Sandra I. Aguiar

Changes in Streptococcus pneumoniae serotypes causing invasive disease with non-universal vaccination coverage of the seven-valent conjugate vaccine

The pneumococcal seven-valent conjugate vaccine (PCV7) has been administered in Portugal since late 2001 through the private sector. To evaluate the impact of PCV7 use, the serotypes and antimicrobial susceptibility of pneumococci causing invasive disease in Portugal during 2003-2005 were determined and compared with available data for the period 1999-2002. Changes in serotype distribution compatible with the introduction of PCV7 were shown for children <or=5 years of age from 2003 onwards and for adults from 2004 onwards. PCV7 use with coverage of 43% of children with four doses in the 2004 birth cohort, although substantially below universal coverage, seems to have contributed to greatly reducing the proportion of invasive infections due to vaccine serotypes 4, 6B, 14 and 23F. Similarly, significant indirect effects on the serotype distribution of pneumococci causing infections in adults were noted, with reductions in the proportion of invasive infections caused by serotypes 4, 5 and 14. These changes were accompanied by an increase in the proportion of two non-vaccine serotypes: 19A isolates in all age groups and 7F isolates in adults. Whereas serotypes 6B, 14 and 19A were associated with multidrug resistance, isolates expressing serotypes 4 and 7F were fully susceptible for the most part. There were no changes in the proportion of resistant isolates within each serotype and, in spite of the changes in serotype prevalence, there was not an overall reduction in the proportion of infections caused by resistant pneumococci.

The presence of the pilus locus is a clonal property among pneumococcal invasive isolates

BackgroundPili were recently recognized in Streptococcus pneumoniae and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. However, pili were found to be encoded by the rlrA islet that was not universally distributed in the species. We examined the distribution of the pilus islet, using the presence of the rlrA gene as a marker for the locus, among a collection of invasive isolates recovered in Portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles (PFGE) and multilocus sequence types.ResultsOnly a minority of the isolates were positive for the presence of the rlrA gene (27%). There was a high correspondence between the serotype and the presence or absence of rlrA (Wallace coefficient, W = 0.778). In particular, there was an association between the presence of rlrA and the vaccine serotypes 4, 6B, 9V and 14 whereas the gene was significantly absent from other serotypes, namely 1, 7F, 8, 12B and 23F, a group that included a vaccine serotype (23F) and serotype 1 associated with enhanced invasiveness. Even within serotypes, there was variation in the presence of the pilus islet between PFGE clones and a higher Wallace coefficient (W = 0.939) indicates that carriage of the islet is a clonal property of pneumococci. Analysis of rlrA negative isolates revealed heterogeneity in the genomic region downstream of the rfl gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages.ConclusionThe pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. Carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be ongoing.

Serotypes 1, 7F and 19A became the leading causes of pediatric invasive pneumococcal infections in Portugal after 7 years of heptavalent conjugate vaccine use

We characterized 353 isolates responsible for pediatric invasive pneumococcal infections (IPD) in Portugal between 2006 and 2008. Serotypes included in the seven-valent conjugate vaccine (PCV7) accounted for 17% of IPD. Serotypes 1, 7F and 19A were the most frequent causes of IPD and the later consolidated as the most frequent serotype among erythromycin and penicillin non-susceptible isolates. Serotype 1 was associated with older children and empyemas, while serotype 19A was associated with IPD in younger (<2 years) children. The higher valency vaccines PCV10 and PCV13 have a potentially superior coverage, 55% and 83% respectively, but non-vaccine serotypes are emerging as important causes of IPD. A decline of resistance with patient age was noted. Comparing with previous data from Portugal, this study showed a continued decline of PCV7 serotypes and that overall resistance has stabilized following the initial decline of the first post-PCV7 years.

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.

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.

Serotype changes in adult invasive pneumococcal infections in Portugal did not reduce the high fraction of potentially vaccine preventable infections.

We determined the serotype and antimicrobial susceptibility of 1100 isolates responsible for adult invasive pneumococcal infections (IPD) in Portugal between 2006 and 2008. Serotypes 3 (13%), 1 (12%), 7F (11%), 19A (10%) and 14 (7%) were the most frequent causes of IPD and the two later serotypes accounted for the majority of erythromycin and penicillin nonsusceptible isolates. Serotype 1 was associated with younger adults whereas serotype 3 was associated with older adults. Despite the availability of the 23-valent polysaccharide vaccine (PPV23) in Portugal since 1996, the proportion of PPV23 preventable IPD remained stable and above 80%. Comparing with previous data from Portugal, we showed a continued decline of the serotypes included in the 7-valent conjugate vaccine (PCV7) in adult IPD and a rise of serotypes included in the 13-valent conjugate vaccine, increasing its potential coverage of adult IPD to 70% in 2008. Penicillin non-susceptibility remained stable (17%) whereas erythromycin resistance (18%) has continued to rise in the post-PCV7 years.

Decreasing incidence and changes in serotype distribution of invasive pneumococcal disease in persons aged under 18 years since introduction of 10-valent and 13-valent conjugate vaccines in Portugal, July 2008 to June 2012

The 10-valent pneumococcal conjugate vaccine (PCV10) became available in Portugal in mid-2009 and the 13-valent vaccine (PCV13) in early 2010. The incidence of invasive pneumococcal disease (IPD) in patients aged under 18 years decreased from 8.19 cases per 100,000 in 2008–09 to 4.52/100,000 in 2011–12. However, IPD incidence due to the serotypes included in the 7-valent conjugate vaccine (PCV7) in children aged under two years remained constant. This fall resulted from significant decreases in the number of cases due to: (i) the additional serotypes included in PCV10 and PCV13 (1, 5, 7F; from 37.6% to 20.6%), particularly serotype 1 in older children; and (ii) the additional serotypes included in PCV13 (3, 6A, 19A; from 31.6% to 16.2%), particularly serotype 19A in younger children. The decrease in serotype 19A before vaccination indicates that it was not triggered by PCV13 administration. The decrease of serotype 1 in all groups, concomitant with the introduction of PCV10, is also unlikely to have been triggered by vaccination, although PCVs may have intensified and supported these trends. PCV13 serotypes remain major causes of IPD, accounting for 63.2% of isolates recovered in Portugal in 2011–12, highlighting the potential role of enhanced vaccination in reducing paediatric IPD in Portugal.

The majority of adult pneumococcal invasive infections in Portugal are still potentially vaccine preventable in spite of significant declines of serotypes 1 and 5.

In Portugal, pneumococcal conjugate vaccines have been administered to children outside of the national immunization plan since 2001. We determined the serotype and antimicrobial susceptibility of 1265 isolates responsible for adult invasive pneumococcal infections (IPD) between 2009 and 2011 and compared the results with previously published data from 1999 to 2008. Serotypes 3 (12.6%), 7F (10.0%), 19A (9.1%), 14 (8.4%), 1 (6.9%) and 8 (6.2%) were the most frequent and together accounted for 53.2% of adult IPD. Serotypes 1 and 5 declined significantly while serotype 34, not included in any vaccine, increased. Taken together, the serotypes included in the 13-valent conjugate vaccine (PCV13) peaked among adult IPD isolates in 2008 (70.2%) and declined since then reaching 53.5% in 2011. The decline in the serotypes included in the 23-valent polysaccharide vaccine since 2007 was also significant but much more modest with 79.2% of the isolates causing IPD in 2011 expressing these serotypes. Since the changes in serotypes causing IPD in adults coincided with the 10-valent and PCV13 introduction in children, it is unlikely that vaccination triggered these changes although it may have accelerated them. The proportion of IPD caused by serotypes included in the 7-valent conjugate vaccine remained stable (19.0%). Both penicillin non-susceptibility and erythromycin resistance increased in the study period, with serotypes 14 and 19A accounting for the majority of resistant isolates.

miR-363-5p regulates endothelial cell properties and their communication with hematopoietic precursor cells

Recent findings have shown that the blood vessels of different organs exert an active role in regulating organ function. In detail, the endothelium that aligns the vasculature of most organs is fundamental in maintaining organ homeostasis and in promoting organ recovery following injury. Mechanistically, endothelial cells (EC) of tissues such as the liver, lungs or the bone marrow (BM) have been shown to produce “angiocrine” factors that promote organ recovery and restore normal organ function. Controlled production of angiocrine factors following organ injury is therefore essential to promote organ regeneration and to restore organ function. However, the molecular mechanisms underlying the coordinated production and function of such “angiocrine” factors are largely undisclosed and were the subject of the present study. In detail, we identified for the first time a microRNA (miRNA) expressed by BM EC that regulates the expression of angiocrine genes involved in BM recovery following irradiation. Using a microarray-based approach, we identified several miRNA expressed by irradiated BMEC. After validating the variations in miRNA expression by semi-quantitative PCR, we chose to study further the ones showing consistent variations between experiments, and those predicted to regulate (directly or indirectly) angiogenic and angiocrine factors. Of the mi-RNA that were chosen, miR-363-5p (previously termed miR-363*) was subsequently shown to modulate the expression of numerous EC-specific genes including some angiocrine factors. By luciferase reporter assays, miR-363-5p is shown to regulate the expression of angiocrine factors tissue inhibitor of metalloproteinases-1 (Timp-1) and thrombospondin 3 (THBS3) at post-transcriptional level. Moreover, miR-363-5p reduction using anti-miR is shown to affect EC angiogenic properties (such as the response to angiogenic factors stimulation) and the interaction between EC and hematopoietic precursors (particularly relevant in a BM setting). miR-363-5p reduction resulted in a significant decrease in EC tube formation on matrigel, but increased hematopoietic precursor cells adhesion onto EC, a mechanism that is shown to involve kit ligand-mediated cell adhesion. Taken together, we have identified a miRNA induced by irradiation that regulates angiocrine factors expression on EC and as such modulates EC properties. Further studies on the importance of miR-363-5p on normal BM function and in disease are warranted.

Use of the 13-valent conjugate vaccine has the potential to eliminate pilus carrying isolates as causes of invasive pneumococcal disease

We examined the distribution of the two pilus island loci (PI-1 and PI-2) in all pediatric invasive pneumococcal disease (IPD) isolates recovered in Portugal over the ten year period 1999-2008 (n=575). While 199 isolates (35%) harbored PI-2, only 98 isolates (17%) harbored PI-1. A decline of isolates carrying PI-1 was observed in the post 7-valent conjugate vaccine (PCV7) period since most PI-1 carrying isolates expressed PCV7 serotypes. On the other hand, the PI-2 increase in the post-PCV7 period was associated mostly with serotypes 1 and 7F. The overall proportion of piliated isolates declined in the period following PCV7 but quickly recovered to pre-PCV7 levels (50%). Only serotypes 19F and 19A contained isolates carrying simultaneously PI-1 and PI-2 and the proportion of isolates carrying both pilus islands remained stable and low. All PI-1 clades were represented in our collection and were associated with multiple serotypes. Over 99% of pilus island carrying isolates belonged to the 13-valent conjugate vaccine serotypes.