Rapid Decrease of 7-Valent Conjugate Vaccine Coverage for Invasive Pneumococcal Diseases in Pediatric Patients in Japan

Abstract

In Japan, the heptavalent pneumococcal conjugate vaccine (PCV7) has been introduced on a voluntary basis since February 2010, and official financial support for children under 5 years started in November 2010. The impact of PCV7 on invasive pneumococcal diseases (IPD) in children is unknown. There are 340 medical institutions that actively participated in our surveillance project throughout Japan. We collected 252 strains from patients with IPD in 2006 (pre-PCV7), 280 strains in 2010 (under 10% immunization achieved), and 128 strains in 2011 (50% to 60% immunization). Serotypes and penicillin-resistance genotypes (g) were compared between these years. Multilocus sequence typing was also carried out on these strains. Due to the official promotion, IPD significantly decreased in 2011 (p<0.001). In particular, meningitis and sepsis caused by vaccine type (VT) strains declined (p=0.033, p<0.001). In less than 2 years, among nonvaccine types (NVT), 15A and 22F increased in 2011 (p=0.015, p=0.015). Coverage by PCV7 decreased from 71.8% in 2006 to 51.6% in 2011. Sequence-type diversities accompanied by evolution to gPRSP occurred in both VT and NVT strains. Reduction of IPD caused by VT strains was accomplished, but a rapid increase of NVT raises concern about a future decrease in the efficacy of PCV7.

Introduction

S treptococcus pneumoniae is a leading etiologic agent in children with severe invasive infections that contribute importantly to morbidity and mortality.¹⁹ Invasive pneumococcal diseases (IPD) caused by penicillin G (PEN)-resistant S. pneumoniae (PRSP) have emerged and spread rapidly worldwide.^2,17

In the United States, heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in February 2000 and added to the immunization schedule for children in October 2000.¹ Subsequent surveillance studies have demonstrated a decrease in the prevalence of pneumococcal infection caused by vaccine type (VT) serotypes and PRSP.^4,22

Currently, children in over 100 countries have undergone PCV7 immunization, followed by substantial regional decreases of IPD.¹⁸ However, increases in pneumococcal infections caused by nonvaccine types (NVT), such as PRSP with serotypes 19A and 6A,¹² suggest that NVTs are emerging and are replacing VT serotypes in some countries.^15,16,21 Other NVT serotypes such as 15A and 35B have been reported to be increasing in the US¹³ and elsewhere.¹⁴

In Japan, PRSP has increased as a causative pathogen among children with respiratory tract infections, acute otitis media, and IPD since the late 1990s.^6,26,27 PCV7 was approved in October 2009 and has been clinically used in infants on a voluntary basis since February 2010, but the vaccination rate was estimated to be under 10% in that year. From November 2010, PCV7 vaccination was encouraged for the children under 5 years old throughout Japan by an official program, the Provisional Special Fund for the Urgent Promotion of Vaccination. As a result, PCV7 immunization was estimated to have reached 50% to 60% in 2011.

In the present study we aimed to clarify the changes in serotypes and genotypes favoring resistance to β-lactam antibiotics in S. pneumoniae isolated from pediatric patients with IPD. A possible relationship between the results of multilocus sequence typing (MLST) and serotype or genotype was also evaluated.

Materials and Methods

Patients and pneumococcal strains

All subjects were pediatric patients under 18 years old with IPD. Isolates from sterile clinical samples such as blood, cerebrospinal fluid, pleural effusion, and joint fluid were examined. Clinical laboratories serving 340 Japanese medical institutions actively participated in this surveillance project after written permission was granted by the laboratory director or hospital director. Surveillance was performed from May 2006 to April 2007 (designated 2006), May 2010 to April 2011 (designated 2010), and May 2011 to April 2012 (designated 2011). The 3 periods, respectively, correspond to the year preceding the introduction of PCV7 (pre-PCV7); the year of voluntary immunization (vol-PCV7; less than 10% immunization achieved); and the year of official promotion (post-PCV7; 50% to 60% immunization).

Clinical isolates were promptly sent to our laboratory, accompanied by a survey form filled out anonymously by the attending physician. The following information was collected from all patients: patient age at onset, sex, specifics of the disease, prognosis, and blood test results, sequel, and outcome. Our survey form was based on the format of the Active Bacterial Core Surveillance case report (ABCs).

Serotypes and antibiotic-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 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 have reported previously.⁷ The lytA gene encoding the autolysin enzyme specific to S. pneumoniae was analyzed similarly. The genes mef (A) and erm (B), which confer resistance to macrolide (ML) antibiotics, were also identified.⁷

Genotype (g) based on molecular analysis is represented here as PEN-susceptible S. pneumoniae (gPSSP) possessing 3 normal pbp genes; PEN-intermediate S. pneumoniae (gPISP), further classified as gPISP (pbp2x), gPISP (pbp1a+pbp2x), or gPISP (pbp2x+pbp2b); or PEN-resistant S. pneumoniae (gPRSP) possessing all 3 abnormal pbp genes. The relationship between susceptibility to parenteral agents among phenotype S. pneumoniae and resistance genotype was described previously.⁷

Results of serotype and resistance genotype analysis for each strain were immediately relayed to the referring pediatrician and local laboratory technicians.

Multilocus sequence typing

MLST was performed on a total of 408 strains obtained in 2010 and 2011 according to the previously described methods,¹¹ with slight modifications. Primers posted on the CDC website (www.cdc.gov/ncidod/biotech/strep/alt-MLST-primers.htm) were used except for the forward primer for the ddl gene.²⁵ MLST and eBURST analyses were performed according to the MLST website (http://spneumoniae.mlst.net).

Statistical analysis

Microsoft Excel 2010 for Statistics (SSRI, Tokyo, Japan) was used for data analyses. Categorical variables were compared using chi-squared tests.

Results

Patient age and capsular serotype

The age distribution of patients with IPD according to the serotype of isolates (VT vs. NVT) and the year when isolated are shown in Table 1.

Table 1.

Vaccine-Type and Nonvaccine Type for 3 Years, According to Age

		Year
Age	Serotype	2006 (n=252)	2010 (n=280)	2011 (n=128)	p-Value
≤1 year	VT^a	113	141	40	<0.001
	NVT^b	38	45	43
2–4 years	VT	60	54	20	0.347
	NVT	16	22	10
≥5 years	VT	8	9	6	0.492
	NVT	17	9	9
Total	VT	181 (71.8)^c	204 (72.9)	66 (51.6)	<0.001
	NVT	71 (28.2)	76 (27.1)	62 (48.4)

VT, serotypes (4, 9V, 18C, 6B, 14, 19F, 23F) included in PCV7.

NVT, serotypes not included in PCV7.

The number of cases is followed by the percentage in parentheses.

VT, vaccine type; NVT, nonvaccine types; PCV7, pneumococcal conjugate vaccine.

Specimens were collected throughout Japan in 2006 (n=252), 2010 (n=280), and 2011 (n=128). The years corresponded to pre-PCV7, vol-PCV7 (PCV7 immunization rate below 10%), and post-PCV7 (50% to 60% PCV7 immunization rate).

The total number of cases in 2011 was significantly lower compared with that in 2006 or 2010, especially in patients under 2 years old (p<0.001). Overall, coverage by PCV7 in the 3 periods decreased from 71.8% in 2006 to 51.6% in 2011. Inversely to the proportion of VT, the proportion of NVT serotypes increased from 28.2% in 2006 to 48.4% in 2011(p<0.001).

Year-to-year changes in VT and NVT prevalence by disease

Table 2 compares the VT and NVT serotype prevalence among the isolates from various types of IPD: meningitis, sepsis and bacteremia, pneumonia, etc., during each of the 3 years studied. Pneumonia was confined to cases in which S. pneumoniae was isolated from blood culture. VT strains decreased significantly in meningitis and sepsis cases through the three periods (p=0.033, p<0.001).

Table 2.

Year-to-Year Changes Vaccine-Type and Nonvaccine Type Prevalence by Disease

Diseases	Serotype	2006 (n=252)	2010 (n=280)	2011 (n=128)	p-Value
Meningitis	VT^a	46	40	13	0.033
	NVT^b	21	18	17
Sepsis and bacteremia	VT	90	98	33	<0.001
	NVT	37	45	40
Pneumonia	VT	38	51	12	0.715
	NVT	10	9	3
Other	VT	7	15	8	0.832
	NVT	3	4	2
Total	VT	181	204	66	<0.001
	NVT	71	76	62

VT, serotypes (4, 9V, 18C, 6B, 14, 19F, 23F) included in PCV7.

NVT, serotypes not included in PCV7.

Year-to-year changes in serotype and resistance genotype

Year-to-year changes in the serotypes and resistance genotypes in children under 5 years old subjected to immunization by the official promotion are shown in Figs. 1 and 2.

FIG. 1.

Changes in the number of heptavalent pneumococcal conjugate vaccine (PCV7) serotypes and those additionally covered by PCV13, and changes in penicillin resistance genotype in the three periods: 2006, 2010, and 2011. These results were limited to isolates from young children under 5 years old. PEN, penicillin G; gPSSP, genotypic PEN-susceptible Streptococcus pneumonia; gPISP, genotypic PEN-intermediate S. pneumonia; gPRSP, genotypic PEN-resistant S. pneumonia.

FIG. 2.

Changes in the number of nonvaccine serotypes (excluding strains covered by PCV13) and in penicillin-resistant genotypes, in the three periods: 2006, 2010, and 2011.

Resistance genotypes were classified according to the presence or absence of 3 abnormal PBP genes, pbp1a, pbp2x, and pbp2b, identified by real-time PCR methods.⁷ All strains of serotypes 6B, 19F, 14, and 23F were gPRSP, gPISP (pbp1a+pbp2x), gPISP (pbp2x+pbp2b), or gPISP (pbp2x). The number of serotype 14 and 19F decreased in 2011, representing the post-PCV7 period (p=0.003, p=0.044).

The numbers of NVT 15A and 15C increased significantly in 2011 (p<0.001, p=0.046). Strains identified as gPRSP, which showed MICs of 0.5 to 4.0 μg/ml for PEN and 1 to 8 μg/ml for cefotaxime, were evident among serotypes 15A, 16F, and 35B.

Changes in the proportion of individual serotypes

Increases and decreases in the proportion of each serotype under 2 years old between 2006 (pre-PCV7) and 2011 (post-PCV7) are shown in Fig. 3. VT serotype 14, which was common among IPD cases, decreased considerably (p=0.007). The serotypes of 6B, 18C, and 23F were little changed. Serotype 19A, which is included in a newer vaccine, PCV13, tended to increase (p=0.056). Other PCV13 serotypes showed minimal changes. Proportions of NVT serotypes, especially serotypes 15A and 22F, increased (both p=0.015).

FIG. 3.

Proportional increases and decreases in each serotype in 2006 and 2011. This result was limited to isolates from young children under 2 years old. Gray, serotypes covered by PCV7; stippled, serotypes additionally covered by PCV13; diagonal line and black, nonvaccine serotypes not covered by PCV13.

Multilocus sequence type

MLST was performed on all VT and NVT strains in 2010 and 2011. As presented in Table 3, the sequence type (ST) and clonal complex (CC) were diversified to number 113 for ST and 35 for CC, respectively. Sixty-six of the STs (61.1%) were registered from Japan.

Table 3.

Relationships Between Multilocus Sequence Types, Serotypes, and Penicillin-Resistance Genotypes, in Invasive Pneumococcal Isolates (n=408)

Serotype	CC	n	Genotype	ST (n: PMEN clone)%
6B	156	50	R	90(30: Spain^6B-2), 95(1), 273(2: Greece^6B-22), 5497(1), 7870(1)%
			2x+2b	5830(1)%
			1a+2x	2983(8), 5497(2), 7824(1)%
			2x	2983(3)%
	242	1	R	242(1: Taiwan^23F-15)%
	490	20	R	902(5), 6413(3), 7492(2), 8345(1), New(1)%
			2x+2b	902(1)%
			2x	2923(5), 5245(1), 6430(1)%
	2224	14	R	2224(6), 7835(1)%
			1a+2x	2224(7)%
	2924	4	2x+2b	8348(1)%
			1a+2x	6183(1)%
			2x	2924(2)%
	3787	6	R	2756(1), 3787(5)%
	7834	2	1a+2x	7834(2)%
	Group 358	1	R	7967(1)%
	singleton	8	R	5232(7), 5244(1)%
14	554	12	R	343(9), 3388(1), 7971(1), 7974(1)%
	230	9	1a+2x	5240(7), 7966(1), 7973(1)%
	15	7	1a+2x	2922(7)%
	156	3	R	5493(1)%
			2x	124(1), 7972(1)%
	199	1	R	876(1)%
	320	1	R	236(1: Taiwan^19F-14)%
	singleton	1	R	New(1)%
19F	320	43	R	236(31: Taiwan^19F-14), 926(1), 1421(1), 1428(1), 1464(1), 7873(1),%
				7991(1), 8341(1), 8342(1), 8344(1), 8349(1)%
			2x	236(2: Taiwan^19F-14)%
	156	1	R	8352(1)%
	242	1		New(1)%
	2924	1	2x	6183(1)%
23F	2924	31	R	1437(28), 6434(1), 7836(1), 7872(1)%
	242	25	R	242(21: Taiwan^23F-15), 1435(1), 1444(1), 7968(1), 8343(1)%
	156	1		338(1: Colombia^23F-26)%
4	490	11	S	246(10), New(1)%
	Group 457	1	S	5872(1)%
9V	156	9	2x	280(6), 5231(3)%
18C	3594	6	S	3594(5), 7829(1)%
6A	3787	11	R	2756(4), 6432(1), 6437(1), 8350(1)%
			1a+2x	3787(1)%
			2x	3113(1), 7969(1), 3787(1)%
	81	4	R	282(2), 81(2: Spain^23F-1)%
	3115	1	R	3115(1)%
	490	1	2x	2923(1)%
	singleton	1	R	7871(1)%
	singleton	1	S	8347(1)%
19A	3111	25	R	3111(7)%
			1a+2x	3111(4)%
			2x	3111(13)%
			S	8339(1)%
	2331	13	2x	2331(9)%
			S	2331(3), 5842(1)%
	156	1	R	156(1: Spain^9V-3)%
	320	1	R	320(1)%
3	180	7	2x	180(6: Netherlands³-31), New(1)%
7F	191	2	S	191(2: Netherlands^7F-39)%
6C	490	8	2x	2923(8)%
	5832	3	2x+2b	5832(3)%
	5241	2	2x+2b	5241(2)%
	2924	2	2x	2924(2)%
	3787	1	2x	3787(1)%
10A	113	1	2x	5236(1)%
11A	99	2	S	99(2)%
12F	1527	2	2b	4846(1)%
			2x+2b	4846(1)%
15A	63	8	R	63(4: Sweden^15A-25), 2105(1), 8354(1)%
			1a+2x	63(2: Sweden^15A-25)%
15B	199	3	1a+2x	199(1: Netherlands^15B-37)%
			2x	199(2: Netherlands^15B-37)%
15C	199	5	1a+2x	199(1: Netherlands^15B-37)%
			2x	199(4: Netherlands^15B-37)%
16F	3117	1	R	8351(1)%
22F	433	6	2x	433(6)%
	2572	1	S	5496(1)%
23A	156	6	2x+2b	338(3: Colombia^23F-26), 5242(1), 6685(1),8340(1)%
24	2572	6	S	2572(1), 5496(5)%
35B	558	1	R	558(1)%
	1816	1	2x	2755(1)%
38	393	3	S	393(3)%
20	4745	1	S	4745(1)%
21	1381	1	2x	1233(1)%
33	717	3	S	717(3)%
34	Group 363	1	S	3116(1)%
37	447	1	S	7970(1)%
NT	2572	1	S	5496(1)%

Bold type indicates STs registered from Japan.

CC, clonal complex; ST, sequence type; PMEN, Pneumococcal Molecular Epidemiology Network.

Considering the associations between ST and serotype in gPRSP, 30 strains of serotype 6B were ST90, including CC156, which was submitted as a Pneumococcal Molecular Epidemiology Network (PMEN) clone of Spain^6B-2, followed by ST2224 in CC2224, ST902 and ST6413 in CC490, and ST5232 (singleton). Most gPRSP in other VT strains were ST343 in serotype 14, which had evolved from Sweden ST554; ST236 in serotype 19F, representing the Taiwan^19F-14 clone; ST1437, which was identified in Japan or ST242 of the Taiwan^23F-15 clone in serotype 23F. Notably, gPRSP strains in serotypes 6A and 19A expanded to 11 STs, including ST3111, which had evolved from the original US strain (Alaska; MIC for PEN, 0.03 μg/ml); ST2756, identified in China; and ST282, which developed from the original Vietnam strain. ST81 (n=2) and ST156 (n=1), recorded as Spain^23F-1 and Spain ^9V-3 clones, respectively, were very few.

Meanwhile, STs of gPRSP in NVT serotypes were mainly seen ST63 in serotype 15A submitted from Sweden^15A-25, originally showing an MIC of 0.12 μg/ml for PEN; ST8351 of serotype 16F, registered in Japan during study; and ST558 in serotype 35B, registered in the US and showing an MIC of 2.0 μg/ml for PEN.

Variation in the STs and genotypes in serotype 6C and STs in serotype 23A were evident. In addition, gPISP (pbp2x) strains with ST199 submitted as the Netherlands^15B-37 clone had evolved to gPISP (pbp1a+2x) by way of pbp1a gene alterations in serotypes 15B and 15C.

Notably, 10 cases of capsular switching suggested the following relationships to ST and serotype as follows: between serotypes 15B and 15C in ST199; 14 and 19F in ST236; 6B and 23F in ST242; 23A and 23F in ST338; 6A and 6B in ST2756; 6A, 6B, and 6C in ST2923 and ST3787; 6B and 6C in ST2924; 22F and 24 in ST5496; and 6B and 19F in ST6183.

On the other hand, 93.6% of all strains isolated in 2010 and in 2011 possessed mef(A) and/or erm(B) genes mediating ML antibiotic resistance.

Discussion

Prevention of IPD, well known for high morbidity and mortality in immunologically immature infants, is an important priority.²⁸ Increases of β-lactam and ML resistances in this pathogen pose ongoing difficulties in selecting therapeutic agents.⁹

Aiming for the prevention of pneumococcal infections in infants, PCV7 was licensed in 2000 and recommended for all children aged 2 to 23 months in the United States.¹ PCV7 was licensed in Europe in 2001, but 6 nations, including the UK, have only included it in their national immunization programs since 2006.²⁰ Currently, PCV7 has been introduced in more than 100 countries as a voluntary vaccination or routine practice for young children.¹⁸ Rapid implementation of PCV7 in young children has resulted in a dramatic reduction in the incidence of IPD and non-IPD in many countries.^15,18,24

However, increases of pneumococcal infections due to NVT after the introduction of PCV7, especially PRSP of serotypes 6A and 19A,¹² have occurred in some countries, even as the prevalence of VT serotypes was decreasing.^15,24

PCV13, including serotypes 6A and 19A, replaced PCV7 in vaccination schedules in the United States in 2010.⁵ Presently, PCV7 is gradually being replaced with PCV13 worldwide.^8,10 Additionally, an increase serotypes of 15A and 35B has been reported in the United States.¹³

As previously described, PCV7 was approved in Japan in 2009; presently, PCV7 immunization of children under 5 years old has been promoted nationwide by the Ministry of Health, Labour and Welfare (Provisional Special Fund for the Urgent Promotion of Vaccination) since November 2010. The immunization rate was estimated to have reached 50% to 60% in 2011. PCV7 will be formally added to the immunization schedule for Japanese infants in 2013.

In the present study, we aimed to investigate the impact of PCV7 on the serotype of the causative S. pneumoniae isolates from children with IPD. Unfortunately, IPD reduction could not be studied in terms of incidence because Japanese data for incidence of IPD per 100,000 persons is not available.

We found that IPD caused by VT strains decreased significantly for serotypes 14 and 19F after promotion of PCV7 vaccination in 2011. Interestingly, the relative decrease in every VT serotype resembled to the kinetics of the serotype-specific immune responses described by Rennels et al.²³ This is reflected by a significant decrease in the onset of IPD in children under 2 years old.

In contrast, NVT serotypes 15A and 22F have increased as causative pathogens. The obvious change from VT to NVT serotypes appears to be a consequence of PCV7 vaccination.

By MLST analysis of VT serotype strains, the already well-known PMEN clone and CCs predominated among gPRSP, such as Spain^6B-2 in 6B, CC490 and CC2224 in 6B, ST343 evolving from Sweden ST554 in serotype 14, Taiwan^19F-14 in 19F, and Taiwan^23F-15 in 23F. The occurrence of many new ST numbers suggest that housekeeping gene(s) evolved by mutation or genetic recombination.

Focusing on gPRSP and gPISP among the NVT serotypes, diversities of STs occurred easily as a result of mutations in housekeeping genes and pbp genes originating in other countries. The PMEN clone Sweden^15A-25 of ST63 was found among serotype 15A with gPRSP in Japan. This had an MIC of 0.12 μg/ml for PEN in 1992, but evolved to PISP showing an MIC of 0.5 μg/ml for PEN in 2008 in France. This worsened to MIC of 2.0 μg/ml for PEN in Japan. A new ST8354 evolved from ST63 by mutations in the gdh gene encoding glucose-6-phosphate dehydrogenase.

Recently, capsular switching occurring between different ST strains has been reported by Brueggemann et al.³ As an example, ST2923 including serotypes 6A, 6B, and 6C in this study suggests that capsular switching occurred readily by recombination of the capsular locus region that was sandwiched between the pbp1a and pbp2x genes, although the original strain was recorded from Bulgaria as serotype 6A and CC490 (ST490).

In conclusion, serotype, genotype, and MLST analyses indicate that spread of microorganisms, especially potential respiratory pathogens occasionally carried as normal flora, is commonplace in the era of globalization. Peumococcal strains first identified abroad were then influenced by antibiotic selection, vaccination status, and population density in subsequent countries, with the emergence of mutations of housekeeping genes and the pbp gene, as well as capsular switching. Prevention and control of pneumococcal infections in young children and adults will require the development of a new vaccine including all pneumococcal serotypes. Further surveillance studies on clinical and molecular epidemiology of IPD caused by S. pneumoniae is needed to determine the impact of future conjugate vaccines on serotype and clone distribution.

Footnotes

Acknowledgments

Our research project was supported by a grant under the category “Research on Emerging and Re-emerging Infectious Diseases (number H21-002 and H22-013)” from the Japanese Ministry of Health, Labour and Welfare (to Dr. K. Ubukata).

Disclosure Statement

No competing financial interests exist.

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