS: From September 2014 to January 2015, 118 mothers/newborn pairs were recruited. Prevalence of maternal HIV infection was 31.4% (37/118). CMV RT-PCR was positive in 3/115 (2.6%) of DUC samples and in 3/96 (6.3%) of NPA samples obtained from neonates. The concordance of the RT-PCR assay through DUC with their correspondent NPA sample was moderate (Kappa = 0.42 and p<0.001. No differences on cCMV prevalence were found among HIV-exposed and unexposed. All (100%) mothers were seropositive for CMV IgG. RT-PCR of EV and B19V in DUC were both negative in all screened cases. No histological specific findings were found in placental tissues. No risk factors associated to vertical transmission of these viral infections were found. CONCLUSIONS: This study indicates the significant occurrence of vertical transmission of CMV in southern Mozambique. Larger studies are needed to evaluate the true burden, clinical relevance and consequences of congenital infections with such pathogens in resource-constrained settings

Detection of Streptococcus pneumoniae and Haemophilus influenzae type B by real-time PCR from dried blood spot samples among children with pneumonia: a useful approach for developing countries

Correction https://doi.org/10.1371/journal.pone.0147678Background: Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco. Methods: Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested. Results: Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001). Conclusion: Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries

Easy Diagnosis of Invasive Pneumococcal Disease

Invasive pneumococcal disease (IPD) causes many cases of severe disease and death among children <5 years of age, mostly in developing countries. Before conjugate vaccines can be introduced in developing countries, information about disease epidemiology is urgently needed. The lack of laboratories equipped to perform pneumococcal serotyping leads to the need to send isolates to reference laboratories. Good sample preservation is necessary to prevent samples from arriving at the laboratory in poor condition. We evaluated the usefulness of multiplex real-time PCR from strains and blood samples kept at room temperature on dried blood spot (DBS) filter paper for detecting and serotyping Streptococcus pneumoniae. DBS screening is a reliable method that requires only a small amount of blood; it is used for the diagnosis of several human disease

Effectiveness of the 13-valent pneumococcal conjugate vaccine in preventing invasive pneumococcal disease in children aged 7-59 months. A matched case-control study

Background The 13-valent pneumococcal conjugate vaccine (PCV13) was licensed based on the results of immunogenicity studies and correlates of protection derived from randomized clinical trials of the 7-valent conjugate pneumococcal vaccine. We assessed the vaccination effectiveness (VE) of the PCV13 in preventing invasive pneumococcal disease (IPD) in children aged 7-59 months in a population with suboptimal vaccination coverage of 55%. Methods The study was carried out in children with IPD admitted to three hospitals in Barcelona (Spain) and controls matched by hospital, age, sex, date of hospitalization and underlying disease. Information on the vaccination status was obtained from written medical records. Conditional logistic regression was made to estimate the adjusted VE and 95% confidence intervals (CI). Results 169 cases and 645 controls were included. The overall VE of ≥1 doses of PCV13 in preventing IPD due to vaccine serotypes was 75.8% (95% CI, 54.1-87.2) and 90% (95% CI, 63.9-97.2) when ≥2 doses before 12 months, two doses on or after 12 months or one dose on or after 24 months, were administered. The VE of ≥1 doses was 89% (95% CI, 42.7-97.9) against serotype 1 and 86.0% (95% CI, 51.2-99.7) against serotype 19A. Serotype 3 showed a non-statistically significant effectiveness (25.9%; 95% CI, -65.3 to 66.8). Conclusions The effectiveness of ≥1 doses of PCV13 in preventing IPD caused by all PCV13 serotypes in children aged 7-59 months was good and, except for serotype 3, the effectiveness of ≥1 doses against the most frequent PCV13 serotypes causing IPD was high when considered individually

Factors influencing the spread of pertussis in households: a prospective study, Catalonia and Navarre, Spain, 2012 to 2013

We aimed to investigate transmission rates of pertussis in household contacts of cases and factors associated with transmission. A prospective epidemiological study was conducted in 2012 and 2013 to determine the incidence of pertussis among household contacts of reported cases in Catalonia and Navarre, Spain. An epidemiological survey was completed for each case and contact, who were followed for 28 days to determine the source of infection (primary case) and detect the occurrence of secondary cases. Odds ratios (ORs) were used to estimate the effectiveness of vaccination and chemoprophylaxis in preventing new cases, using the formula (1 − OR) × 100. For the 688 primary cases, a total of 2,852 contacts were recorded. The household transmission rate was 16.1% (459/2,852) and rose according to the age (> 18 years) and lack of immunisation of the primary cases, and also the age (0–18 years), family relationship (siblings and children), lack of vaccination and chemoprophylaxis of contacts. Pertussis vaccine effectiveness in preventing new cases was 65.0% (95% confidence interval (CI): 11.6 to 86.2) for full vaccination (≥ 4 doses) and 59.7% (95% CI: −6.8 to 84.8) for incomplete vaccination (< 4 doses). The effectiveness of chemoprophylaxis was 62.1% (95% CI: 40.3 to 75.9). To reduce household transmission, contacts should be investigated to detect further cases and to administer chemoprophylaxis. The current vaccination status of cases and contacts can reduce household transmission.This study was supported by the Ministry of Science and Innovation, Institute of Health Carlos III (Project PI11/02557) and Fondo Europeo de Desarrollo Regional (FEDER-Una manera de hacer Europa)

Nasopharyngeal Microbiota in Children With Invasive Pneumococcal Disease: Identification of Bacteria With Potential Disease-Promoting and Protective Effects

Background and Aims: The risk of suffering from some infectious diseases can be related to specific microbiota profiles. Specifically, the nasopharyngeal microbiota could play a role as a risk or protective factor in the development of invasive disease caused by S. pneumoniae.Methodology: We analyzed the nasopharyngeal microbiota of children with invasive pneumococcal disease (IPD) and that of healthy controls matched by age, sex, and seasonality from Catalonia, Spain. Epidemiological, microbiological and clinical variables were considered to compare microbiota profiles, analyzed by sequencing the V1–V4 region of the 16S rRNA gene.Results: Twenty-eight children with IPD (median age 43 months) and 28 controls (42.6 months) were included in the study. IPD children presented a significantly higher bacterial diversity and richness (p &lt; 0.001). Principal coordinate analysis revealed three different microbiota profiles: microbiota A, dominated by the genus Dolosigranulum (44.3%); Microbiota B, mostly represented by Streptococcus (36.9%) and Staphylococcus (21.3%) and a high diversity of anaerobic genera including Veillonella, Prevotella and Porphyromonas; and Microbiota C, mainly containing Haemophilus (52.1%) and Moraxella (31.4%). The only explanatory factor for the three microbiotas was the classification of children into disease or healthy controls (p = 0.006). A significant negative correlation was found between Dolosigranulum vs. Streptococcus (p = 0.029), suggesting a potential antagonistic effect against pneumococcal pathogens.Conclusions: The higher bacterial diversity and richness in children with IPD could suggest an impaired immune response. This lack of immune competence could be aggravated by breastfeeding &lt;6 months and by the presence of keystone pathogens such as Porphyromonas, a bacterium which has been shown to be able to manipulate the immune response, and that could favor the overgrowth of many proteolytic anaerobic organisms giving rise to a dramatic dysbiosis. From an applied viewpoint, we found suggestive microbiota profiles associated to IPD or asymptomatic colonization that could be used as disease biomarkers or to pave the way for characterizing health-associated inhabitants of the respiratory tract. The identification of beneficial bacteria could be useful to prevent pneumococcal infections by integrating those microorganisms in a probiotic formula. The present study suggests not only respiratory tract samples, but also breast milk, as a potential source of those beneficial bacteria