Luís Januário

Relationships Between Rhinitis Symptoms, Respiratory Viral Infections and Nasopharyngeal Colonization With Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus in Children Attending Daycare

Background: Nasal bacterial colonization is often dubbed “asymptomatic.” We hypothesized that rhinitis, common in preschool children, is associated with bacterial colonization and that respiratory viruses, which cause rhinitis, interact with bacteria in ways which promote transmission. Methods: Five hundred eighty-five children (4.2–73.6 months) attending daycare had clinical information, a rhinitis score and nasal swabs collected in February 2009. Swabs in soya tryptone glucose glycerine broth were cultured for Streptococcus pneumoniae (Sp), Haemophilus influenzae (Hi) and Staphylococcus aureus and analyzed by real-time polymerase chain reaction for respiratory viruses, both semiquantitatively. Results: Rhinitis symptoms, carriage of Sp and Hi and viral infection fell, whereas S. aureus carriage rates rose with age. Significant, age-independent associations between rhinitis symptoms and detection of Hi (P < 0.033) and Hi colonization density (P < 0.027) were observed. Of the 42% with detected viruses, most (78%) had picornavirus infection. There was a significant age-independent association between viral infection (and viral load, picornavirus infection and picornaviral load) and detection of Sp (P = 0.020, 0.035, 0.005, 0.014) and between viral infection and viral load and Sp colonization density (P = 0.024, 0.028). Conclusions: Hi may promote its own transmission by inducing or amplifying rhinitis in children. There is a close quantitative relationship between respiratory viral infection, including picornavirus infection and Sp colonization. These findings have implications for understanding disease pathogenesis and formulating prevention strategies using vaccines.

Progressive changes in pneumococcal carriage in children attending daycare in Portugal after 6 years of gradual conjugate vaccine introduction show falls in most residual vaccine serotypes but no net replacement or trends in diversity

OBJECTIVES To track ongoing trends in pneumococcal (Sp) serotype carriage under the selection pressure of moderate pneumococcal conjugate vaccine (PCV) use, children in a community in Portugal were studied in the same months in 3 consecutive years. METHODS Nasopharyngeal specimens were collected (children aged 3 months to <7 years) in 8 urban daycare centers in February 2008 (n=561) and 2009 (n=585). Sp isolates were serotyped. RESULTS While demographics were similar in 2008-2009 and a previously reported sample in 2007, PCV coverage (at least one dose) in the children studied rose from 76.5% to 84% although national coverage was lower than this. Sp carriage fell from 61% to 51% with a concomitant fall in PCV7 serotype carriage from 12.1% to 4.3%. Remaining PCV7 serotypes declined to near (23F) or totally (6B, 14) undetectable levels except 19F which persisted unchanged in around 4% of children. Although carriage of 3 and 6C rose, there was no net increase in non-PCV7 serotypes and no progressive trend in serotype diversity. CONCLUSIONS Ecological changes induced by PCVs where uptake is moderate appear to be different from high usage settings. We report falling Sp carriage due to PCV7 serotype disappearance with persistence of 19F and no ongoing net replacement after several years of PCV7 use and slowly rising uptake.

Case control study of rotavirus vaccine effectiveness in Portugal during 6 years of private market use.

Background: Although recommended by the vaccine committee of the Portuguese Paediatric Society, rotavirus vaccines have not been included in the routine immunization schedule. They have been available privately since 2006 with estimated coverage reaching approximately 30%. However, unlike other European countries using the vaccine, sentinel surveillance has detected fluctuations but no clear trends in the rate of gastrointestinal disease presentations. In this study, we set out to establish the real world effectiveness of rotavirus immunization in this low vaccine coverage setting. Methods: We carried out a test-negative case control study on a population of children attending a regional pediatric hospital, between 2006 and 2012, with symptoms of acute gastroenteritis and producing a stool sample for routine rotavirus testing. We calculated exposure odds ratio (ratio of odds of antecedent vaccination among cases compared with controls) to derive vaccine effectiveness ([1 − adjusted odds ratio]/100) against both hospital attendance and admission. Results: Vaccine effectiveness against attendance with rotavirus acute gastroenteritis was 83.7% (95% confidence interval: 73.9–89.8) and against hospital admission was 96.1% (95% confidence interval: 83.8–99.1). No significant difference between the 2 available vaccines was detected. Conclusion: Both rotavirus vaccines offer a high degree of individual protection in this population.

The evolving epidemiology of rotavirus gastroenteritis in central Portugal with modest vaccine coverage

BACKGROUND Rotavirus (RV) vaccines have been available on the private market in Portugal since 2006, with an estimated coverage rising from 16 to 42% between 2007 and 2010. OBJECTIVES To assess trends, surveillance of children presenting with acute gastroenteritis (AG) to a large paediatric emergency service (ES) in the central region of Portugal was conducted yearly during the winter-spring seasons. STUDY DESIGN Stool samples, collected throughout five epidemic seasons (January-June, 2006 to 2010) from children ≤ 36 months of age attending the ES with AG, were tested for RV by immunochromatographic rapid test and positive samples were genotyped. RESULTS A total of 6145 AG cases were identified: 1956 (32%) provided a stool sample (range: 28% in 2008-37% in 2009). The proportion of AG subjects who tested positive for RV fluctuated over the five surveillance seasons (49%, 39%, 25%, 26% and 39%, respectively) as did the distribution of co-circulating RV genotypes. There were no consistent changes in seasonality or age distribution and the proportion of admitted AG subjects who tested RV-positive did not show progressive trends over time. CONCLUSIONS Our results demonstrate fluctuations in RVAG incidence with no clear progressive trends or seasonal RV shifts among our surveillance subjects over five years, in the context of limited rotavirus vaccine coverage. Significant annual changes in genotype distributions were detected. Higher vaccine coverage may be necessary than at present for consistent impact on disease.

Multiple Streptococcus pneumoniae Serotypes in Aural Discharge Samples from Children with Acute Otitis Media with Spontaneous Otorrhea

ABSTRACT Among 55 children with cultures positive for acute otitis media with spontaneous otorrhea, 28 (51%) had cultures positive for aural Streptococcus pneumoniae, and in 10 of these, two distinct strains were detected, in which 5 had pairs of strains that were both capsule-bearing serotypes. Such cases were more likely to have cultures positive for other otopathogens than those with only one pneumococcus present.

Density Distribution of Pharyngeal Carriage of Meningococcus in Healthy Young Adults: New Approaches to Studying the Epidemiology of Colonization and Vaccine Indirect Effects.

Background: Improved understanding of Neisseria meningitidis (Nm) carriage biology and better methods for detection and quantification would facilitate studies of potential impact of new vaccines on colonization and transmission in adolescents. Methods: We performed plate cultures on 107 oropharyngeal swabs stored frozen in skim milk tryptone glucose glycerol (STGG) broth and previously positive for Nm. We compared quantitative polymerase chain reaction (qPCR) detection of Nm in 601 STGG-swabs with culture. Using qPCR (n = 87), a log-phase broth culture standard curve and semiquantitative plate cultures (n = 68), we measured density of carriage. We compared qPCR genogrouping of DNA extracts from STGG-swabs and from plate culture lawns (n = 110) with purified isolates (n = 80). Results: Swab storage resulted in only 10% loss of culture sensitivity. Direct sodC qPCR Nm detection yielded more positives (87/601, 14.5%) than culture (80/601, 13.3%). Most samples (57/110) positive by culture were also positive by qPCR and vice versa, but discrepancies (single positives) were frequent among low-density samples. sodC qPCR was positive in 79/80 isolates but in only 65 by ctrA qPCR. Density both by culture and qPCR varied across 4 orders of magnitude with the majority being low (<50 bacteria-gene copies/mL) and a minority being high (>1000). Genogrouping qPCRs yielded more positive results when performed on DNA extracts from lawn cultures. Conclusions: We provide the first description of the distribution of Nm carriage density. This could be important for understanding transmission dynamics and population-level effectiveness of adolescent vaccine programs. Storage of swabs frozen in STGG for batched laboratory analysis facilitates carriage studies and direct sodC qPCR for Nm combined with qPCR genogrouping of lawn culture extracts provides accurate, detailed description of colonization.

Oropharyngeal Carriage of Meningococcus in Portugal by Group and Clonal Complex 6 Years After Adolescent Vaccine Campaign.

Portugal introduced (2+1) conjugate Meningococcal group C vaccine in 2006 with high coverage catch up to 18 years and has given only 1 dose at 1 year since 2012. Among 601 student oropharyngeal swabs, meningococcal carriage rate was 13.3% (A-0%, B-5.3%, C-0.3%, W-0.2%, X-0.2% and Y-1.7%). C and W strains were of potentially disease-causing clonal complexes (cc) but not the hyperinvasive cc11.

Resurgence of Serotype 19F Carriage in Preschool Children in Portugal in the Context of Continuing Moderate Conjugate Pneumococcal Vaccine Uptake

TO THE EDITOR—Reduction to near extinction of pneumococcal (Streptococcus pneumoniae [Sp]) vaccine serotype nasal colonization has been reported over a period of around 5–7 years of pneumococcal conjugate vaccine (PCV) use [1, 2]. PCVs have been used in private practice in Portugal since 2001. They are not included in the universal national immunization program. After licensure of the 7-valent vaccine (PCV7, Wyeth), estimated coverage, based on sales information, increased from 32% in 2002 to 65% in 2005 and 79% in 2007, then fell slightly to 75% in 2008 (oral communication, Pfizer). In April 2009, the 10-valent vaccine (PCV10, GSK) became available and some infants received it. In January 2010, the 13-valent vaccine (PCV13, Pfizer) replaced PCV7 and is the vaccine that has been used almost exclusively since then (estimated coverage in 2010 and 2011 was approximately 65%; oral communication, Pfizer). Both schedules (2 + 1 and 3 + 1) have been used. Previously, we reported carriage rates by serotype in cross-sectional studies performed annually in preschool children attending day care centers (DCCs) in Coimbra, a city in the central region of Portugal, between 2007 and 2009 [3, 4]. Among the children studied, the proportion who had received at least 1 dose of PCV7 vaccine showed a significant upward trend over the 3 years (76.5%, 82.4%, and 84%) [4]. The proportion of PCV7 serotypes (by Quellung reaction) showed a significant downward trend over time, apart from 19F, which persisted, detected in 4.1% of all children studied in 2009 [4]. Studies from other countries suggest that 19F is slower to disappear than other PCV types [5, 6]. In February–March 2010 we swabbed the nasopharynges of 586 children attending the same urban DCCs in Coimbra. The study was approved by the Ethics Committee of Centro Hospitalar de Coimbra. Parents or guardians provided written informed consent for their child to participate. The mean age was 41.5 months (standard deviation, 18.1; range, 6.3–74.5); 326 (56%) children were male, and 507 (86.5%) had received at least 1 dose of PCV7 or PCV10 Among those aged ≥18 months, 78 (15.2%) and 338 (65.6%) had completed either a full 2 + 1 or 3 + 1 schedule, respectively. Nasopharyngeal swabs were inoculated into skim-milk tryptone glucose glycerol broth and stored at −80°C prior to culture. Standard microbiological techniques for the isolation and identification of Sp were used as described previously [7]. The Sp carriage rate was 58.5% (343/586). Molecular serotyping was undertaken, using a microarray-based method to determine cps gene content from genomic DNA hybridization, capable of detecting multiple serotypes in a single sample [8]. Excluding nontypeable (NT) signals (n = 7 NT only, n = 76 NT with other serotypes), which, using this methodology can include non-Sp streptococci, 73 children (12.5%) carried >1 Sp serotype. A total of 11.3% had PCV7/10 vaccine serotypes: 19F, 8.9% (52); 7F, 2.2% (13, including 2 with 19F); 1, 5, and 18C (1 each). Serotype 19F was more commonly detected than any other serotype. There was sole colonization by 19F in 34 children (5.8%) and by 19F with other serotype(s) in 18 (3.1%), among whom in 11 (1.9%) 19F was both the predominant serotype and represented >50% of the bacterial DNA detected in the sample (Figure 1). Accordingly, 45 children (7.7%) carried 19F as the only or the predominant serotype, a clear rise. Twenty cases of invasive disease due to 19F were reported to the national surveil-

Paediatric Invasive Pneumococcal Disease Before Universal Vaccination: 1995 - 2015

INTRODUCTION Pneumococcal conjugate vaccine was introduced in the private market in Portugal in 2001, reaching over the years a moderately high coverage. In July 2015, it was included in the National Immunisation Program. The aim of this study was to characterize invasive pneumococcal disease in a pediatric hospital before universal use of the vaccine. MATERIAL AND METHODS Retrospective analysis of medical records of all children with Streptococcus pneumoniae identified by culture and/or molecular biology (available since 2008), in products obtained from sterile sites, from January 1995 to June 2015. We evaluated demographic, clinical and microbiological data. Serotype results are available since 2004. RESULTS Over those 20 years, 112 invasive pneumococcal disease cases were identified, with a median age of 15 months (1 month - 15 years). The median number of cases /year was 4, the highest between 2001 - 2002 (8/year) and 2007 - 2012 (7 - 11/year). The identification occurred mostly in blood culture (72), cerebrospinal fluid (24), pleural fluid (11) an others (5). The most frequent diagnoses were pneumonia (38%), occult bacteraemia (34%) and meningitis (21%). Over the period under review, there was an increase of pneumonia and slight increase of OB, with meningitis cases remaining relatively unchanged. DISCUSSION In the last two decades, there was no reduction in the number of cases of invasive pneumococcal disease. There was an increase in isolates from pneumonia and occult bacteraemia that might be due to the introduction of molecular biological methods for Streptococcus pneumoniae detection. Vaccine serotypes were predominant. CONCLUSION This retrospective analysis before universal vaccination will contribute to evaluate the impact of vaccination in the Portuguese pediatric population.