Impact of pneumococcal conjugate vaccine (PCV7 and PCV13) on pneumococcal invasive diseases in Italian children and insight into evolution of pneumococcal population structure.

BACKGROUND: The use of PCV7 for children immunization was gradually implemented in the Italian regions starting from 2006 and was replaced by PCV13 in 2010-2011. In this study we aimed to assess the PCV impact on invasive pneumococcal diseases (IPD) incidence, serotype distribution and antibiotic resistance in Italian children under 5years old. METHODS: All IPD cases in children from 5 Italian regions (Emilia-Romagna, Lombardia, A. P. Bolzano, A. P. Trento, and Piemonte) reported through the nationwide surveillance system during 2008-2014 were included in this study. Pneumococcal isolates were subjected to serotyping, antibiotic susceptibility testing, and clonal analysis according to standard methods. RESULTS: During the study period overall IPD incidence decreased from 7.8 cases/100,000 inhabitants in 2008 to 3.0 cases/100,000 in 2014 (61% decrease, P<0.001). In particular, from 2008 to 2014, PCV7-type IPD decreased from 2.92 to 0.13 cases/100,000 inhabitants (95% decrease, P<0.001) while PCV13-non-PCV7 type IPD decreased from 3.2 to 0.89 cases/100,000 inhabitants (72% decrease, P=0.008). Conversely, non-vaccine serotype (NVS) IPD increased overtime, becoming more common than PCV13 serotype IPD in 2013-2014. Emergent NVS 24F and 12F were the most prevalent in 2014. Antibiotic resistance testing revealed an overall increasing trend in penicillin resistance, from 14% in 2008 to 23% in 2014. Erythromycin resistance showed a downward trend, from 38% in 2008 to 27% in 2014. While in 2008 PCV13 serotypes were the major responsible for antibiotic resistance, during the following years antimicrobial resistance due to NVS increased, mainly as a result of expansion of pre-existing clones. CONCLUSIONS: Both PCVs led to a substantial decrease in vaccine-related IPD incidence in the children population. However NVS-related IPD increased, becoming the most prevalent in the last two-years period. Continuous surveillance is an essential tool to monitor evolution of pneumococcal population causing IPD in children.

Invasive pneumococcal disease in children and adults in seven Italian regions after the introduction of the conjugate vaccine, 2008-2014.

OBJECTIVE: To describe the trend of invasive pneumococcal disease in the years 2008-2014; to verify the impact of the conjugate vaccine and monitor the occurrence of serotype replacement. DESIGN: Prospective observational study based on data from the national surveillance for invasive bacterial diseases coordinated by the Istituto superiore di sanità. SETTING AND PARTICIPANTS: Seven Italian regions (A.P. Bolzano, A.P. Trento, Emilia-Romagna, Friuli-Venezia Giulia, Lombardia, Piemonte, Veneto), accounting for 43% of the national population. MAIN OUTCOME MEASURES: Number of cases and incidence of invasive pneumococcal diseases: global, stratified by age groups and by serotypes included or not in the PCV13. RESULTS: In 2008-2014, in the 0-4 age group IPD incidence for all serotypes decreased from 7.1 to 2.9/100,000; incidence for vaccine serotypes (VT) decreased from 5.5 to 1.1/100,000, while incidence for non-vaccine serotypes (NVT) increased from 1.6 to 2.0/100,000 (2.5 in 2013). In the >64 age group, IPD incidence increased from 5.3 to 7.5/100,000; VT incidence decreased from 3.9 to 3.2 (4.9 in 2010 and 4.3 in 2013), whereas NVT incidence increased from 1.4 to 4.4/100,000. CONCLUSION: Use of the conjugate vaccine has reduced the number of cases of IPD by VT in children; the increase in IPD by NVT, above all in older age groups, suggests a serotype replacement.

Characterization of Streptococcus pneumoniae clones from paediatric patients with cystic fibrosis.

The role of Streptococcus pneumoniae in cystic fibrosis (CF) is poorly understood. The pneumococcal population has changed over time after the introduction of the heptavalent conjugate vaccine (PCV7) and, more recently, the 13-valent conjugate vaccine (PCV13). Although serotypes and clones causing invasive pneumococcal disease or colonizing healthy children have been extensively analysed, little is known so far on the serotypes and clones of pneumococci in CF patients. The aim of this work was to investigate serotypes, antibiotic susceptibilities, genotypes and biofilm production of CF pneumococcal isolates. Overall, 44 S. pneumoniae strains collected from 32 paediatric CF patients from January 2010 to May 2012 in a large Italian CF Centre were tested for antimicrobial susceptibility testing by Etest, serotyped by the Quellung reaction and genotyped by a combination of different molecular typing methods, including pbp gene restriction profiling, pspA restriction profiling and sequencing, PFGE and multilocus sequence typing. Biofilm production by pneumococcal strains was also assessed. Penicillin non-susceptibility was 16 %. High resistance rates (>56 %) were observed for erythromycin, clindamycin and tetracycline. The most frequent serotype recovered was serotype 3 (31.8 %). The coverage of PCV7 and PCV13 was 6.8 and 47.7 %, respectively. More than 80 % of CF strains belonged to Pneumococcal Molecular Epidemiology Network (PMEN) reference clones, the most common being Netherlands(3)-ST180 (28.2 %), and Greece(21)-30/ST193 (15.4 %). All strains produced biofilm in vitro, although with large variability in biofilm formation efficiency. No correlation was found between biofilm levels and serotype, clone or antibiotic resistance. The high isolation rate of antibiotic-resistant serotype 3 pneumococci from CF patients suggests that PCV13 could increase protection from pneumococcal colonization and infection.

Pneumococcal carriage in young children one year after introduction of the 13-valent conjugate vaccine in Italy.

BACKGROUND: In mid 2010, the 7-valent pneumococcal conjugate vaccine (PCV7) was replaced by the 13-valent conjugate vaccine (PCV13) for childhood immunization in Italy. Our objective in this study was to obtain a snapshot of pneumococcal carriage frequency, colonizing serotypes, and antibiotic resistance in healthy children in two Italian cities one year after PCV13 was introduced. METHODS: Nasopharyngeal swabs were obtained from 571 children aged 0-5 years from November 2011-April 2012. Pneumococcal isolates were serotyped and tested for antimicrobial susceptibility. Penicillin and/or erythromycin non-susceptible isolates were analyzed by Multi Locus Sequence Typing (MLST). RESULTS: Among the children examined, 81.2% had received at least one dose of PCV7 or PCV13 and 74.9% had completed the recommended vaccination schedule for their age. Among the latter, 57.3% of children had received PCV7, 27.1% PCV13, and 15.6% a combination of the two vaccines. The overall carriage rate was 32.9%, with children aged 6-35 months the most prone to pneumococcal colonization (6-23 months OR: 3.75; 95% CI: 2.19-6.43 and 24-35 months OR: 3.15, 95%CI: 2.36-4.22). A total of 184 pneumococcal isolates were serotyped and divided into PCV7 (5.4%), PCV13 (18.0%), and non-PCV13 (82.0%) serotypes. Serotypes 6C, 24F, and 19A were the most prevalent (10.3%, 8.6%, and 8.1%, respectively). The proportion of penicillin non-susceptible (MIC >0.6 mg/L) isolates was 30.9%, while 42.3% were erythromycin resistant. Non-PCV13 serotypes accounted for 75.4% and 70.8% of the penicillin and erythromycin non-susceptible isolates, respectively. CONCLUSIONS: Our results revealed low rates of PCV7 and PCV13 serotypes in Italian children, potentially due to the effects of vaccination. As the use of PCV13 continues, its potential impact on vaccine serotypes such as 19A and cross-reactive serotypes such as 6C will be assessed, with this study providing a baseline for further analysis of surveillance isolates.

Increase of pneumococcal serotype 19A in Italy is due to expansion of the piliated clone ST416/CC199.

The emergence of Streptococcus pneumoniae serotype 19A, following use of the heptavalent pneumococcal conjugate vaccine (PCV7), has been favoured by multiple antibiotic resistance of this serotype and by other unknown factors. The aim of this study was to examine 19A isolates from invasive pneumococcal diseases (IPD) obtained before and after PCV7 implementation to ascertain which characteristics, including the presence of pili, might have favoured the emergence of this serotype in Italy. All S. pneumoniae isolates from IPD collected at the Italian National Institute of Health in the years 2001-2003 and 2006-2009 were serotyped. The 19A isolates were submitted to antimicrobial susceptibility testing by Etest and were genotyped by a combination of pulsed field gel electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST). The presence of the pilus islets PI-1 and PI-2 was detected by PCR assays targeting a marker gene in each islet. The proportion of 19A isolates from IPD significantly increased from 4 % in 2001-2003 to 12 % in 2006-2009. This was largely due to the expansion of a clone characterized by sequence type (ST) 416, clonal complex (CC) 199, already present in Italy before PCV7 implementation. This clone included isolates susceptible to penicillin and containing PI-1 genes. Other CCs contributed to the emergence of serotype 19A: CC63 and CC193, already present in 2001-2003, and new-emerging CCs or clones such as CC230, CC320 and ST5204, that include drug-resistant and/or pilus-positive isolates. The expansion of serotype 19A in Italy might have been favoured not only by antibiotic resistance, but also by other bacterial factors such as the presence of pili.

Serotype and clonal evolution of penicillin-nonsusceptible invasive Streptococcus pneumoniae in the 7-valent pneumococcal conjugate vaccine era in Italy.

The percentage of invasive penicillin-nonsusceptible pneumococci (PNSSP) isolated in Italy in the seven-valent pneumococcal conjugate vaccine (PCV7) era moderately increased in comparison to the pre-PCV7 era. Increase of nonvaccine serotypes was observed among PNSSP. The most frequent PNSSP clones were the same as those identified in the pre-PCV7 era, although they were present in different proportions. Clonal expansion, emergence of new clones, and acquisition of penicillin resistance by established clones contributed to the maintenance of penicillin resistance.

Antibiotic-resistant invasive pneumococcal clones in Italy.

A total of 105 multiple-antibiotic-resistant invasive pneumococcal isolates recovered in Italy from 2001 to 2003 were genetically characterized. Of these, 40 were penicillin-nonsusceptible (PNSSP) and 65 were penicillin-susceptible (PSSP) Streptococcus pneumoniae strains. Among the PNSSP isolates, 8 and 11 different restriction profiles were obtained for the pbp2b and pbp2x genes, respectively. Clonal groups were established on the basis of analysis of both pulsed-field gel electrophoresis (PFGE) types and multilocus sequence typing (MLST). Several international clones, such as Spain(23F)-1/ST81, Spain(6B)-2/ST90, Spain(9V)-3/ST156, and Sweden(15A)-25/ST63 [corrected] were identified among the PNSSP isolates. Other, smaller clones, such as the minor Spanish 19F clone/ST88 and Denmark(14)-32/ST230, were also found. Among the PSSP isolates, clones related to England(14)-9/ST9, Greece(6B)-22/ST273, and Portugal(19F)-21/ST177 were found. In addition, two large clones comprised nonvaccine serotypes. One, comprising serotype 3 isolates, corresponded to the clone Netherlands(3)-31/ST180; the other, comprising serotype 15B/C isolates, ST474, was not related to any previously described clone. Two small clusters related to the newly described clones Greece(21)-30/ST193 and Netherlands(15B)-37/ST199 included isolates with unrelated PFGE profiles. An unusual finding was the inability to obtain the MLST allelic profile for an isolate of serotype 19A, belonging to the Sweden(15A)-25/ST63 [corrected] clone, due to a large deletion of the xpt gene. Capsular switching was observed among both PNSSP and PSSP isolates and involved also serotypes not included in the 7-valent pneumococcal conjugate vaccine (PCV7), such as serotypes 15B/C and 19A. Since antibiotic-resistant nonvaccine serotype clones are present in Italy, continuous monitoring of pneumococcal epidemiology should be carried out in the PCV7 era.

Genetic diversity of invasive strains of Haemophilus influenzae type b before and after introduction of the conjugate vaccine in Italy.

We determined the genotypes of 95 invasive Haemophilus influenzae type b (Hib) strains collected before and after introduction of widespread Hib vaccination in Italy. No substantial change in genetic diversity was highlighted by pulsed-field gel electrophoresis. However, an upward temporal trend in proportion of strains possessing multiple copies of the capsulation b locus was detected (P = .03).

Evolution of erythromycin resistance in Streptococcus pneumoniae in Italy.

OBJECTIVES: To evaluate erythromycin resistance in recent invasive isolates of Streptococcus pneumoniae in Italy, to study the phenotypic and genotypic characteristics of the isolates, and to compare data with those obtained in a previous survey. METHODS: Invasive pneumococcal isolates were obtained from 56 laboratories throughout the country, in 2001-2003. Isolates were serotyped and antimicrobial susceptibilities determined by Sensititre panels and Etest. A new PCR was performed to detect erythromycin resistance genes. Typing methods for selected erythromycin-resistant isolates included PFGE and multilocus sequence typing (MLST). RESULTS: One hundred and fifty-five isolates out of 444 (34.9%) were resistant to erythromycin: 95 isolates (21.4%) carried erm(B), 56 (12.6%) carried mef(A) and three carried both genes. One isolate, carrying neither erm(B) nor mef(A), showed a point mutation in domain V of the 23S rRNA genes. The mef(A)-positive isolates carried subtype mef(A) (47 isolates), subtype mef(E) (nine isolates), and both subtype mef(E) and erm(B) (three isolates). All subtype mef(A) strains, except two, belonged to serotype 14, appeared to be clonally related by PFGE and related to the England14-9 clone by MLST. The two isolates belonging to other serotypes showed different genetic backgrounds. CONCLUSIONS: Erythromycin resistance in S. pneumoniae has increased in the last few years in Italy. erm(B) is still the predominant resistance determinant; however, the increase in erythromycin resistance (34.9% versus 28.8% of the previous years) is mainly due to an increase in the proportion of isolates carrying the efflux pump mef(A), whereas the proportion of isolates carrying erm(B) has not changed.

Inferring the potential success of pneumococcal vaccination in Italy: serotypes and antibiotic resistance of Streptococcus pneumoniae isolates from invasive diseases.

To evaluate the potential impact of antipneumococcal vaccination in Italy, Streptococcus pneumoniae isolates from invasive disease were collected from 65 laboratories in the years 1997-2000. Of the 503 isolates examined, 15% were from children <5 years and 34% from adults > or = 65 years. The most frequent serogroups were, in ranking order, 14, 19, 6, and 23. Overall, 93.8% of the isolates belonged to serogroups enclosed in the 23-valent polysaccharide vaccine. Among children isolates, serotypes 14, 6B, and 23F comprised 60% of the isolates; overall, 72% of the isolates belonged to serotypes included in the heptavalent conjugate vaccine. Penicillin nonsusceptible isolates (10%) belonged to a limited number of serogroups, being more common in serogroups 19 and 9 and in the nonvaccine serogroups 24 and 35. Erythromycin-resistant isolates (29%) belonged to several serogroups, more frequently to serogroups 14, 6, and 19. Both vaccines are potentially able to prevent the majority of resistant infections in the respective age groups in Italy.

Phenotypic and genotypic characterization of two penicillin-susceptible serotype 6B Streptococcus pneumoniae clones circulating in Italy.

Twenty-nine penicillin-susceptible serotype 6B strains isolated from patients with invasive diseases and from healthy carriers were examined by different genotyping methods. Ten groups were identified on the basis of the pulsed-field gel electrophoresis (PFGE) profiles, and two of these contained multiple isolates and were analyzed further. PFGE group 1 comprised 12 isolates, the majority of which had a multiresistant phenotype (resistance to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole), corresponding to that of a clone previously described in the Mediterranean area and related to penicillin-resistant clone Spain(6B)-2. The pbp2b, pbp2x, dhf, and pspA genes of the isolates had identical restriction profiles; and the partial sequence of pspA was identical to that of clone Spain(6B)-2. In all isolates the resistance determinants erm(B) and tet(M) were inserted in a Tn1545-like element; 11 isolates carried cat as part of the integrated plasmid pC194. Multilocus sequence typing (MLST) performed with two isolates confirmed that their profiles corresponded to that of the Mediterranean clone. PFGE group 2 comprised nine strains, of which the majority showed no antibiotic resistance. Their pspA profiles were different, and the partial sequences obtained for two representative isolates indicated the presence of PspA proteins of different clades. The MLST profile of one strain was identical to that of a serotype 6B strain from the United Kingdom, while two other isolates were novel one-allele variants. This clone appears to be related (five of seven identical alleles) to two other internationally disseminated clones, Hungary(19A)-6 and Poland(23F)-16, both of which are penicillin resistant. The presence of antibiotic-susceptible isolates of this clone suggests that traits other than antibiotic resistance can make a clone successful.

Genotypes of invasive pneumococcal isolates recently recovered from Italian patients.

We examined 73 recent invasive pneumococcal isolates within selected areas of Italy for genotypic variability. Thirty-three genomic macrorestriction types were found, three of which represented multiple serotypes. Restriction fragment patterns of pbp2b, pbp2x, and pspA were conserved within the majority of isolates that shared macrorestriction types. Of the nine macrorestriction types found among the 22 penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) isolates, seven comprised isolates with allelic profiles showing five to seven allelic matches to profiles in the multilocus sequence typing database (www.mlst.net); however, three of the seven profiles represented serotypes not previously associated with these clonal clusters. Two PNSP macrorestriction types represented new clones with unique allelic profiles. Allelic profiles obtained from isolates of 3 of the 25 macrorestriction types found among the 51 penicillin-susceptible S. pneumoniae (PSSP) isolates were closely related to previously described profiles. One PSSP isolate was a novel type 24F isolate related to the multiresistant clone France(9V)-3. This work reports new PNSP strains and new serotype-clone associations.