Determination of saccharide content in pneumococcal polysaccharides and conjugate vaccines by GC-MSD

A simple and sensitive gas chromatographic method was designed for quantitative analysis of Streptococcus pneumoniae capsular polysaccharides, activated polysaccharides, and polysaccharide conjugates. Pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F polysaccharide or conjugate were subjected to methanolysis in 3N hydrochloric acid in methanol followed by re-N-acetylation and trimethylsilylation. Derivatized samples were chromatographed and detected using gas chromatography with mass selective detector. Gas chromatographic results were compared with colorimetric values with agreement of 92 to 123% over the range of all samples tested. Monosaccharides released during methanolysis included hexoses, uronic acids, 6-deoxy-hexoses, amino sugars, and alditols. Quantitative recovery of monosaccharides was achieved for all serotypes by the use of a single methanolysis, derivatization, and chromatography procedure. Response factors generated from authentic monosaccharide standards were used for quantitation of pneumococcal polysaccharides and conjugates with confirmation of peak assignments by retention time and mass spectral analysis. This method allows saccharide quantitation in multivalent pneumococcal vaccine intermediates and final drug products with low-level detection (10 pg) and peak purity.     

Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10

Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.     

Serotype distribution of Streptococcus pneumoniae in north-western Greece and implications for a vaccination programme

Serotypes and antibiotic sensitivities were determined for 338 strains of Streptococcus pneumoniae from children of north-western Greece with invasive pneumococcal disease (IPD), acute otitis media (AOM) and nasopharyngeal carriage. The most common serotypes among the isolates from IPD were 14 and 19F, while 3, 19F, 9V and 14 were the major cause of AOM. In these groups, the heptavalent conjugate vaccine for pneumococci (7vPCV) seems to cover 90.5% of the serotypes isolated from children less than 2 years old. Serotypes 23F and 6B were the most prevalent in carrier strains. Overall, 23.7% of the isolates were penicillin nonsusceptible (PNS), 97% were fully susceptible to cefotaxime, 29% were resistant to erythromycin, 11.2% to co-trimoxazole and 1.2% to clindamycin.     

MALDI-TOF MS对肺炎链球菌鉴定与分型的应用

目的探讨MALDI-TOF MS对肺炎链球菌鉴定和质谱分型的应用价值。方法收集2009年1月至2013年5月温州医科大学附属第二医院临床分离的112株肺炎链球菌标本,采用Optochin敏感试验和全自动细菌分析仪对收集的菌株进行鉴定验证,并用Microflex MALDI-TOF质谱仪进行分析鉴定。根据质谱图的相似性进行细菌同源聚类树分析并构建质谱分型模型,采用荚膜肿胀试验对参与分型的菌株进行血清型比较。结果除20株不符合检测条件之外,92株临床菌株和1株标准株经质谱分析均为肺炎链球菌,选取的60株菌株以0.5的差异水平,将60株肺炎链球菌分为18个质谱型别,在这些菌株的血清分型中有19F、19A、23F、23A、3和14六个血清型别,分布于不同的MALDI-TOF MS分型中,其中19F有18株,占30%(18/60),分布在6种不同的MALDI-TOF MS分型中,也有3型血清型较为集中地分布于相应的MALDI-TOF MS一个型别里。结论 MALDI-TOF MS能快速、准确、简便地鉴定肺炎链球菌,且能达到种的水平。对比血清型,按照0.5差异水平,建立的18个质谱分型部分的型别与血清型有一致性,但也存有差异。     

Serotypes and Patterns of Antibiotic Resistance in Strains Causing Invasive Pneumococcal Disease in Children Less than 5 Years of Age

Objective

The serotypes and patterns of antibiotic resistance of Streptococcus pneumoniae (S. pneumoniae) strains that cause invasive pneumococcal disease (IPD) in infants were analyzed to provide guidance for clinical disease prevention and treatment.

Methods

The clinical features of confirmed IPD were evaluated in 61 patients, less than 5 years of age, who were admitted to our hospital between January 2009 and December 2011. The serotypes and antibiotic resistance of strains of S.pneumoniae were determined using the capsular swelling method and the E-test.

Results

A total of 61 invasive strains were isolated. The serotype distribution of those isolates were 19A (41.0%), 14 (19.7%), 19F (11.5%), 23F (9.8%), 8 (4.9%), 9V (4.9%), 1 (3.3%), and 4, 6B, and 20 (each 1.6%). The percentage of S. pneumoniae strains resistant to erythromycin, clindamycin, and cotrimoxazole were 100%, 86.9%, and 100%, respectively. The percentage of S. pneumoniae strains resistant to penicillin, amoxicillin/clavulanic acid, cefuroxime, ceftriaxone, cefotaxime, cefepime, and meropenem were 42.6%, 18.0%, 82.0%, 18.0%, 13.1%, 13.1%, and 36.1%, respectively. The percentage of multidrug-resistant strains was 95.6%. Strains of all serotypes isolated in this study were highly resistant to erythromycin, cotrimoxazole, and clindamycin. Strains with serotype 19A had the highest rates of resistance.

Conclusions

Serotype 19A strains were most frequently isolated from children with IPD treated in our hospital. The strains causing IPD are highly resistant to antibiotics.     

Analysis of the 5' portion of the type 19A capsule locus identifies two classes of cpsC, cpsD, and cpsE genes in Streptococcus pneumoniae.

Analysis of the sequence data obtained from the 5' portion of the Streptococcus pneumoniae type 19A capsular polysaccharide biosynthesis locus (cps19a) revealed that the first seven genes are homologous to the first seven genes in the type 19F (cps19f) locus. The former genes were designated cps19aA to -G and were 70 to 90% identical to their cps19f counterparts. Southern hybridization analysis of the cps loci from various S. pneumoniae serotypes with probes specific for the cps19aC, cps19aD, and cps19aE genes indicated a hybridization pattern complementary to that previously reported for cps19fC, cps19fD, and cps19fE. That is, all serotypes tested contained high-stringency homologues of either the cps19aC to -E genes or the cps19fC to -E genes, but not both. On this basis S. pneumoniae cps loci can be divided into two distinct classes. Long-range PCR was used to amplify the cps regions between cpsB and aliA from a variety of pneumococcal serotypes. Direct sequencing of the 5' end of these PCR products, and phylogenetic analysis of the sequence data, confirmed the presence of the two distinct classes of cpsC. Whereas members within one class are greater than 95% identical to each other, the DNA sequence identity between the two classes is only approximately 70%.     

Genetic analysis of the capsular polysaccharide synthesis locus in 15 Streptococcus suis serotypes

The capsular polysaccharide (CPS) synthesis locus of 13 Streptococcus suis serotypes (serotype 1, 3, 4, 5, 7, 8, 9, 10, 14, 19, 23, 25 and 1/2) was sequenced and compared with that of serotype 2 and 16. The CPS synthesis locus of these 15 serotypes falls into two genetic groups. The locus is located on the chromosome between orfZ and aroA. All the translated proteins in the CPS synthesis locus were clustered into 127 homology groups using the tribemcl algorithm. The general organization of the locus suggested that the CPS of S.?suis could be synthesized by the Wzy-dependent pathway. The capsule of serotypes 3, 4, 5, 7, 9, 10, 19 and 23 was predicted to be amino-polysaccharide. Sialic acid was predicted to be present in the capsule of serotypes 1, 2, 14, 16 and 1/2. The characteristics of the CPS synthesis locus suggest that some genes may have been imported into S.?suis (or their ancestors) on multiple occasions from different and unknown sources.     

Serotype distribution of invasive Streptococcus pneumoniae in Canada during the introduction of the 13-valent pneumococcal conjugate vaccine, 2010

A baseline serotype distribution was established by age and region for 2058 invasive Streptococcus pneumoniae isolates collected during the implementation period of the 13-valent pneumococcal conjugate vaccine (PCV13) program in many parts of Canada in 2010. Serotypes 19A, 7F, and 3 were the most prevalent in all age groups, accounting for 57% in <2 year olds, 62% in 2-4 year olds, 45% in 5-14?year olds, 44% in 15-49?year olds, 41% in 50-64?year olds, and 36% in ≥65?year olds. Serotype 19A was most predominant in Western and Central Canada representing 15% and 22%, respectively, of the isolates from those regions, whereas 7F was most common in Eastern Canada with 20% of the isolates. Other prevalent serotypes include 15A, 23B, 12F, 22F, and 6C. PCV13 serotypes represented 65% of the pneumococci isolated from?<2 year olds, 71% of 2-4 year olds, 61% of 5-14 year olds, 60% of 15-49 year olds, 53% of 50-64 year olds, and 49% of the?≥65?year olds. Continued monitoring of invasive pneumococcal serotypes in Canada is important to identify epidemiological trends and assess the impact of the newly introduced PCV13 vaccine on public health.     

Sequence diversity within the capsular genes of Streptococcus pneumoniae serogroup 6 and 19

The main virulence factor of Streptococcus pneumoniae is the capsule. The polysaccharides comprising this capsule are encoded by approximately 15 genes and differences in these genes result in different serotypes. The aim of this study was to investigate the sequence diversity of the capsular genes of serotypes 6A, 6B, 6C, 19A and 19F and to explore a possible effect of vaccination on variation and distribution of these serotypes in the Netherlands. The complete capsular gene locus was sequenced for 25 serogroup 6 and for 20 serogroup 19 isolates. If one or more genes varied in 10 or more base pairs from the reference sequence, it was designated as a capsular subtype. Allele-specific PCRs and specific gene sequencing of highly variable capsular genes were performed on 184 serogroup 6 and 195 serogroup 19 isolates to identify capsular subtypes. This revealed the presence of 6, 3 and a single capsular subtype within serotypes 6A, 6B and 6C, respectively. The serotype 19A and 19F isolates comprised 3 and 4 capsular subtypes, respectively. For serogroup 6, the genetic background, as determined by multi locus sequence typing (MLST) and multiple-locus variable number of tandem repeat analysis (MLVA), seemed to be closely related to the capsular subtypes, but this was less pronounced for serogroup 19 isolates. The data also suggest shifts in the occurrence of capsular subtypes within serotype 6A and 19A after introduction of the 7-valent pneumococcal vaccine. The shifts within these non-vaccine serotypes might indicate that these capsular subtypes are filling the niche of the vaccine serotypes. In conclusion, there is considerable DNA sequence variation of the capsular genes within pneumococcal serogroup 6 and 19. Such changes may result in altered polysaccharides or in strains that produce more capsular polysaccharides. Consequently, these altered capsules may be less sensitive for vaccine induced immunity.     

Comparative Genetics of Capsular Polysaccharide Biosynthesis in Streptococcus pneumoniae Types Belonging to Serogroup 19

The genetic basis for the structural diversity of capsule polysaccharide (CPS) in Streptococcus pneumoniae serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19 cps loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (cps19aI and cps19fI). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two cps loci (cps19cS), which is likely to encode a glucosyl transferase. The arrangement of the genes within the cps19 loci is highly conserved, with 13 genes (cps19A to -H and cps19K to -O) common to all four serogroup 19 members. These cps genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19 cps loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the cps19 gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.     

Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Isolates Causing Invasive Diseases from Shenzhen Children’s Hospital

Objective

To provide guidance for clinical disease prevention and treatment, this study examined the epidemiology, antibiotic susceptibility, and serotype distribution of Streptococcus pneumoniae (S. pneumoniae) associated with invasive pneumococcal diseases (IPDs) among children less than 14 years of age in Shenzhen, China.

Materials and Methods

All the clinical strains were isolated from children less than 14 years old from January 2009 to August 2012. The serotypes and antibiotic resistance of strains of S. pneumoniae were determined using the capsular swelling method and the E-test.

Results

A total of 89 strains were isolated and 87 isolates were included. The five prevailing serotypes were 19F (28.7%), 14 (16.1%), 23F (11.5%), 19A (9.2%) and 6B (6.9%). The most common sequence types (ST) were ST271 (21.8%), ST876 (18.4%), ST320 (8.0%) and ST81 (6.9%) which were mainly related to 19F, 14, 19A and 23F, respectively. The potential coverage by 7-, 10-, and 13-valent pneumococcal conjugate vaccine were 77.0%, 77.0%, and 89.7%, respectively. Among the 87 isolates investigated, 11.5% were resistant to penicillin, and for meningitis isolates, the resistance rate was 100%. Multi-drug resistance (MDR) was exhibited by 49 (56.3%) isolates. Eighty-four isolates were resistance to erythromycin, among which, 56 (66.7%) carried the ermB gene alone and 28 (33.3%) expressed both the ermB and mefA/E genes.

Conclusions

The potential coverage of PCV13 is higher than PCV7 and PCV10 because high rates of serotypes 19A and 6A in Shenzhen. The clinical treatment of IPD needs a higher drug concentration of antibiotics. Continued surveillance of the antimicrobial susceptibility and serotypes distribution of IPD isolates may be necessary.     

Vaccination response to tetanus toxoid and 23-valent pneumococcal vaccines following administration of a single dose of abatacept: a randomized,open-label,parallel group study in healthy subjects

The effect of abatacept, a selective T-cell co-stimulation modulator, on vaccination has not been previously investigated. In this open-label, single-dose, randomized, parallel-group, controlled study, the effect of a single 750 mg infusion of abatacept on the antibody response to the intramuscular tetanus toxoid vaccine (primarily a memory response to a T-cell-dependent peptide antigen) and the intramuscular 23-valent pneumococcal vaccine (a less T-cell-dependent response to a polysaccharide antigen) was measured in 80 normal healthy volunteers. Subjects were uniformly randomized to receive one of four treatments: Group A (control group), subjects received vaccines on day 1 only; Group B, subjects received vaccines 2 weeks before abatacept; Group C, subjects received vaccines 2 weeks after abatacept; and Group D, subjects received vaccines 8 weeks after abatacept. Anti-tetanus and anti-pneumococcal (Danish serotypes 2, 6B, 8, 9V, 14, 19F and 23F) antibody titers were measured 14 and 28 days after vaccination. While there were no statistically significant differences between the dosing groups, geometric mean titers following tetanus or pneumococcal vaccination were generally lower in subjects who were vaccinated 2 weeks after receiving abatacept, compared with control subjects. A positive response (defined as a twofold increase in antibody titer from baseline) to tetanus vaccination at 28 days was seen, however, in ≥ 60% of subjects across all treatment groups versus 75% of control subjects. Similarly, over 70% of abatacept-treated subjects versus all control subjects (100%) responded to at least three pneumococcal serotypes, and approximately 25–30% of abatacept-treated subjects versus 45% of control subjects responded to at least six serotypes.     

Impact of Preceding Flu-Like Illness on the Serotype Distribution of Pneumococcal Pneumonia

Background

Even though the pathogenicity and invasiveness of pneumococcus largely depend on capsular types, the impact of serotypes on post-viral pneumococcal pneumonia is unknown.

Methods and Findings

This study was performed to evaluate the impact of capsular serotypes on the development of pneumococcal pneumonia after preceding respiratory viral infections. Patients with a diagnosis of pneumococcal pneumonia were identified. Pneumonia patients were divided into two groups (post-viral pneumococcal pneumonia versus primary pneumococcal pneumonia), and then their pneumococcal serotypes were compared. Nine hundred and nineteen patients with pneumococcal pneumonia were identified during the study period, including 327 (35.6%) cases with post-viral pneumococcal pneumonia and 592 (64.4%) cases with primary pneumococcal pneumonia. Overall, serotypes 3 and 19A were the most prevalent, followed by serotypes 19F, 6A, and 11A/11E. Although relatively uncommon (33 cases, 3.6%), infrequently colonizing invasive serotypes (4, 5, 7F/7A, 8, 9V/9A, 12F, and 18C) were significantly associated with preceding respiratory viral infections (69.7%, P<0.01). Multivariate analysis revealed several statistically significant risk factors for post-viral pneumococcal pneumonia: immunodeficiency (OR 1.66; 95% CI, 1.10–2.53), chronic lung diseases (OR 1.43; 95% CI, 1.09–1.93) and ICI serotypes (OR 4.66; 95% CI, 2.07–10.47).

Conclusions

Infrequently colonizing invasive serotypes would be more likely to cause pneumococcal pneumonia after preceding respiratory viral illness, particularly in patients with immunodeficiency or chronic lung diseases.     

Predicted functions and linkage specificities of the products of the Streptococcus pneumoniae capsular biosynthetic loci

The sequences of the capsular biosynthetic (cps) loci of 90 serotypes of Streptococcus pneumoniae have recently been determined. Bioinformatic procedures were used to predict the general functions of 1,973 of the 1,999 gene products and to identify proteins within the same homology group, Pfam family, and CAZy glycosyltransferase family. Correlating cps gene content with the 54 known capsular polysaccharide (CPS) structures provided tentative assignments of the specific functions of the different homology groups of each functional class (regulatory proteins, enzymes for synthesis of CPS constituents, polymerases, flippases, initial sugar transferases, glycosyltransferases [GTs], phosphotransferases, acetyltransferases, and pyruvyltransferases). Assignment of the glycosidic linkages catalyzed by the 342 GTs (92 homology groups) is problematic, but tentative assignments could be made by using this large set of cps loci and CPS structures to correlate the presence of particular GTs with specific glycosidic linkages, by correlating inverting or retaining linkages in CPS repeat units with the inverting or retaining mechanisms of the GTs predicted from their CAZy family membership, and by comparing the CPS structures of serotypes that have very similar cps gene contents. These large-scale comparisons between structure and gene content assigned the linkages catalyzed by 72% of the GTs, and all linkages were assigned in 32 of the serotypes with known repeat unit structures. Clear examples where very similar initial sugar transferases or glycosyltransferases catalyze different linkages in different serotypes were also identified. These assignments should provide a stimulus for biochemical studies to evaluate the reactions that are proposed.     

Construction of otherwise isogenic serotype 6B, 7F, 14, and 19F capsular variants of Streptococcus pneumoniae strain TIGR4

The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was "backcross" transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 x 10(6) to 8 x 10(6) CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.     

Analysis of Campylobacter jejuni capsular loci reveals multiple mechanisms for the generation of structural diversity and the ability to form complex heptoses

We recently demonstrated that Campylobacter jejuni produces a capsular polysaccharide (CPS) that is the major antigenic component of the classical Penner serotyping system distinguishing Campylobacter into >60 groups. Although the wide variety of C. jejuni serotypes are suggestive of structural differences in CPS, the genetic mechanisms of such differences are unknown. In this study we sequenced biosynthetic cps regions, ranging in size from 15 to 34 kb, from selected C. jejuni strains of HS:1, HS:19, HS:23, HS:36, HS:23/36 and HS:41 serotypes. Comparison of the determined cps sequences of the HS:1, HS:19 and HS:41 strains with the sequenced strain, NCTC11168 (HS:2), provides evidence for multiple mechanisms of structural variation including exchange of capsular genes and entire clusters by horizontal transfer, gene duplication, deletion, fusion and contingency gene variation. In contrast, the HS:23, HS:36 and HS:23/36 cps sequences were highly conserved. We report the first detailed structural analysis of 81-176 (HS:23/36) and G1 (HS:1) and refine the previous structural interpretations of the HS:19, HS:23, HS:36 and HS:41 serostrains. For the first time, we demonstrate the commonality and function of a second heptose biosynthetic pathway for Campylobacter CPS independent of the pathway for lipooligosaccharide (LOS) biosynthesis and identify a novel heptosyltransferase utilized by this alternate pathway. Furthermore, we show the retention of two functional heptose isomerases in Campylobacter and the sharing of a phosphatase for both LOS and CPS heptose biosynthesis.     

猪链球菌荚膜多糖合成相关基因簇保守区的克隆与分析

【目的】为了解猪链球菌各血清型荚膜多糖合成相关基因保守区的功能与基因进化关系,【方法】在分析已知的猪链球菌1、2、7、9型荚膜多糖合成相关基因簇序列,及其各orf与猪链球菌33个血清型基因组DNA杂交结果的基础上,提出猪链球菌荚膜多糖合成相关基因簇具有与肺炎链球菌相似的盒样结构的假设。并采用PCR、测序和Southern印迹杂交等方法验证这些假设。【结果】结果显示,猪链球菌的荚膜多糖合成相关基因簇确存在与肺炎链球菌相似的盒样结构,5’端的前4个调节相关基因同源性极高,基因簇两端都有保守的侧翼基因,且在3’端的侧翼序列中找到了适于扩增荚膜多糖合成相关基因簇中血清型特异性区域的下游引物所在基因(aroA)。分析发现,各血清型的orfY、orfX、cpsA、cpsB、cpsC、cpsD和aroA的亲缘关系较近。     

Capsular serotype and antibiotic resistance of Streptococcus pneumoniae isolates in Malaysia

Background

Streptococcus pneumoniae is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, that has since become a major public health concern. In this study, the serotypes distribution of pneumococcal isolates was investigated to predict the efficacy of the 7-valent pneumococcal conjugate vaccine (PCV7) among the Malaysian populations.

Methodology/Principal Findings

A total of 151 clinical isolates were serotyped using multiplex PCR assays. Out of them, there were 21.2% penicillin-resistant, 29.1% penicillin-intermediate, and 49.7% penicillin-susceptible S.pneumoniae strains. Serotypes detected among the Malaysian isolates were 1, 3, 10A, 11A/11D, 12F/12A, 14, 15A, 15B/15C, 16F, 18C/18B/18A/18F, 19A, 19F, 23F, 35B, 35F/47F, 6A/6B, 7C/7B/40, 7F/7A, 9V/9A, and 34. Serotype 19F and 23F were the two most prevalent serotypes detected. Serotypes are highly associated with invasiveness of isolates (p = 0.001) and penicillin susceptibility (p<0.001). Serotype 19F was observed to have increased resistance against penicillin while serotype 19A has high invasive tendency. Age of patients was an important factor underlying the pneumococcal serotypes (p = 0.03) and clinical sites of infections (p<0.001). High prevalence of pneumococcal isolates were detected among children <5 years old at nasopharyngeal sites while elderly adults ≥60 years old were at increased risk for pneumococcal bacteremia.

Conclusion/Significance

Current study revealed that a number of serotypes, especially those associated with high penicillin resistance, have been formulated in the PCV7. Therefore, the protections expected from the routine use of PCV7 would be encouraging for the Malaysian. However, it is not possible to predict serotypes that might become predominant in the future and hence continued surveillance of circulating serotypes will be needed.     

Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae

Serotype 19F variants of the major Spanish multiresistant serotype 23F clone of Streptococcus pneumoniae have been proposed to have arisen by recombinational exchanges at the capsular biosynthetic locus. Members of the Spanish multiresistant serotype 23F clone and the serotype 19F variants were confirmed to be essentially identical in overall genotype, as they were indistinguishable by REP-PCR, and had identical sequences at three polymorphic housekeeping genes. Eight serotype 19F variants were studied and all had large recombinational replacements at the capsular biosynthetic locus. In all cases, one of the recombinational cross-over points appeared to be upstream of dexB which flanks one end of the capsular locus, and in six of the variants the other cross-over point was downstream of aliA , which flanks the other end of the locus. In two strains a recombinational cross-over point between the introduced serotype 19F capsular region and that of the Spanish serotype 23F clone could be clearly identified, within cpsN in one strain and within cpsM in the other. The differences in the recombinational junctions and sequence polymorphisms within the introduced capsular genes, suggested that the eight serotype 19F variants emerged on at least four separate occasions. Changes in capsular type by recombination may therefore be relatively frequent in pneumococci and this has implications for the long-term efficacy of conjugate pneumococcal vaccines that will protect against only a limited number of serotypes.     

A Randomized Clinical Trial of the Immunogenicity of 7-Valent Pneumococcal Conjugate Vaccine Compared to 23-Valent Polysaccharide Vaccine in Frail,Hospitalized Elderly

Background

Elderly people do not mount strong immune responses to vaccines. We compared 23-valent capsular polysaccharide (23vPPV) alone versus 7-valent conjugate (PCV7) vaccine followed by 23vPPV 6 months later in hospitalized elderly.

Methods

Participants were randomized to receive 23vPPV or PCV7-23vPPV. Antibodies against serotypes 3, 4, 6A, 6B, 9V, 14, 18C, 19A, 19F, 23F were measured by enzyme-linked immunosorbent (ELISA) and opsonophagocytic (OPA) assays at baseline, 6 months and 12 months.

Results

Of 312 recruited, between 40% and 72% of subjects had undetectable OPA titres at baseline. After one dose, PCV7 recipients had significantly higher responses to serotypes 9V (both assays) and 23F (OPA only), and 23vPPV recipients had significantly higher responses to serotype 3 (ELISA), 19F and 19A (OPA only). In subjects with undetectable OPA titres at baseline, a proportionately greater rise in OPA titre (P<0.01) was seen for all serotypes after both vaccines. The GMT ratio of OPA was significantly higher at 12 months in the PCV7-23vPPV group for serotypes 6A, 9V, 18C and 23F. OPA titre levels for these serotypes increased moderately after 6 months, whereas immunity waned in the 23vPPV only arm.

Conclusion

We did not show overwhelming benefit of one vaccine over the other. Low baseline immunity does not preclude a robust immune response, reiterating the importance of vaccinating the frail elderly. A schedule of PCV7-23vPPV prevents waning of antibody, suggesting that both vaccines could be useful in the elderly. Follow up studies are needed to determine persistence of immunity.

Trial Registration

The Australian Clinical Trials Registry ACTRN12607000387426