Streptococcus pneumoniae has natural competence for acquiring exogenous DNA, thus showing rapid evolution in response to clinical interventions as antibiotic consumption and vaccination. The World Health Organization reported in 2007 that inclusion of the 7-valent pneumococcal conjugate vaccine (PCV7) in national immunization programs should be seen as a priority, and it also encouraged countries to conduct appropriate surveillances for monitoring the impact of vaccination.1 Because serotypes may act as quasi species, and because the impact of clinical interventions on natural serotype evolution and nonsusceptibility is serotype-dependent,2 analyses of effects over time should be aimed at evolution of incidence of invasive pneumococcal disease (IPD) globally and by serotype, age group and clinical presentation.3–5
PCV7 was available in Spain in October 2001, but it was available only in the private market for healthy children. The vaccine use increased from 2002 onwards, with reported vaccine coverage in 2006 being less than 50%, assuming complete vaccination schedules.6 Selective PCV7 vaccination has resulted in a low impact in the incidence of IPD in children in Spain7,8 and has been linked to an increase in the prevalence of non-PCV7 serotypes.2 In the Madrid autonomous community (approximately 6 million inhabitants), PCV7 was included in the systematic childhood vaccination calendar in November 2006 for universal vaccination of children younger than 24 months, with doses at 2, 4, 6 and 18 months of age. In 2009, PCV7 coverage in Madrid with this universal vaccination was 94.47%9 in the target population (children younger than 24 months). In June 2010, the 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in the childhood vaccination calendar, replacing PCV7, with a catch-up program for children aged 18 to 24 months.
The aim of this study was to describe incidence of invasive pneumococcal disease in children requiring hospitalization in Madrid over a 4-year period (2007 to 2011), with analysis by age, serotype and clinical presentation.
PATIENTS AND METHODS
A prospective, active, clinical surveillance study of laboratory-confirmed IPD was performed in all hospitals with pediatric departments located in the autonomous region of Madrid, Spain, over the course of 4 years (2007 to 2011). Analyses were performed by epidemiological year as follows: first period, May 2007 to April 2008; second period, May 2008 to April 2009; third period, May 2009 to April 2010; and fourth period, May 2010 to April 2011. Because new hospitals have been constructed in this region in past years, the first period included 20 centers, the second period included 22 centers, the third included 23 centers, and the fourth period included a total of 26 centers. The study population consisted of all children (younger than age 15 years) with IPD requiring hospitalization. Only those children with laboratory-confirmed IPD by culture or polymerase chain reaction (PCR) were finally considered in the study. IPD was defined as presence of Streptococcus pneumoniae in normal sterile fluids such as blood, pleural fluid and cerebrospinal fluid. Basic demographic data (age, gender, PCV7 vaccination status) and clinical presentation were recorded. Local Research Ethics Committees approved the study protocol.
Samples were sent to the clinical microbiology laboratory at each center for microbiological culture or PCR detection. All pneumococcal isolates were sent to a single reference laboratory (Microbiology Department of the Universitary Clinic Hospital in Madrid) for serotyping by Quellung reaction. Pleural and cerebrospinal fluids not yielding positive culture also were sent to the reference laboratory for PCR detection of pneumolysin (ply) and autolysin (lyt) genes.10,11 Pneumococci confirmed by PCR were serotyped by real-time PCR assay using the LightCycler SYBR green format followed by melting-curve analysis, as previously described,12 detecting serotypes 1, 3, 4, 5, 6, 7F, 14, 19A and 19F. Molecular typing of the isolates was performed by using the DiversiLab Microbial Typing System (bioMérieux, Marcy l’Etoile, France) that is based on repetitive sequence-based PCR, which amplifies the regions between the noncoding repetitive sequences in bacterial genomes.13 All DNA samples were amplified using the DiversiLab Enterococcus kit for DNA fingerprinting. Detection of repetitive sequence-based PCR products was implemented using the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA), which uses microfluidics chip-based DNA electrophoresis. Analysis was performed with the DiversiLab software version 3.3. The resulting DNA fingerprint patterns profiles were compared by using the Pearson correlation index. A correlation of ≥95 was applied as the cut-off. Multilocus sequence typing was performed as previously described.14 The assignment of alleles at each locus was performed using the software available at the pneumococcal multilocus sequence typing Web site.15 Susceptibility to penicillin, amoxicillin, cefotaxime and erythromycin was determined by microdilution according to Clinical and Laboratory Standards Institute recommendations.16 Current Clinical and Laboratory Standards Institute breakpoints17 were considered for susceptibility interpretation. Isolates with intermediate or high-level resistance were defined as nonsusceptible.
Incidence rates were calculated as the number of cases per 100,000 inhabitants using population data for children in Madrid (for each study period, age-specific and for total children 15 years of age or younger) from Instituto Nacional de Estadistica.18 Trends of incidence rates over time were explored using the lineal regression command (SPSS V14; SPSS, Chicago, IL), with time periods as independent variables and rates as dependent variables. Comparisons of incidence rates between the different periods were performed with the EPIDAT version 3.1.
From May 2007 to April 2011, a total of 614 pediatric laboratory-confirmed IPD cases were identified: 209 parapneumonic pneumococcal empyema (PPE), 191 bacteremic pneumonia, 75 primary bacteremia, 72 meningitis, 38 IPDs secondary to otic foci (SOF) and 29 other IPDs (septic arthritis from different sites, peritonitis, cerebral abscess, periorbital cellulitis and others). A total of 87 PPEs were diagnosed based on positive cultures (determining serotype by Quellung) and 122 were diagnosed based on positive PCR results (with negative cultures and serotyping performed by real-time PCR). All but one (PCR-positive) of the 72 meningitis cases were diagnosed based on positive culture.
Incidence Rates by Clinical Presentations
Table 1 shows incidence rates of clinical presentations by study period. Globally, incidence rates of IPD decreased from 17.15 in 2007-2008 and 2008-2009 to 11.34 in 2010-2011, with significant (P<0.01) differences between the incidence rate in the last period and previous periods. When analyzing clinical presentations in separate, incidence rates of bacteremic pneumonia (R2=0.966; β=1.132; P=0.017) and meningitis (R2=0.898, β=0.505; P=0.052) showed decreasing linear trends over time, with significant (P<0.05) differences in incidence rates in 2007-2008 and 2008-2009 compared with 2010-2011 for both presentations. No linear trends were found for primary bacteremia, SOF, or PPE, although the incidence rate of PPE in 2010-2011 was significantly lower than in 2009- 2010 (4.14 vs. 6.73; P=0.019).
In the target population for vaccination (younger than 24 months), incidence rates of global IPD were lower in 2010-2011 (24.92) than in previous periods (38.13 in 2007-2008, 50.19 in 2008-2009 and 43.55 in 2009-2010); differences were significant (P<0.005) for 2010-2011 compared with 2008-2009 and compared with 2009-2010.
Incidence Rates by Age Groups
Table 2 shows number of IPDs and incidence rates by age groups (younger than 12 months, 12 months to younger than 24 months, 24 months to younger than 5 years and 5 years of age or older). No linear trends over time were found. In the group of children younger than 12 months, incidence rates in 2008-2009 significantly decreased compared with those in 2010-2011 (57.69 vs. 30.41; P= 0.016). In children aged 12 months to younger than 24 months, significant reductions in incidence rates were found in 2010-2011 compared with those in 2008-2009 (19.52 vs. 42.52; P=0.017) and compared with those in 2009-2010 (19.52 vs. 49.75; P=0.002).
Incidence Rates by Serotype
Pooling isolates from all study periods, serotypes included in PCV13 were the most prevalent (78.7%), specifically serotypes 1 (28.8%), 19A (22.5%), 5 (9.9%), 7F (7.5%), 3 (5.2%) and grouped PCV7 serotypes (3.6%). In respiratory infections (bacteremic pneumonia and PPE), the most prevalent serotype was serotype 1 (accounting for 42.4% cases of bacteremic pneumonia and 41.6% cases of PPE), whereas serotype 19A was the most prevalent in SOF (68.4% cases), meningitis (27.8% cases) and primary bacteremia (36.0% cases). Each non-PCV13 serotype represented ≤2% isolates individually. Nontypeable isolates by the PCR method used represented 6.0% cases. Table 3 shows per-period incidence rates of IPDs caused by grouped PCV7 serotypes, grouped PCV13 serotypes and, individually, by extra added serotypes in PCV13 (serotypes 1, 3, 5, 6A, 7F and 19A). Over time, linear decreasing trends were found for incidence rates of IPDs caused by PCV7 serotypes (R2=0.891; β=0.256; P=0.056) and serotype 7F (R2=0.904; β=0.268; P=0.049). Although the linear trend (R2=0.831; β=-1.030) was not significant (P=0.088), incidence rates of IPDs by serotype 5 in 2009-2010 (0.40) or in 2010-2011 (0.59) were significantly (P<0.020) lower than in 2007-2008 (3.58) or in 2008-2009 (1.75). Incidence of IPDs caused by serotype 19A in 2007-2008 increased in 2009-2010 (2.42 vs. 4.82; P=0.008), with a marked reduction in 2010-2011, when the incidence rate was significantly lower than in 2009-2010 (2.76 vs. 4.82; P=0.024). Incidence rates of IPDs caused by PCV13 serotypes were significantly (P≤0.004) lower in 2010-2011 (8.78) than they were in previous periods (incidence rates ranging from 13.16 to 13.76).
Table 4 shows incidence rates of IPDs caused by serotypes included in PCV13 by clinical presentations and study periods. Incidence rates of bacteremic pneumonia (R2=0.918; β=0.833; P=0.042) decreased over time, following a linear trend, with significant differences between the first and the last periods (5.47 vs. 2.76; P=0.004). The lower incidence rate in 2010-2011 was related to reductions in the incidence of bacteremic pneumonia caused by serotypes 1 (1.58 vs. 2.11) and 19A (0.39 vs. 1.00) when compared with incidences in 2009-2010. A decreasing linear trend also was found for incidence rates of meningitis over time (R2=0.957; β=0.295; P=0.022). The incidence rate of PPE by PCV13 serotypes was significantly lower in 2010-2011 than in 2009-2010 (3.55 vs. 5.52; P=0.049), again related to reductions in the incidence of PPE by serotypes 1 (2.07 vs. 2.81) and 19A (0.69 vs. 1.51). No reductions in incidence rates of SOF by PCV13 serotypes were found because of the lack of reduction in the incidence rate of SOF caused by serotype 19A.
Clonal Patterns and Susceptibility
Genotyping of culture-isolated strains showed a monoclonal pattern for serotype 1 (pattern 6) as follows: 91% of 66 isolates in 2007 to 2009; 81.8% of 44 isolates in 2009-2010; and 100% of 31 isolates in 2010-2011. Serotype 19A presented mainly a biclonal pattern, with most isolates belonging to pattern 20 (ST276; 44.8% of 58 isolates in 2007-2009, 30.0% of 40 isolates in 2009-2010 and 26.9% of 26 isolates in 2010-2011) or pattern 23 (ST320; 36.2% of 58 isolates in 2007-2009, 50.0% of 40 isolates in 2009-2010 and 57.7% of 26 isolates in 2010-2011).
No differences in rates of nonsusceptibility to penicillin (16.8% in 2007 to 2009, 14.2% in 2009-2010 and 16.4% in 2010-2011), cefotaxime (12.2% in 2007 to 2009, 13.3% in 2009-2010 and 9.4% in 2010-2011) and erythromycin (27.9% in 2007-2009, 32.3% in 2009-2010 and 31.9% in 2010-2011) were found during the study periods.
The HERACLES studies, a yearly clinical surveillance of all pediatric laboratory-confirmed IPD requiring hospitalization in the autonomous region of Madrid started in May 2007, 6 months after introduction of PCV7 to the pediatric calendar for universal vaccination. In the first 1-year period, the incidence rate of global IPD was 17.15. This rate was not reduced in the two following periods despite the incidence of IPD by PCV7 serotypes showing a significant decreasing trend. The lack of reduction of global IPD attributable to the increase in non-PCV7 IPD also has been reported in other countries mainly attributable to, among others, serotypes 1, 7F and 19A.19,20 Serotype and resistance patterns over time can be described, but determining the responsibility of clinical interventions or of natural secular trends in these variations is not simple. In this sense, it was reported that serotypes 3, 4 and 8 showed a constant trend during the three decades (1979 to 2007) in Spain,2 despite their high antimicrobial susceptibility and their inclusion (serotype 4) or not (serotypes 3 and 8) in the PCV7 formulation. In the present study, serotype 5, previously linked to community outbreaks,9 showed high frequency in the first period but significantly lower incidence rates in the third period, despite this serotype is not included in PCV7. Another interesting case in our study is serotype 7F. Incidence rates of IPD by serotype 7F (not included in PCV7) showed a significant continuous decrease from 2007 on, thus reinforcing the idea of the difficulties in determining the responsibility of the different therapeutic and preventive measures or of natural secular trends in serotype variations. In this sense, using data from different countries, it also has been hypothesized that increases in serotype 1 are not directly related to PCV721 but rather to cyclical changes.2,9 In the present study, IPD by serotype 1 showed the highest rates in the four periods without significant differences between periods or a significant trend, although incidence of serotype 1 IPD increased from 2007 to 2010. In this period, incidence of IPDs by serotype 19A showed a statistically significant increase, and this serotype has been deemed responsible for the lack of reduction of global IPD after PCV7 introduction in other countries.19,22–24
Although there was no reduction in the incidence of global IPD during the period from 2007 to 2010, a marked decrease was found in 2010-2011. This reduction was attributable to reductions in the incidence of global IPD in populations targeted by PCV13 (children aged younger than 12 months and those 12 months to younger than 24 months) in 2010-2011 (compared with previous periods). In 2010-2011, there has been a marked increase in reductions in incidence rates of bacteremic pneumonia and meningitis (with the most frequent presentations in children younger than 12 months)25 observed after PCV7 introduction by reducing incidences rates of cases caused by serotypes 1 and 19A. The reduction of cases by serotypes 1 and 19A in 2010-2011 also is responsible for the reversion in this period of the increase in the incidence rate of PPE over the course of the period from 2007 to 2010. These facts were concomitant with the switch from PCV7 to PCV13 in the vaccination calendar. A previous study reported PCV13 effectiveness for serotype 19A, reversing the previous increase in cumulative cases after the introduction of PCV7.22 The reduction in the incidence rate of IPD by serotype 19A in the last period in Madrid is important because penicillin-resistant 19A had become the most worrisome serotype because of the emergence in our country of two multidrug-resistant clones (sequence types ST276 and ST320).3,26,27 The increase in the incidence rate of IPD by serotype 19A has occurred in our country in areas where PCV7 was introduced in the vaccination calendar, such as Madrid, and in areas where conjugate vaccines were not included (only available for private market), such as Barcelona, with recent reports of continuous increase in IPD in children younger than 5 years of age mainly attributable to serotypes 1, 3 and 19A.28 In 2009, in Barcelona, 71.7% IPDs were caused by PCV13 serotypes.29 Serotype 19A also expanded in countries with low PCV7 coverage, such as Taiwan,30 or before PCV7 use, mainly attributable to ST320, such as in South Korea.31 The reduction in IPD by 19A in Madrid found in the present study is promising, although it was related to a reduction in ST276, but not in ST320, that increased among 19A isolates in 2010-2011. Nevertheless, it has been postulated that 2 to 3 years of universal vaccinations are necessary before disease rates stabilize and higher decreases compared with baseline are shown.22,23,32 Protection against serotype 19A is critical in our country, and decision analytic models evaluating health and economic outcomes have shown that universal PCV13 vaccination would be a cost-effective intervention.33,34
Nevertheless, one important fact that might have influenced our results is that only four time periods were studied, and only one period was after the switch from PCV7 to PCV13. However, the high number of IPD cases (all hospitalized children in our region included) and the fact that conjugate vaccines are included in the childhood vaccination calendar, thus providing universal vaccination, are strengths of the study. Further HERACLES study periods analyzing additional years after PCV13 introduction will increase our knowledge of epidemiological effects derived from the switch from PCV7 to PCV13.
In conclusion, early data after the switch from PCV7 to PCV13 suggest that introduction of PCV13 in vaccination calendars is especially important in areas showing high incidence rates of IPD by serotype 19A and its associated multidrug-resistant clones. Continuous laboratory-based IPD surveillance is essential to monitor the impact of conjugate vaccines and the switch of vaccines on pediatric calendars and must explore not only effects on global incidence of IPD over time but also sequence type, serotype and clinical presentation levels to establish future strategies for preventive measures.
Members of the HERACLES study group: A. Delgado-Iribarren, M. Bueno (H. Universitario Fundación de Alcorcón); A. Alhambra, M.T. García (H. Sanchinarro); A. Rivas-Castillo, D. Monclús (H. San Rafael); A. Gutiérrez (H. La Paz); B. Hernández (H. Niño Jesús); M. Zafra, J. Jaqueti (H. de Fuenlabrada); C. Betriu, E. Culebras, F. González, I. Rodriguez-Avial (H. Clínico San Carlos); C. Calvo, I. Wilhelmi (H. Severo Ochoa); C. Serrano, T. Montoya (H. de la Zarzuela); E. Bouza, E. Cercenado (H. Gregorio Marañón and CIBER of Respiratory Diseases, CIBERES); M.A. Meseguer (H. Ramón y Cajal); F. Sanz, S. Negreira, I. Sánchez (H. 12 de Octubre); I. Gadea, M. Bernacer (Fundación Jiménez Díaz); I. Romero, A. Alhambra (H. de Torrelodones); J.L. Gómez-Garcés, M.A. Roa (H. de Móstoles); J.T. Ramos, M. Sánchez (H. de Getafe); J.C. Sanz (LRSP); M.L. García-Picazo, S. Gallego (H. de El Escorial); M. Marco, V. Buezas (H. Gómez Ulla); M.J. Cilleruelo, M.I. Sánchez (H. Puerta de Hierro); M. Beltrán, M. Penín (H. Príncipe de Asturias); S. Salso, V. Soler (H. de Montepríncipe); A. Rodriguez (H. del Sureste); J. Clemente (H. del Henares); J.L. Ruibal (H. Santa Cristina); B. Agundez (H. Infanta Leonor); A. Cañete (H. Infanta Sofia); C. Garcia-Vao (H. del Tajo); A. Sanchez (BR. Salud); A. García-Sampedro, C. Balseiro, M. del Amo (Pfizer S.A.); J.J. Granizo, M.J. Giménez, L. Aguilar (Univ. Complutense, Madrid, Spain).
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incidence rates; invasive pneumococcal disease; 13-valent pneumococcal conjugate