Sónia Nunes

Changes in pneumococcal serotypes and antibiotypes carried by vaccinated and unvaccinated day-care centre attendees in Portugal, a country with widespread use of the seven-valent pneumococcal conjugate vaccine

The seven-valent pneumococcal conjugate vaccine (PCV7) has been available in Portugal since June 2001, but is not included in the National Vaccination Plan. Its impact on colonization is unknown. A point-prevalence study to evaluate PCV7 usage was carried out in 2006 among day-care centre attendees from the Lisbon area. Pneumococcal carriage rates, serotypes, and antibiotypes were determined and compared with results from a similar study conducted in 2001 before vaccine approval. In 2001 and 2006, 717 and 571 children, respectively, were enrolled. In 2006, 45.9% of the participants were appropriately vaccinated and 11.5% were incompletely vaccinated. Carriage of pneumococci remained stable (64.9% in 2001; 68.7% in 2006). Vaccine types (VT) decreased from 53.1% of all pneumococci to 11.2% (p <0.001). Serotype replacement was observed among vaccinated and unvaccinated children. Non-vaccine types (NVT) 1, 6C, 7F, 15A, 16F, 21, 23A, 29, and non-typeable (NT) strains increased significantly; serotype 19A increased, but not significantly. Rates of resistance to penicillin, erythromycin, clindamycin and tetracycline remained stable (p >0.05) due to significant increases in intermediate resistance to penicillin (from 5.5% to 17.8%), erythromycin (from 9.2% to 21.8%), clindamycin (from 6.4% to 19.3%) and tetracycline (from 8.3% to 15.8%) among NVT. Whereas in 2001 resistance among NVT was mostly associated with serotype 19A and NT strains, in 2006 resistance was also found among serotypes 6C, 15A, 24F and 33F. In conclusion, dramatic shifts in serotypes of colonizing pneumococci were observed among vaccinated and unvaccinated children. Rates of antibiotic resistance remained unchanged due to a balance between reduction in VT and an increase in antimicrobial-resistant NVT.

High Rates of Transmission of and Colonization by Streptococcus pneumoniae and Haemophilus influenzae within a Day Care Center Revealed in a Longitudinal Study

ABSTRACT Day care centers (DCCs) are unique settings where young children are at increased risk for colonization by pneumococci and Haemophilus influenzae. Although point prevalence studies in DCCs are frequent, only a few longitudinal studies on the dynamics of colonization have been published. We conducted a 1-year longitudinal study with 11 sampling periods on nasopharyngeal carriage of pneumococci and H. influenzae among 47 children who attended a single DCC. All isolates were antibiotyped and genotyped by pulsed-field gel electrophoresis. Pneumococci were also serotyped. Of the 414 samples obtained, 61.4% contained pneumococci, and 87% contained H. influenzae. Only 8.3% of the samples were negative for both species. Twenty-one pneumococcal clones and 47 H. influenzae clones were identified. Introduction of clones occurred during all year. Ninety-eight percent and 96% of all pneumococcal and H. influenzae isolates, respectively, belonged to clones shared by more than one child. Children were sequentially colonized with up to six pneumococcal clones (mean, 3.6) and five serotypes and nine H. influenzae clones (mean, 7.1). Clones with increased capacity for transmission and/or prolonged colonization were identified in both species. These two fitness properties appeared to be independent. In conclusion, among DCC attendees, a high rate of acquisition and turnover of strains was observed, and all children were overwhelmingly colonized by clones shared with others. DCCs are units where permanent introduction of new clones occurs, and attendees, as a whole, provide a pool of hosts where the fittest clones find privileged opportunities to persist and expand.

Assessment of Band-Based Similarity Coefficients for Automatic Type and Subtype Classification of Microbial Isolates Analyzed by Pulsed-Field Gel Electrophoresis

ABSTRACT Pulsed-field gel electrophoresis (PFGE) has been the typing method of choice for strain identification in epidemiological studies of several bacterial species of medical importance. The usual procedure for the comparison of strains and assignment of strain type and subtype relies on visual assessment of band difference number, followed by an incremental assignment to the group hosting the most similar type previously seen. Band-based similarity coefficients, such as the Dice or the Jaccard coefficient, are then used for dendrogram construction, which provides a quantitative assessment of strain similarity. PFGE type assignment is based on the definition of a threshold linkage value, below which strains are assigned to the same group. This is typically performed empirically by inspecting the hierarchical cluster analysis dendrogram containing the strains of interest. This approach has the problem that the threshold value selected is dependent on the linkage method used for dendrogram construction. Furthermore, the use of a linkage method skews the original similarity values between strains. In this paper we assess the goodness of classification of several band-based similarity coefficients by comparing it with the band difference number for PFGE type and subtype classification using receiver operating characteristic curves. The procedure described was applied to a collection of PFGE results for 1,798 isolates of Streptococcus pneumoniae, which documented 96 types and 396 subtypes. The band-based similarity coefficients were found to perform equally well for type classification, but with different proportions of false-positive and false-negative classifications in their minimal false discovery rate when they were used for subtype classification.

Analysis of Invasiveness of Pneumococcal Serotypes and Clones Circulating in Portugal before Widespread Use of Conjugate Vaccines Reveals Heterogeneous Behavior of Clones Expressing the Same Serotype

ABSTRACT To estimate the invasive disease potential of serotypes and clones circulating in Portugal before extensive use of the seven-valent pneumococcal conjugate vaccine, we analyzed 475 invasive isolates recovered from children and adults and 769 carriage isolates recovered from children between 2001 and 2003. Isolates were serotyped and genotyped by pulsed-field gel electrophoresis, and a selection of isolates were also characterized by multilocus sequence typing. We found that the diversities of serotypes and genotypes of pneumococci responsible for invasive infections and carriage were identical and that most carried clones could also be detected as causes of invasive disease. Their ability to do so, however, varied substantially. Serotypes 1, 3, 4, 5, 7F, 8, 9N, 9L, 12B, 14, 18C, and 20 were found to have an enhanced propensity to cause invasive disease, while serotypes 6A, 6B, 11A, 15B/C, 16F, 19F, 23F, 34, 35F, and 37 were associated with carriage. In addition, significant differences in invasive disease potential between clones sharing the same serotype were found among several serotypes, namely, 3, 6A, 6B, 11A, 14, 19A, 19F, 22F, 23F, 34, and NT. This heterogeneous behavior of the clones was found irrespective of the serotypes overall invasive disease potential. Our results highlight the importance of the genetic background when analyzing the invasive disease potential of certain serotypes and provide an important baseline for its monitoring following conjugate vaccine use. Continuous surveillance should be maintained, and current research should focus on uncovering the genetic determinants that contribute to the heterogeneity of invasive disease potential of clones sharing the same serotype.

Trends in Drug Resistance, Serotypes, and Molecular Types of Streptococcus pneumoniae Colonizing Preschool-Age Children Attending Day Care Centers in Lisbon, Portugal: a Summary of 4 Years of Annual Surveillance

ABSTRACT Of the nasopharyngeal cultures recovered from 942 day care center (DCC) attendees in Lisbon, Portugal, 591 (62%) yielded Streptococcus pneumoniae during a surveillance performed in February and March of 1999. Forty percent of the isolates were resistant to one or more antimicrobial agents. In particular, 2% were penicillin resistant and 20% had intermediate penicillin resistance. Multidrug resistance to macrolides, lincosamides, and tetracycline was the most frequent antibiotype (17% of all isolates). Serotyping and molecular typing by pulsed-field gel electrophoresis were performed for 202 out of 237 drug-resistant pneumococci (DRPn). The most frequent serotypes were 6B (26%), 14 (22%), 19F (16%), 23F (10%), and nontypeable (12%). The majority (67%) of the DRPn strains were representatives of nine international clones included in the Pneumococcal Molecular Epidemiology Network; eight of them had been detected in previous studies. Fourteen novel clones were identified, corresponding to 26% of the DRPn strains. The remaining 7% of the strains were local clones detected in our previous studies. Comparison with studies conducted since 1996 in Portuguese DCCs identified several trends: (i) the rate of DRPn frequency has fluctuated between 40 and 50%; (ii) the serotypes most frequently recovered have remained the same; (iii) nontypeable strains appear to be increasing in frequency; and (iv) a clone of serotype 33F emerged in 1999. Together, our observations highlight that the nasopharynxes of children in DCCs are a melting pot of successful DRPn clones that are important to study and monitor if we aim to gain a better understanding on the epidemiology of this pathogen.

Temporal trends and molecular epidemiology of recently described serotype 6C of Streptococcus pneumoniae.

ABSTRACT We studied the epidemiology of the recently described serotype 6C of Streptococcus pneumoniae among a collection of carriage isolates recovered between 1996 and 2007 in Portugal. Of 4,064 isolates, 106 (2.6%) were of serotype 6C, 17.9% of which were multidrug resistant. The strains were genetically diverse.

Denmark14-230 Clone as an Increasing Cause of Pneumococcal Infection in Portugal within a Background of Diverse Serotype 19A Lineages

ABSTRACT Pneumococci of serotype 19A are increasingly found to be the cause of infection in various geographic regions. We have characterized the serotype 19A isolates (n = 288) found among pneumococci responsible for infections (n = 1,925) and pneumococci recovered from asymptomatic carriers (n = 1,973) in Portugal between 2001 and 2006. We show that despite the existence of serotype 19A clones that have a greater potential to cause invasive disease or an enhanced colonization capacity, the lineage that is increasing as a cause of infection in Portugal is a multiresistant clone that is competent at both. The expanding Denmark14-230 clone found in Portugal is disseminated in other Mediterranean countries, where it is also increasingly responsible for invasive infections in both children and adults. The lineages driving the rise of serotype 19A infections in Asia and the United States (sequence type 320 [ST320] and ST199) are either absent or account for only a small proportion of isolates in Portugal. These data highlight the importance of locally circulating clones with the ability to compete in the nasopharyngeal niche in the emergence of the serotype 19A lineages which are an increasing cause of infection in various geographic regions.

Multiple-Locus Variable Number Tandem Repeat Analysis for Streptococcus pneumoniae: Comparison with PFGE and MLST

In the era of pneumococcal conjugate vaccines, surveillance of pneumococcal disease and carriage remains of utmost importance as important changes may occur in the population. To monitor these alterations reliable genotyping methods are required for large-scale applications. We introduced a high throughput multiple-locus variable number tandem repeat analysis (MLVA) and compared this method with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLVA described here is based on 8 BOX loci that are amplified in two multiplex PCRs. The labeled PCR products are sized on an automated DNA sequencer to accurately determine the number of tandem repeats. The composite of the number of repeats of the BOX loci makes up a numerical profile that is used for identification and clustering. In this study, MLVA was performed on 263 carriage isolates that were previously characterized by MLST and PFGE. MLVA, MLST and PFGE (cut-off of 80%) yielded 164, 120, and 87 types, respectively. The three typing methods had Simpsons diversity indices of 98.5% or higher. Congruence between MLST and MLVA was high. The Wallace of MLVA to MLST was 0.874, meaning that if two strains had the same MLVA type they had an 88% chance of having the same MLST type. Furthermore, the Wallace of MLVA to clonal complex of MLST was even higher: 99.5%. For some isolates belonging to a single MLST clonal complex although displaying different serotypes, MLVA was more discriminatory, generating groups according to serotype or serogroup. Overall, MLVA is a promising genotyping method that is easy to perform and a relatively cheap alternative to PFGE and MLST. In the companion paper published simultaneously in this issue we applied the MLVA to assess the pneumococcal population structure of isolates causing invasive disease in the Netherlands before the introduction of the 7-valent conjugate vaccine.

Streptococcus pneumoniae nasopharyngeal carriage in children attending day-care centers in the central region of Portugal, in the era of 7-valent pneumococcal conjugate vaccine.

The 7-valent pneumococcal conjugate vaccine became available in Portugal in 2001. Although not included in the national immunization program, vaccination coverage is high (over 60%). We studied for the first time the rates of nasopharyngeal (NP) carriage of pneumococci, antibiotic resistance patterns and serotypes among children attending day-care centers (DCCs) in Coimbra, a city in the Central Region of Portugal. Between January and February 2007, a cross-sectional study was conducted among children aged 6 months to 6 years attending eight DCCs. NP swabs were obtained from 507 children: 76.7% had received at least one dose of 7-valent pneumococcal conjugate vaccine and 64.3% were age-appropriately vaccinated. The global pneumococcal carriage rate was 61.3%. Colonization proportions varied with age and number of children attending each DCC. Serotyping revealed that 20.7% of the pneumococci were vaccine types (VTs), 70.8% were non-VTs, and 8.5% were nontypeable. Serotype 19F was the second most frequent serotype being detected in 10.5% of the samples. While global NP carriage was not associated with vaccination status, non-VTs were predominant among vaccinated children, who had significantly lower prevalence of VT. Of all isolates, 15.7% had penicillin minimum inhibitory concentrations that ranged between 0.12 and 2 microg/ml. The proportion of resistant strains was significantly higher among VT and unvaccinated children. In conclusion, the rates of vaccination and prevalence of pneumococcal NP were high. Rates of antimicrobial resistance were similar to those found in studies conducted in Oeiras and Lisbon. This study is a platform for future surveillance activities.

Clonal Evolution Leading to Maintenance of Antibiotic Resistance Rates among Colonizing Pneumococci in the PCV7 Era in Portugal

ABSTRACT The introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) in Portugal led to extensive serotype replacement among carriers of pneumococci, with a marked decrease of PCV7 types. Although antimicrobial resistance was traditionally associated with PCV7 types, no significant changes in the rates of nonsusceptibility to penicillin, resistance to macrolides, or multidrug resistance were observed. This study aimed to investigate the mechanisms leading to maintenance of antimicrobial resistance, despite marked serotype replacement. We compared, through molecular typing, 252 antibiotic-resistant pneumococci recovered from young carriers in 2006 and 2007 (era of high PCV7 uptake) with collections of isolates from 2002 and 2003 (n=374; low-PCV7-uptake era) and 1996 to 2001 (n=805; pre-PCV7 era). We observed that the group of clones that has accounted for antimicrobial resistance since 1996 is essentially the same as the one identified in the PCV7 era. The relative proportions of such clones have, however, evolved substantially overtime. Notably, widespread use of PCV7 led to an expansion of two Pneumococcal Molecular Epidemiology Network (PMEN) clones expressing non-PCV7 capsular variants of the original strains: Sweden15AST63 (serotypes 15A and 19A) and Denmark14ST230 (serotypes 19A and 24F). These variants were already in circulation in the pre-PCV7 era, although they have now become increasingly abundant. Emergence of novel clones and de novo acquisition of resistance contributed little to the observed scenario. No evidence of capsular switch events occurring after PCV7 introduction was found. In the era of PCVs, antimicrobial resistance remains a problem among the carried pneumococci. Continuous surveillance is warranted to evaluate serotype and clonal shifts leading to maintenance of antimicrobial resistance.