Streptococcus pneumoniae is a major cause of invasive infections in young infants and children. Among >90 serotypes, only a limited number account for pneumococcal diseases. Serotype incidence can vary by patient age, geographic region, and time of surveillance. Since the introduction of 7-valent conjugate vaccine (PCV7) in the United States, a decrease in the incidence of invasive pneumococcal disease (IPD) caused by vaccine serotypes has been observed in pediatric and nonpediatric age groups ( 1 , 2 ). However, the incidence of IPD caused by nonvaccine serotypes (including serotype 19A) increased 1.5-fold in 2002 compared to that in 1999 ( 2 , 3 ). To date, replacement for IPD has been observed for serotypes 3, 15, 19A, 22F, 33F, and 35, with the increase in 19A being the most prevalent. ( 2 – 7 ). Recently, Singleton et al. reported that serotype 19A was responsible for 28.3% of IPD in rural Alaska Native children 3 antimicrobial drug classes. The mefA and ermB genes were detected by PCR using the primers mefA (forward, 5′-AGT ATC ATT AAT CAC TAG TGC-3′; reverse, 5′-TTC TTC TGG TAC TAA AAG TGG-3′) and ermB (forward, 5′-GAA AAG GTA CTC AAC CAA ATA-3′; reverse, 5′-GTA ACG GTA CTT AAA TTG TTT AC-3′) ( 20 ). PCRs were performed with 35 amplification cycles: 30 s at 94°C, 30 s at 50°C, and 1 min 30 s at 72°C, followed by a final extension at 72°C for 10 min. Statistical Analysis Statistical analysis was performed by using SPSS software version 13.0 (SPSS, Chicago, IL, USA). Serotype proportion in each period was compared using the χ2 or Fisher exact test, as appropriate. The Mantel-Haenszel χ2 test was used for trend analysis. Results Changes in Serotype Distributions From 1991 through 2006, 538 strains of S. pneumoniae were obtained from various clinical specimens. Of these, 158 (29%) were from invasive isolates; 124 blood, 15 cerebrospinal fluid, 6 pleural fluid, 5 ascites, and 8 other sterile deep-seated tissues (e.g., bone and joint fluid). The remaining 380 (71%) were from noninvasive isolates; 110 (pharyngeal swab), 91 (transtracheal aspirate), 81 (sputum), 69 (middle ear fluid), 15 (urine), and 14 (open pus). The most common serotypes were 19F (113, 21%), 23F (96, 17.8%), 19A (58, 10.8%), 6B (50, 9.3%), 6A (43, 8%), 14 (40, 7.4%), and 9V (24, 4.5%); these 7 serotypes accounted for 79% of the total isolates. Overall, PCV7 serotypes accounted for 64.1% of total isolates and 62.7% of invasive isolates. Table 1 shows the serotype distributions of invasive and noninvasive isolates by age group. For invasive isolates, PCV7 serotype coverage was 67% among children 60 months (Table 1). During 2001–2003, just before PCV7 was introduced in South Korea, overall PCV7 coverage rates for PCV7 serotypes and PCV7-related serotypes were 54% and 10% among the 138 invasive isolates and 57% and 13% among the 380 noninvasive isolates. The proportion of serotype 19A isolates increased from 0% (0/40) during 1991–1994 to 18% (7/39) during 2001–2003 among the 138 invasive isolates. Similarly, from 1995–1997 to 1998–2000, the proportion of serotype 19A isolates increased from 3% (1/33) to 17% (14/81) among noninvasive isolates (Figure 1). Table 1 Distributions of serotypes among 538 isolates from children in South Korea, by age group, 1991–2006* Serotype No. (%) invasive isolates† No. (%) noninvasive isolates† 60 mo Total 60 mo Total PCV7 serotypes 44 (67) 28 (68) 24 (47) 96 (61) 113 (65) 81 (71) 42 (45) 236 (62) 19F 8 (12) 6 (15) 4 (8) 18 (11) 50 (29) 33 (29) 12 (13) 95 (25) 23F 16 (24) 6 (15) 4 (8) 26 (16) 32 (18) 20 (18) 18 (19) 70 (18) 6B 8 (12) 3 (7) 7 (14) 18 (11) 19 (11) 10 (9) 3 (3) 32 (8) 14 7 (11) 10 (24) 5 (10) 22 (14) 8 (5) 7 (6) 3 (3) 18 (5) 9V 4 (6) 3 (7) 3 (6) 10 (6) 4 (2) 8 (7) 2 (2) 14 (4) 4 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (2) 4 (4) 6 (2) 18C 1 (2) 0 (0) 1 (2) 2 (1) 0 (0) 1 (1) 0 (0) 1 (0) PCV7-related serotypes 3 (5) 6 (15) 7 (14) 16 (10) 15 (9) 14 (12) 9 (9) 38 (10) 6A 3 (5) 5 (12) 6 (12) 14 (9) 12 (7) 10 (9) 7 (7) 29 (8) 23A 0 (0) 1 (2) 1 (2) 2 (1) 3 (2) 4 (4) 0 (0) 7 (2) 9 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (2) 2 (1) 19A 11 (16) 2 (5) 0 (0) 13 (8) 27 (16) 8 (7) 10 (11) 45 (12) Non-PCV7 serotypes 8 (12) 5 (12) 20 (39) 33 (21)‡ 18 (11) 11 (10) 32 (35) 61 (16) Total 66 (100) 41 (100) 51 (100) 158 (100) 173 (100) 114 (100) 93 (100) 380 (100) *PVC7, 7-valent conjugate vaccine.
†Percentages have been rounded.
‡Thirteen serogroups were included among 33 invasive isolates as follows: 15 (6 strains), 24F (6), 10 (4), 34 (3), 35 (3), 1 (2), 3 (2), 12F (2), 5 (1), 11A (1), 13 (1), 20 (1), and 27 (1). Figure 1 Distribution of serotypes with regard to 7-valent conjugate vaccine (PCV7) among 538 isolates encountered during five 3-year periods from 1991 through 2006, South Korea. A) Invasive isolates. B) Noninvasive isolates. Among the 107 invasive isolates from children 3 antimicrobial drug classes. Erythromycin MICs of CC271 isolates were >256 µg/mL and were all positive for mef and erm. However, 5 singlets that were unrelated to CC271 showed different patterns of antimicrobial drug susceptibility and presence of mef/erm determinants (Table 2). Four serotype 19A isolates representing ST1374 appeared to be less resistant to 2 β-lactam antimicrobial drugs than CC271 isolates. ST 1374 strains were also highly resistant to erythromycin (MIC >256 µg/mL) and were positive for ermB only. Strains of ST2394, ST2395, and ST2399 showed a lower degree of erythromycin resistance (MIC = 2–8 μg/mL) than CC271 and ST1374 and contained mefA only. One serotype, 19F strain of ST1203, did not express mef or erm. Serotypes 19A and 19F strains with CC271 were shown to be closely related to the internationally established clone, Taiwan19F-14, which is multidrug resistant and carries mefA/ermB macrolide–resistance determinants defined by the pneumococcal molecular epidemiology network ( 22 ). Table 2 Antimicrobial susceptibility and mef and erm prevalence of serogroup 19 pneumococcal isolates from children in South Korea, 1991–2006, according to sequence type CC or ST* (no. strains) Serotypes Multidrug resistance† MIC50 (range) in µg/mL for each antimicrobial drug mef/erm determinants Penicillin Cefotaxime Erythromycin CC271-related‡ (116) 19A, 19F Yes 1.5 (1.0–3.0) 1.0 (0.75–4.0) >256 (>256) mef+, erm+ ST1203 (1) 19F No 0.5 0.25 0.5 mef ST1374 (4) 19A Yes 0.06 (0.04–0.06) 0.12 (0.09–0.12) >256 (>256) mef ST2394 (1) 19A Yes 1.0 0.5 2.0 mef+, erm ST2395 (3) 19F Yes 4.0 (4.0) 2.0 (1.0–2.0) 2.0 (2.0) mef+, erm ST2399 (1) 19F Yes 0.12 0.25 8.0 mef+, erm *CC, clonal complex; ST, sequence type.
†Resistant to at least 3 antimicrobial drug classes.
‡CC271-related sequence types: ST320 (59 isolates), ST271 (14), ST236 (14), ST283 (7), ST1451 (1), ST1464 (10), ST2395 (3), ST2695 (3), and 1 isolate of each of ST1412, ST1417, ST2396, ST2397, ST2398, ST2694, ST2696, ST2697, and ST2698. Discussion We found that before PCV7 introduction in South Korea, the proportion of serotype 19A isolates increased from 0% in 1991 to 26% in 2003 but 19F isolates decreased during the same period. Our study also demonstrated that multidrug-resistant ST320 isolates containing mef/erm determinants were responsible for the expansion of serotype 19A. In the United States, serotype 19A is now the most important cause of IPD by replacement serotypes ( 4 , 8 , 23 , 24 ). Contrary to what we report for South Korea, the increase in the United States was documented after widespread use of PCV7 ( 4 – 6 ). Increase in non-PCV7 serotypes, i.e., serotype replacement, has been noted in carriage studies and the pre-licensure clinical trials ( 11 , 25 , 26 ). After widespread use of PCV7 in young children, replacement of serotypes for IPD has been described for several serotypes in previous studies ( 4 – 6 ). Of these, an increase in the incidence of IPD cases caused by serotype 19A was quite high. Therefore, the increasing prevalence of IPDs caused by serotype 19A among the vaccine target group is of considerable concern. Our findings of an increase in serotype 19A disease before conjugate vaccine introduction calls into question the role vaccine may play in the emergence of serotype 19A disease and suggests that other factors are important. Of the factors contributing to the increase in serotype 19A, the MLST findings in our study point to a homogeneous pattern of ST320. ST320 of serotype 19A might have originated from ST271 or ST236 strains that have been prevalent among serotype 19F since 1993 ( 20 ) or could have been introduced from other countries. Thus, single clonal expansion of ST320, related to a multidrug-resistant internationally prevalent clone, Taiwan19F-14 (that also carries mefA/ermB determinants), was most likely responsible for the prevaccine increase observed for serotype 19A. Antimicrobial drug use may provide selective pressure that would give this highly resistant strain an advantage over other strains. A study in the United States demonstrated an increasing prevalence of mefA/ermB; 17% percent of 221 children had been colonized by mefA/ermB containing serogroup 19 pneumococci strains after receiving at least 1 dose of PCV7; the major clonal type was related to Taiwan19F-14 ( 27 ). In Alaska, the increase observed in 19A colonization and IPD seems to be related to CC172 clonal expansion ( 8 ). In contrast, recent genetic analysis of 19A strains isolated after PCV7 use in the United States showed that diverse mechanisms were involved in the expansion of 19A strains, expansion of preexisting predominant CC199, capsular switching of PCV7 types (4, 6, 14, and 9V), and appearance of multiple unrelated multidrug-resistant CCs among serotype 19A strains (CC271, including ST1451 and ST320, CC156, and CC1296) ( 4 ). Capsular switching of PCV7 serotypes under selective pressure by vaccine use is one of the mechanisms underlying the expansion of serotype 19A ( 4 , 11 – 13 , 28 ). However, there is no evidence of capsular switching as a contributing factor to the increase in serotype 19A in our study. We also found that serotype 19F gradually decreased in proportion and diversified to include several newer descendants that differ from the founder by only 1 or 2 of 7 alleles but ST320 was not a major genetic structure among serotype 19F strains ( 18 ). Thus, it appears that ST320 has a selective advantage in serotype 19A strains, whereas ST320 did not seem to have expansion merit in the close serotype 19F strains. This finding suggests that the properties of particular clonal or capsular types are likely important determining factors of the potential of pneumococci to influence disease type and severity ( 29 , 30 ). Further studies are necessary to explain why certain sequence types exhibit different selective pressures according to serotype, even for close serotypes such as 19A and 19F. This study has several limitations. First, pneumococcal isolates were collected at a single center, and thus, may not represent the national situation. However, no surveillance system has been established for IPD in South Korea. Nevertheless, at least 2 studies have demonstrated a recent increase in serotype 19A isolates among children in daycare centers (3% in 2002 and 11% in 2004) ( 14 , 31 ). In addition, the number of serotype 19A isolates from children and adults at another tertiary South Korean hospital showed an increase over the same period ( 32 ). Second, the number of 19A isolates was relatively small, and it is possible that uncommon strains possessing minor clones were not detected. Thus, our finding of a clonal expansion of ST320 among serotype 19A strains may be an overstatement. However, MLST analysis showed that all 7 of the 19A strains from colonized children who were identified from a previous study ( 14 ) were of ST320 (H.J. Lee, unpub. data). The present study has implications for future pneumococcal immunization programs. In particular, the demonstration of an increase in 19A before the use of PCV7 suggests that PCV7 vaccination may not be entirely responsible for the observed increase of serotype 19A. Had the vaccine been introduced in 2000, as in the United States, the increase of serotype 19A would have been attributed to serotype replacement after PCV7 introduction. Nevertheless, whether minor multidrug-resistant clones of serotype 19A (appearing after the introduction of PCV7 in the United States) possess an advantage to increase with time is a concern ( 23 , 29 ). We demonstrated that multidrug-resistant ST320 strains among serotype 19A have selective advantage in terms of expansion over ST1374, which were less resistant to β-lactams in a country where antimicrobial drug therapy is frequently used. Reports on PCV7 efficacy indicate negligible cross-protection for serotype 19A ( 11 ). In addition, a recent increase in the antimicrobial drug resistance of invasive 19A isolates and the increase in colonization by serogroup 19 strains carrying mef/erm determinants raise the possibility of potential increases in the prevalence of this clone. Thus, potential for colonization because of widespread antimicrobial drug use and resistance may interact and provide the selective advantage necessary for serotype expansion, which may be the situation in South Korea. Similarly, the influence of population characteristic dynamics, i.e., HIV infection or poverty and overcrowding, as well as PCV7 introduction, may combine with the necessary factor for serotype replacement and play an important role in serotype expansion, which may be the situation in Alaska. It is too early to determine the effect of PCV7 on expansion of serotype 19A in South Korea. PCV7 was introduced in South Korea in November 2003 when PCV7 serotype coverage was 56% among invasive isolates in children <5 years of age. In 2007, PCV7 coverage is ≈30% of the target group ( 21 ). At this time it is difficult to predict the effect of low vaccination coverage on serotype expansion/replacement. Therefore, surveillance is essential to monitor antimicrobial drug resistance, serotype expansion, and serotype replacement as early indications of an increase in pneumococcal disease by non-PCV7 or PCV7-related serotypes.
