Impact of molecular methods in the analysis of the invasiveness of Streptococcus pneumoniae

[eng] This doctoral thesis proves the necessity of applying molecular techniques in direct sample for a more accurate study of the Invasive Disease Potential (IDP) of Streptococcus pneumoniae and for its epidemiological surveillance. These methods allow a better detection and serotype identification of the pneumococcal population in both Invasive Pneumococcal Disease (IPD) and nasopharyngeal carriers. However, to better understand the behavior of the different serotypes a complete analysis of the clonal types of pneumococci (invasive and carriers) should be also performed. In the first two studies the IDP of pneumococcal serotypes found in our pediatric population was estimated. In the first study, traditional methods based only in culture were used for both the detection of Streptococcus pneumoniae and the identification of the capsular type; while in the second study, the same estimation was performed but this time adding molecular techniques in direct sample for both the detection and serotyping. When comparing the results of both studies, the addition of molecular techniques in direct sample doubled the number of pneumococci identified causing IPD, and in the nasopharynx of healthy children. The great increase in the pneumococcus detection revealed a different distribution of the serotypes circulating in the population of the area, when compared with the data that had been obtained only by culture, which was translated in a huge impact in the IDP of these serotypes. In the carrier’s population an important proportion in the increase of serotypes that were able to be identified in carriers was due to the detection of multiple colonies in one sample, with up to four different serotypes distinguished in the same sample. The analysis of the carriers’ population also revealed a rate of co-colonization of 26.4% in the children population of our area. The better estimation of the ranking order of the most common serotypes in IPD and in the nasopharynx also allowed a more accurate analysis of the effects of PCV7 in the study time period. The results from the third study provided additional information about the invasive disease potential of serotypes causing IPD in our area due to the clonal study of invasive strains. In addition, it provided information about the evolution of serotypes causing disease after the introduction of PCV13 in 2010, in both children and adults. Although the most common serotypes found in the study were still PCV13 serotypes (with a special concern for the increase of the ST156 serotype 14), probably due to the low vaccine coverage in our area, a significant decrease was observed. In contrast, a significant increase in the proportion of non-PCV13 serotypes was revealed. Worth mentioning, is the increase experienced by serotype 12F (in high association with ST989) and, when analyzed by age groups, the increase of serotype 24F in children less than 2 years old (the major clone being the multi drug resistant ST230). But most importantly was the data obtained in the serotypes analysis of their clonal composition. Despite the capsule is the most important factor in virulence, strains expressing the same serotype but belonging to different clonal types has been found to present different invasiveness. Certain clonal types may present some characteristics that are likely to be advantageous for invasiveness, like antibiotic resistance. The study of the genetic population of the serotypes offers a more complete knowledge of the pneumococcal invasiveness, explains temporal trends and differences in geographical areas, and provides useful information for the prediction of which serotypes could be replacing the ones included in the vaccines. For a better interpretation of the pneumococcal behavior, the analysis of the clonal types associated with a serotype should be performed along with the capsular identification.[cat] Aquesta tesis doctoral demostra la necessitat d’aplicar tècniques moleculars en mostra directa per a poder realitzar un estudi més acurat del potencial invasiu de Streptococcus pneumoniae, i per a la seva vigilància epidemiològica. Aquests mètodes permeten una millor detecció i un millor serotipatge de la població pneumocòccica, tant en la malaltia pneumocòccica invasiva (MPI) com en els portadors nasofaringis. Tot i això, per realment entendre millor el comportament dels diferents serotips també s’hauria de realitzar un anàlisi complet dels tipus clonals que presenten els pneumococs (tant en malaltia com en portadors). Als dos primers estudis es va calcular el potencial invasiu dels serotips pneumocòccics trobats en la població pediàtrica de Catalunya (Espanya). Al primer estudi es van utilitzar mètodes tradicionals basats en el cultiu tant per la detecció de Streptococcus pneumoniae com per la identificació del tipus capsular; mentre que en el segon estudi, es va realitzar el mateix anàlisi però en aquest cas afegint les tècniques moleculars en mostra directa tant per la detecció com pel serotipatge. Al comparar els resultats d’ambdós estudis, l’addició de les tècniques moleculars en mostra directa va duplicar el número de pneumococs identificats causant MPI i presents a la nasofaringe de nens sans. Aquesta millora en la detecció del pneumococ també es va traduir en un gran impacte en el càlcul del potencial invasiu d’aquest serotips. En la població de portadors la capacitat de detectar múltiples colònies en una mateixa mostra va tenir un paper rellevant en l’increment de serotips que es van poder identificar en el segon estudi. Els resultats del tercer estudi van aportar informació addicional sobre el potencial invasiu dels serotips causant MPI a la nostra àrea gràcies a l’estudi clonal de les soques invasives. A més, van aportar informació sobre l’evolució dels serotips causant malaltia després de la introducció de la PCV13 al 2010, tant en nens com en adults. L’anàlisi dels tipus clonals sent expressats pels serotips pneumocòccics podria explicar perquè alguns tipus capsulars que es troben normalment colonitzant asimptomàticament han experimentat un canvi en la seva invasivitat convertint-se en les principals causes de la MPI

Prevalence and clonal distribution of pcpA, psrP and Pilus-1 among pediatric isolates of Streptococcus pneumoniae

Streptococcus pneumoniae is the leading cause of vaccine-preventable deaths globally. The objective of this study was to determine the distribution and clonal type variability of three potential vaccine antigens: Pneumococcal serine-rich repeat protein (PsrP), Pilus-1, and Pneumococcal choline binding protein A (PcpA) among pneumococcal isolates from children with invasive pneumococcal disease and healthy nasopharyngeal carriers. We studied by Real-Time PCR a total of 458 invasive pneumococcal isolates and 89 nasopharyngeal pneumococcal isolates among children (total = 547 strains) collected in Barcelona, Spain, from January 2004 to July 2010. pcpA, psrP and pilus-1 were detected in 92.8%, 51.7% and 14.4% of invasive isolates and in 92.1%, 48.3% and 18% of carrier isolates, respectively. Within individual serotypes the prevalence of psrP and pilus-1 was highly dependent on the clonal type. pcpA was highly prevalent in all strains with the exception of those belonging to serotype 3 (33.3% in serotype 3 isolates vs. 95.1% in other serotypes; P<.001). psrP was significantly more frequent in those serotypes that are less apt to be detected in carriage than in disease; 58.7% vs. 39.1% P<.001. Antibiotic resistance was associated with the presence of pilus-1 and showed a negative correlation with psrP. These results indicate that PcpA, and subsequently Psrp and Pilus-1 together might be good candidates to be used in a next-generation of multivalent pneumococcal protein vaccine

Serotypes and Clonal Diversity of Streptococcus pneumoniae Causing Invasive Disease in the Era of PCV13 in Catalonia, Spain

The aim of this study was to study the serotypes and clonal diversity of pneumococci causing invasive pneumococcal disease in Catalonia, Spain, in the era of 13-valent pneumococcal conjugate vaccine (PCV13). In our region, this vaccine is only available in the private market and it is estimated a PCV13 vaccine coverage around 55% in children. A total of 1551 pneumococcal invasive isolates received between 2010 and 2013 in the Molecular Microbiology Department at Hospital Sant Joan de Déu, Barcelona, were included. Fifty-two serotypes and 249 clonal types defined by MLST were identified. The most common serotypes were serotype 1 (n = 182; 11.7%), 3 (n = 145; 9.3%), 19A (n = 137; 8.8%) and 7F (n = 122; 7.9%). Serotype 14 was the third most frequent serotype in children < 2 years (15 of 159 isolates). PCV7 serotypes maintained their proportion along the period of study, 16.6% in 2010 to 13.4% in 2013, whereas there was a significant proportional decrease in PCV13 serotypes, 65.3% in 2010 to 48.9% in 2013 (p<0.01). This decrease was mainly attributable to serotypes 19A and 7F. Serotype 12F achieved the third position in 2013 (n = 22, 6.4%). The most frequent clonal types found were ST306 (n = 154, 9.9%), ST191 (n = 111, 7.2%), ST989 (n = 85, 5.5%) and ST180 (n = 80, 5.2%). Despite their decrease, PCV13 serotypes continue to be a major cause of disease in Spain. These results emphasize the need for complete PCV13 vaccination

Prevalence and clonal distribution of pcpA, psrP and Pilus-1 among pediatric isolates of Streptococcus pneumoniae

Streptococcus pneumoniae is the leading cause of vaccine-preventable deaths globally. The objective of this study was to determine the distribution and clonal type variability of three potential vaccine antigens: Pneumococcal serine-rich repeat protein (PsrP), Pilus-1, and Pneumococcal choline binding protein A (PcpA) among pneumococcal isolates from children with invasive pneumococcal disease and healthy nasopharyngeal carriers. We studied by Real-Time PCR a total of 458 invasive pneumococcal isolates and 89 nasopharyngeal pneumococcal isolates among children (total = 547 strains) collected in Barcelona, Spain, from January 2004 to July 2010. pcpA, psrP and pilus-1 were detected in 92.8%, 51.7% and 14.4% of invasive isolates and in 92.1%, 48.3% and 18% of carrier isolates, respectively. Within individual serotypes the prevalence of psrP and pilus-1 was highly dependent on the clonal type. pcpA was highly prevalent in all strains with the exception of those belonging to serotype 3 (33.3% in serotype 3 isolates vs. 95.1% in other serotypes; P<.001). psrP was significantly more frequent in those serotypes that are less apt to be detected in carriage than in disease; 58.7% vs. 39.1% P<.001. Antibiotic resistance was associated with the presence of pilus-1 and showed a negative correlation with psrP. These results indicate that PcpA, and subsequently Psrp and Pilus-1 together might be good candidates to be used in a next-generation of multivalent pneumococcal protein vaccine

Prevalence and clonal distribution of pcpA, psrP and Pilus-1 among pediatric isolates of Streptococcus pneumoniae

Streptococcus pneumoniae is the leading cause of vaccine-preventable deaths globally. The objective of this study was to determine the distribution and clonal type variability of three potential vaccine antigens: Pneumococcal serine-rich repeat protein (PsrP), Pilus-1, and Pneumococcal choline binding protein A (PcpA) among pneumococcal isolates from children with invasive pneumococcal disease and healthy nasopharyngeal carriers. We studied by Real-Time PCR a total of 458 invasive pneumococcal isolates and 89 nasopharyngeal pneumococcal isolates among children (total = 547 strains) collected in Barcelona, Spain, from January 2004 to July 2010. pcpA, psrP and pilus-1 were detected in 92.8%, 51.7% and 14.4% of invasive isolates and in 92.1%, 48.3% and 18% of carrier isolates, respectively. Within individual serotypes the prevalence of psrP and pilus-1 was highly dependent on the clonal type. pcpA was highly prevalent in all strains with the exception of those belonging to serotype 3 (33.3% in serotype 3 isolates vs. 95.1% in other serotypes; P<.001). psrP was significantly more frequent in those serotypes that are less apt to be detected in carriage than in disease; 58.7% vs. 39.1% P<.001. Antibiotic resistance was associated with the presence of pilus-1 and showed a negative correlation with psrP. These results indicate that PcpA, and subsequently Psrp and Pilus-1 together might be good candidates to be used in a next-generation of multivalent pneumococcal protein vaccine

Serotypes and Clonal Diversity of Streptococcus pneumoniae Causing Invasive Disease in the Era of PCV13 in Catalonia, Spain

The aim of this study was to study the serotypes and clonal diversity of pneumococci causing invasive pneumococcal disease in Catalonia, Spain, in the era of 13-valent pneumococcal conjugate vaccine (PCV13). In our region, this vaccine is only available in the private market and it is estimated a PCV13 vaccine coverage around 55% in children. A total of 1551 pneumococcal invasive isolates received between 2010 and 2013 in the Molecular Microbiology Department at Hospital Sant Joan de Déu, Barcelona, were included. Fifty-two serotypes and 249 clonal types defined by MLST were identified. The most common serotypes were serotype 1 (n = 182; 11.7%), 3 (n = 145; 9.3%), 19A (n = 137; 8.8%) and 7F (n = 122; 7.9%). Serotype 14 was the third most frequent serotype in children < 2 years (15 of 159 isolates). PCV7 serotypes maintained their proportion along the period of study, 16.6% in 2010 to 13.4% in 2013, whereas there was a significant proportional decrease in PCV13 serotypes, 65.3% in 2010 to 48.9% in 2013 (p<0.01). This decrease was mainly attributable to serotypes 19A and 7F. Serotype 12F achieved the third position in 2013 (n = 22, 6.4%). The most frequent clonal types found were ST306 (n = 154, 9.9%), ST191 (n = 111, 7.2%), ST989 (n = 85, 5.5%) and ST180 (n = 80, 5.2%). Despite their decrease, PCV13 serotypes continue to be a major cause of disease in Spain. These results emphasize the need for complete PCV13 vaccination