Changes in Capsule and Drug Resistance of Pneumococci after Introduction of PCV7, Japan, 2010–2013

Drastic changes threaten overall effectiveness of this vaccine.

We aimed to clarify changes in serotypes and genotypes mediating β-lactam and macrolide resistance in Streptococcus pneumoniae isolates from Japanese children who had invasive pneumococcal disease (IPD) after the 7-valent pneumococcal conjugate vaccine (PCV7) was introduced into Japan; 341 participating general hospitals conducted IPD surveillance during April 2010-March 2013. A total of 300 pneumococcal isolates were collected in 2010, 146 in 2011, and 156 in 2012. The proportion of vaccine serotypes in infectious isolates decreased from 73.3% to 54.8% to 14.7% during the 3 years. Among vaccine serotype strains, genotypic penicillin-resistant S. pneumoniae strains also declined each year. Among nonvaccine serotype strains, 19A, 15A, 15B, 15C, and 24 increased in 2012. Increases were noted especially in genotypic penicillin-resistant S. pneumoniae isolates of serotypes 15A and 35B, as well as macrolide resistance mediated by the erm(B) gene in 15A, 15B, 15C, and 24.
I nvasive pneumococcal disease (IPD), such as meningitis, sepsis, and empyema, substantially contributes to illness and death in children (1,2). After increasing numbers of cases caused by penicillin (PEN)-resistant Streptococcus pneumoniae (PRSP) emerged and rapidly spread worldwide during the 1990s (3,4), the need for a vaccine effective in infants became clear. In the United States, a 7-valent pneumococcal conjugate vaccine (PCV7) was introduced in 2000 and made available for routine use in all children 2-23 months of age and in children 24-59 months of age at risk for pneumococcal infection (5). Subsequent surveillance studies demonstrated a marked decrease in prevalence of pneumococcal infection caused by vaccine serotypes, including PRSP (6)(7)(8). In particular, PCV7 appears to have decreased incidence of meningitis caused by vaccine serotypes (9), and cases caused by non-PCV7 serotype strains, such as PRSP with serotype 19A, have increased in the United States (8,10,11). Such changes suggest that nonvaccine serotypes are replacing vaccine serotypes in some countries (12)(13)(14).
A next-generation 13-valent pneumococcal conjugate vaccine (PCV13) was licensed for use in the United States in 2010 (15). PCV13 has been approved in 128 countries, and children in 83 countries have undergone routine PCV13 vaccination. Recently, Richter et al. reported that an increase of type 19A was halted by introduction of PCV13, whereas serotype 35B increased; coverage provided by PCV13 was effective in only 41.4% of children <5 years of age in 2010 and 2011 (16). Furthermore, Kaplan et al. reported a slight increase in serotype 33F (17).
In Japan, PRSP has increased rapidly as a cause of respiratory tract infections, acute otitis media, and IPD in children since the late 1990s (18,19). PCV7 received final approval in October 2009 and has been used clinically in infants on a voluntary basis since February 2010. Since November 2010, PCV7 use has been encouraged for children <5 years of age throughout Japan by an official program, the Provisional Special Fund for the Urgent Promotion of Vaccination. As a result, estimated rates of PCV7 vaccination for such children were <10% in 2010, 50%-60% in 2011, and 80%-90% in 2012.
PCV7 was incorporated into the routine vaccination schedule for children in Japan beginning in April 2013; before then, however, its coverage rate against IPD had decreased rapidly from 71.8% in 2006 to 51.6% in 2011 (20).
Most recently, PCV13 was approved by the government in June 2013, later replacing PCV7 as a routine vaccination in November 2013. The purpose of our study was to clarify changes during April 2010-March 2013 of serotypes and genotypes mediating β-lactam and macrolide resistance in S. pneumoniae isolates from children <18 years of age who had IPD before and after PCV7 introduction.

Patients and Pneumococcal Strains
All study participants were children <18 years of age who had IPD. Pneumococcal isolates from normally sterile clinical samples, such as blood, cerebrospinal fluid, pleural effusions, and joint fluid, were collected and examined.
Medical institutions that had a microbiology laboratory and a pediatric department with hospitalization facilities were permitted to participate actively in surveillance. An estimated 25% of all general hospitals in Japan participated. Participating hospitals were distributed nearly uniformly throughout Japan ( Figure 1). These hospitals took part in the surveillance project after the laboratory director or hospital director granted permission in writing. A questionnaire collecting information for every case-patient was completed anonymously in accordance with the ethical guidelines for conducting epidemiologic studies in Japan.
Pneumococcal isolates were collected nationwide during 3 periods. The first surveillance period was April 2010-March 2011, when voluntary vaccination with PCV7 was estimated to be <10% (vol-PCV7: 2010). The second period was April 2011-March 2012, when the estimated vaccination rate was 50%-60% because of the Urgent Promotion of Vaccination incentive (post-PCV7: 2011). The third period was April 2012-March 2013, when the vaccination rate was 80%-90% just before introduction of PCV13 (pre-PCV13: 2012). We collected a total of 602 isolates from 341 general hospitals: 300 in 2010, 146 in 2011, and 156 in 2012.

Serotypes and Antimicrobial Drug-Resistant Genotypes
Serotypes of all isolates were determined by the capsular quellung reaction using antiserum purchased from the Statens Serum Institute (Copenhagen, Denmark). Alterations in 3 penicillin-binding protein (PBP) genes mediating β-lactam resistance in S. pneumoniae-pbp1a (PBP1A), pbp2x (PBP2X), and pbp2b (PBP2B)-were identified by real-time PCR methods that we developed and reported previously (21). The PCR system detected the presence of amino acid substitution(s) in conserved amino acid motif(s), such as serine-threonine-methionine-lysine, in each PBP. The genes mef (A) and erm (B), which confer resistance to macrolide antimicrobial drugs, also were identified by real-time PCR (21).
Genotype (g) based on molecular analysis is represented here as penicillin-susceptible S. pneumoniae (gPSSP) possessing 3 normal pbp genes; penicillin-intermediate S. pneumoniae (gPISP), further classified as gPISP (pb-p2x), gPISP (pbp1a+pbp2x), or gPISP (pbp2x+pbp2b); or penicillin-resistant S. pneumoniae (gPRSP), possessing all 3 abnormal pbp genes (22). Relationships between susceptibilities to β-lactam agents among phenotypes of S. pneumoniae and resistance genotype were described previously (21). Genotypes involving macrolide resistance are represented here as the following: macrolide-susceptible S. pneumoniae not possessing any genes (MLS); intermediate macrolide resistance to 14-  For each strain transferred to our laboratory, we promptly identified capsular type by quellung reaction, and resistance genotype was determined by real-time PCR. Results were returned immediately to medical staff at the referring hospital. Medical personnel caring for the patients considered this surveillance and results reporting system very helpful, and it was continued for 3 years.

Multilocus Sequence Typing
Multilocus sequence typing for the strains collected was performed according to previously described methods with slight modifications (23). Primers listed on the website of the Centers for Disease Control and Prevention (http:// www.cdc.gov/ncidod/biotech/strep/alt-MLST-primers. htm) were used. Multilocus sequence typing and eBURST analyses were performed according to published methods (http://spneumoniae.mlst.net).

Statistical Analysis
We assessed statistical significance of differences in serotype distribution between the 3 periods, age groups, and specific infectious diseases. We used χ 2 tests or the Fisher exact test, using Ekuseru-Toukei 2012 software for statistics (Social Survey Research Information Co., Ltd., Tokyo, Japan).

Patient Age and Coverage Rate by PCV7
Estimated rates of vaccination with PCV7 were <10% in 2010 (voluntary-PCV7) but rose to 50%-60% with funding in 2011 (post-PCV7) and to 80%-90% with enhanced implementation of PCV7 just before the transition from PCV7 to PCV13 in 2012 (pre-PCV13). S. pneumoniae isolates from patients with IPD, collected every year, decreased by half in 2011 and 2012 from those in 2010. In particular, vaccine serotypes decreased significantly among patients <4 years of age (p<0.001) but not among those >5 years of age (p = 0.733) ( Table 1). For all patients, coverage rate by PCV7 decreased rapidly from 73.3% in 2010 to 54.8% in 2011 to 14.7% in 2012, and prevalence proportion of nonvaccine serotypes increased for 3 years (p<0.001).

Year-to-Year Changes in Vaccine and Nonvaccine Serotype Prevalence Proportion by Disease
We compared year-to-year changes in prevalence of vaccine and nonvaccine serotypes of S. pneumoniae in patients who had meningitis; sepsis and bacteremia; pneumonia; and other invasive infections, including cellulitis, arthritis, endocarditis, and empyema during each of the 3 years studied (Table 2). Pneumonia cases were included only when S. pneumoniae was isolated from blood cultures. Vaccine serotype strains decreased significantly in patients with meningitis (p = 0.006), sepsis and bacteremia (p<0.001), and pneumonia (p<0.001).

Correlation between Serotype and Genotype
We also determined correlations between changes of serotypes and resistance genotypes in all 602 isolates for each period (Figure 2). These resistance genotypes were classified according to real-time PCR results concerning 3 PBP genes: pbp1a, pbp2x, and pbp2b.

Patient Age and Vaccination History in 2012
Only 23 patients had infections caused by strains of vaccine serotypes (14.7%), 21 of whom had not received PCV7 (Table 3). Of the remaining 2 patients, a 1-year-old child infected with a strain of serotype 6B had received 2 doses of vaccine. The other patient, 5 years of age, was infected with a strain of serotype 19F; vaccination history was unknown.

Discussion
Introduction of PCV7 to prevent pneumococcal infections in children was credited with dramatic declines of the incidence of IPD in the United States (6,7), European Union countries (24,25), and many other nations (26).
During this implementation, increases in pneumococcal infections caused by serotype 19A and other nonvaccine serotypes, including those comprising many penicillinnonsusceptible strains, has raised problems in clinical practice (10,11,27). Large-scale longitudinal surveillance showed that the rate of coverage by PCV7 decreased from 70% during 1999-2000 to 4.3% during 2008-2009 (16).
In light of these observations, PCV7 was replaced with PCV13 in the United States in 2010 (15). After the change to PCV13, penicillin-nonsusceptible strains and serotype 15A or 35B strains increased as they did after PCV7 was introduced (16).
In Japan, PCV7 vaccination of children <5 years of age began at the end of 2010 under the Provisional Special Fund for the Urgent Promotion of Vaccination. This measure led to routine vaccination with PCV7 beginning in April 2013 until the vaccine was changed to PCV13 in November 2013. Nationwide, the estimated rate of PCV7 vaccination for children <5 years of age in 2012 and 2013 was 80%-90% and >90%, respectively. We examined the influence of PCV7 against IPD infection in children in detail with active cooperation from 341 clinical laboratories at general hospitals. We therefore believe that our data are highly likely to reflect changes in trends of serotypes and strains causing IPD after PCV7 introduction in Japan. Unfortunately, we could not calculate a precise incidence of IPD per 100 000 children.
The decrease of IPD cases caused by strains of vaccine serotypes after promotion of PCV7 contributed to a reduction in overall IPD by half. Furthermore, dramatic reductions in number of cases caused by serotypes 6B, 14, 19F, and 23Fincluding mainly gPISP and gPRSP-have been beneficial.
However, the relative proportion of IPD caused by nonvaccine serotypes, such as 15A, 15B, 15C, 19A, 22F, 24, and 35B, increased year by year, and the PCV7 coverage rate fell drastically from 73.3% in 2010 to 14.7% in 2012. The impact of routine PCV13 vaccination implemented in December 2013 is likely to be much less than that from PCV7 because coverage already had declined to 39.1% in 2012. However, infections caused by serotype 3, included in PCV13, are expected to decrease. Although IPD caused by serotype 3 occurs infrequently among children in Japan, serotype 3 remains important as a pathogen causing acute otitis media in children and pneumonia or empyema in elderly persons.
Two findings in our study stand out. First, gPRSP strains were confirmed in serotypes 15A, 15C, 16F, and 35B. In particular, strains of serotypes 15A and 35B in 2012 all represented gPRSP. According to multilocus sequence typing analysis, serotype 15A in this study belonged to ST63 of CC63, registered as Sweden 15A-25 in the Pneumococcal Molecular Epidemiology Network clone. Serotype 35B also belonged to ST558 of CC558 from the United States. In addition, gPRSP of serotype 15C corresponded to ST83 of CC81, previously reported from Taiwan. Only 1 serotype 16F strain representing gPRSP was newly identified as ST8351 of CC3117 in the present study. Increasing migration associated with economic development is spreading pneumococcal strains worldwide.
Our finding that vaccine serotype strains are being rapidly replaced by strains of nonvaccine serotypes, such as serotypes 15C and 24, was associated with increased high  macrolide resistance mediated by the erm(B) gene. In this context, wide use of 14-membered and azalide macrolides for children, in addition to adults, will be a major factor favoring resistance. Excessive use of macrolides in Japan also has resulted in substantial increases of macrolideresistant Mycoplasma pneumoniae (28) and macrolideresistant S. pyogenes strains (29). This alarming problem suggests a need to strictly control macrolide use, beginning as soon as possible.
We could not analyze relationships between capsular type and death in children because deaths among children were extremely low (<2.2% every year). The low death rate reflects equal and easy access to hospitals because of Japan's universal health insurance system.
Recently, recombination of the cps gene encoding capsular polysaccharides among pneumococcal strains with different capsular types, called capsular switching, was observed by Brueggemann et al. (30). Capsular switching also is associated with recombination of PBP genes, considering that pbp1a and pbp2x genes which mediate β-lactam resistance, are positioned at both ends of the cps region. Dissemination of vaccine and excessive use of antimicrobial agents could favor S. pneumoniae with a new capsular type in the future.
In conclusion, sustained surveillance on a national and international scale is needed to control pneumococcal infections, especially considering the multifaceted consequences of vaccination programs. Also, controlling the use of antimicrobial agents is urgently needed to avoid increases in the resistant pathogens.
Our study was funded in part by a grant under the category, "Research on Emerging and Re-emerging Infectious Diseases" (H22-013), from the Japanese Ministry of Health, Labour and Welfare to K.U. Dr Chiba is a microbiologist at Keio University School of Medicine. Her research interests include molecular epidemiology, particularly pathogens causing respiratory infection.