13-valent pneumococcal conjugate vaccine (PCV13).

The thirteen valent pneumococcal conjugate vaccine (PCV13, Prevenar 13(TM)) is the broader coverage successor to the highly effective seven valent vaccine (PCV7, Prevenar(TM)) which has reduced rates of pneumococcal disease in many countries. Despite the success of PCV7, pneumococcal disease due to non-PCV7 serotypes remains a threat in many settings, in particular many developing countries with a high burden of pneumococcal disease where serotype 1 and 5 are among the most common serotypes. Disease due to certain non-PCV7 serotypes, in particular serotype 19A has also begun to increase in incidence in countries with widespread use of PCV7. PCV13 consists of thirteen pneumococcal capsular polysaccharides individually conjugated to the diphtheria-derived protein carrier CRM(197). In addition to serotypes 4, 6B, 9V, 14, 18C, 19F and 23F included in PCV7, PCV13 also includes serotypes 1, 3, 5, 6A, 7F and 19A. PCV13 was licensed on the basis of non-inferiority trials and has proved to be at least as safe and effective as PCV7. PCV13 replaced PCV7 in the childhood immunisation schedules of the USA and UK in 2010 and is being rolled out to an increasing number of developing countries during 2011. Here we review the current literature regarding this vaccine, describing safety, efficacy, global serotype coverage and use and future directions.

PclA, a pneumococcal collagen-like protein with selected strain distribution, contributes to adherence and invasion of host cells.

Analysis of Streptococcus pneumoniae sequenced genomes revealed a region present only in selected strains consisting of two ORFs: a putative cell wall anchored protein and a putative transcriptional regulator. The cell wall anchored protein contains large regions of collagen-like repeats, the number of which varies between strains. We have therefore named this protein PclA for pneumococcal collagen-like protein A. The second gene, spr1404, encodes a putative transcriptional regulator. We examined the strain distribution of these two genes among a collection of clinical isolates from invasive pneumococcal disease and found them to be present in 39% of the strains examined. Strains were either positive for both genes or lacked both, with the two genes always present together in the same location of the genome. RT-PCR analysis revealed that pclA is transcribed in vitro, even in the absence of spr1404. Single deletion mutants lacking either gene were not attenuated in a mouse model of invasive pneumonia. However, the pclA mutant was defective in adherence and invasion of host cells in vitro.

Patients with Chronic Obstructive Pulmonary Disease harbour a variation of Haemophilus species.

H. haemolyticus is often misidentified as NTHi due to their close phylogenetic relationship. Differentiating between the two is important for correct identification and appropriate treatment of infective organism and to ensure any role of H. haemolyticus in disease is not being overlooked. Speciation however is not completely reliable by culture and PCR methods due to the loss of haemolysis by H. haemolyticus and the heterogeneity of NTHi. Haemophilus isolates from COPD as part of the AERIS study (ClinicalTrials - NCT01360398) were speciated by analysing sequence data for the presence of molecular markers. Further investigation into the genomic relationship was carried out using average nucleotide identity and phylogeny of allelic and genome alignments. Only 6.3% were identified as H. haemolyticus. Multiple in silico methods were able to distinguish H. haemolyticus from NTHi. However, no single gene target was found to be 100% accurate. A group of omp2 negative NTHi were observed to be phylogenetically divergent from H. haemolyticus and remaining NTHi. The presence of an atypical group from a geographically and disease limited set of isolates supports the theory that the heterogeneity of NTHi may provide a genetic continuum between NTHi and H. haemolyticus.

The rise and fall of pneumococcal serotypes carried in the PCV era.

Streptococcus pneumoniae is a major cause of meningitis, sepsis and pneumonia worldwide. Vaccination using pneumococcal conjugate vaccines (PCV) has therefore been part of the UK's childhood immunisation programme since 2006. Here we describe pneumococcal carriage rates in children under five years of age attending the paediatric department of a large UK hospital in response to vaccine implementation over seven winter seasons from 2006 to 2013. S. pneumoniae (n=696) were isolated from nasopharyngeal swabs (n=2267) collected during seven consecutive winters, October to March, 2006/7 to 2012/13. This includes the period immediately following the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) in 2006 in addition to pre- and post-PCV13 introduction in 2010. We show a decrease in PCV13 vaccine serotypes (VT) in the three years following PCV13 vaccine implementation (2010/11 to 2012/13). Serotype 6A represented the only observed VT following PCV13 implementation with all others (including PCV7 serotypes) absent from carriage. Overall pneumococcal carriage, attributable to non-VT (NVT), was consistent across all sampling years with a mean of 31·1%. The ten most frequently isolated NVTs were 6C, 11A, 15B, 23B, 15A, 21, 22F, 35F, 23A and 15C. Fluctuations in the prevalence of each were however noted. Comparing prevalence at 2006/07 with 2012/13 only 15A was shown to have increased significantly (p value of 0·003) during the course of PCV implementation. These data support the increasing evidence that the primary effect of PCVs is due to population immunity by reducing or eliminating the carriage of invasive VT serotypes. With IPD being increasingly attributed to non-vaccine serotypes, surveillance of carriage data continues to act as an early warning system for vaccine design and public health policy that require continual data of both carried pneumococcal serotypes and IPD attributed serotype data.

Pneumococcal conjugate vaccine implementation in middle-income countries.

BACKGROUND: Since 2000, the widespread adoption of pneumococcal conjugate vaccines (PCVs) has had a major impact in the prevention of pneumonia. Limited access to international financial support means some middle-income countries (MICs) are trailing in the widespread use of PCVs. We review the status of PCV implementation, and discuss any needs and gaps related to low levels of PCV implementation in MICs, with analysis of possible solutions to strengthen the PCV implementation process in MICs. MAIN BODY: We searched PubMed, PubMed Central, Ovid MEDLINE, and SCOPUS databases using search terms related to pneumococcal immunization, governmental health policy or programmes, and MICs. Two authors independently reviewed the full text of the references, which were assessed for eligibility using pre-defined inclusion and exclusion criteria. The search terms identified 1,165 articles and the full texts of 21 were assessed for suitability, with eight articles included in the systematic review. MICs are implementing PCVs at a slower rate than donor-funded low-income countries and wealthier developed countries. A significant difference in the uptake of PCV in lower middle-income countries (LMICs) (71%) and upper middle-income countries (UMICs) (48%) is largely due to an unsuccessful process of "graduation" of MICs from GAVI assistance, an issue that arises as countries cross the income eligibility threshold and are no longer eligible to receive the same levels of financial assistance. A lack of country-specific data on disease burden, a lack of local expertise in economic evaluation, and the cost of PCV were identified as the leading causes of the slow uptake of PCVs in MICs. Potential solutions mentioned in the reviewed papers include the use of vaccine cost-effectiveness analysis and the provision of economic evidence to strengthen decision-making, the evaluation of the burden of disease, and post-introduction surveillance to monitor vaccine impact. CONCLUSION: The global community needs to recognise the impediments to vaccine introduction into MICs. Improving PCV access could help decrease the incidence of pneumonia and reduce the selection pressure for pneumococcal antimicrobial resistance.

Pneumococci causing invasive disease in children prior to the introduction of pneumococcal conjugate vaccine in Scotland.

This study aimed to determine the serotypes and sequence types (STs) of pneumococci causing paediatric invasive disease in Scotland prior to the introduction of pneumococcal conjugate vaccines (PCVs). All invasive pneumococci isolated between 2000 and 2004 from children aged less than 5 years in Scotland were used. The isolates were characterized by serotyping and multi-locus sequence typing. Two hundred and seventeen pneumococci were characterized into 22 different serogroups/types, the most common, in rank order, being 14, 19F, 6B, 18C, 23F, 9V, 4, 1, 19A and 6A. They were further genotyped into 77 different STs, the three most common being 9, 162 and 176. Common serotypes possessed multiple STs, but pneumococci of a particular clone were mostly associated with a particular serotype. The seven most common serotypes are included in the 7-valent polysaccharide conjugate vaccine (PCV7). Serotype coverage for PCV7 was 76.5% in those aged less than 5 years but increased to 88.9% for those aged 1 year. The introduction of PCV7 into the childhood immunization schedule would reduce the burden of pneumococcal disease in children, although continued surveillance of invasive pneumococcal disease will be required before, during and after the introduction of PCVs.

Comparative Genomics of Carriage and Disease Isolates of Streptococcus pneumoniae Serotype 22F Reveals Lineage-Specific Divergence and Niche Adaptation.

Streptococcus pneumoniae is a major cause of meningitis, sepsis, and pneumonia worldwide. Pneumococcal conjugate vaccines have been part of the United Kingdom's childhood immunization program since 2006 and have significantly reduced the incidence of disease due to vaccine efficacy in reducing carriage in the population. Here we isolated two clones of 22F (an emerging serotype of clinical concern, multilocus sequence types 433 and 698) and conducted comparative genomic analysis on four isolates, paired by Sequence Type (ST) with one of each pair being derived from carriage and the other disease (sepsis). The most compelling observation was of nonsynonymous mutations in pgdA, encoding peptidoglycan N-acetylglucosamine deacetylase A, which was found in the carriage isolates of both ST433 and 698. Deacetylation of pneumococcal peptidoglycan is known to enable resistance to lysozyme upon invasion. Althought no other clear genotypic signatures related to disease or carriage could be determined, additional intriguing comparisons between the two STs were possible. These include the presence of an intact prophage, in addition to numerous additional phage insertions, within the carriage isolate of ST433. Contrasting gene repertoires related to virulence and colonization, including bacteriocins, lantibiotics, and toxin--antitoxin systems, were also observed.

Vaccine preventable meningitis in Malaysia: epidemiology and management.

Worldwide bacterial meningitis accounts for more than one million cases and 135,000 deaths annually. Profound, lasting neurological complications occur in 9-25% of cases. This review confirms the greatest risk from bacterial meningitis is in early life in Malaysia. Much of the disease burden can be avoided by immunization, particularly against Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae. Despite inclusion of the Hib vaccine in the National Immunisation Programme and the licensure of pneumococcal vaccines, these two species are the main contributors to bacterial meningitis in Malaysia, with Neisseria meningitidis and Mycobacterium tuberculosis, causing a smaller proportion of disease. The high Hib prevalence may partly be due to dated, small-scale studies limiting the understanding of the current epidemiological situation. This highlights the need for larger, better quality surveillance from Malaysia to evaluate the success of Hib immunization and to help guide immunization policy for vaccines against S. pneumoniae and N. meningitidis.

Current methods for capsular typing of Streptococcus pneumoniae.

Streptococcus pneumoniae is a major respiratory tract pathogen causing pneumococcal disease mainly in children aged less than five years and in the elderly. Ninety-eight different capsular types (serotypes) of pneumococci have been reported, but pneumococcal conjugate vaccines (PCV) include polysaccharide antigens against only 7, 10 or 13 serotypes. It is therefore important to track the emergence of serotypes due to the clonal expansion of non-vaccine serotypes. Increased numbers of carried and disease-causing pneumococci are now being analysed as part of the post-PCV implementation surveillance studies and hence rapid, accurate and cost-effective typing methods are important. Here we describe serotyping methods published prior to 10th November 2014 for pneumococcal capsule typing. Sixteen methods were identified; six were based on serological tests using immunological properties of the capsular epitopes, eight were semi-automated molecular tests, and one describes the identification of capsular type directly from whole genome data, which also allows for further intra and inter-genome analyses. There was no single method that could be recommended for all pneumococcal capsular typing applications. Although the Quellung reaction is still considered to be the gold-standard, laboratories should take into account the number of pneumococcal isolates and the type of samples to be used for testing, the time frame for the results and the resources available in order to select the most appropriate method. Most likely, a combination of phenotypic and genotypic methods would be optimal to monitor and evaluate the impact of pneumococcal conjugate vaccines and to provide information for future vaccine formulations.

Distribution of carried pneumococcal clones in UK children following the introduction of the 7-valent pneumococcal conjugate vaccine: a 3-year cross-sectional population based analysis.

The success of Streptococcus pneumoniae (pneumococcus) in both colonisation and disease is associated with the increased prevalence of genetic clones expressing virulence factors that assist host invasion. We studied the distribution of pneumococcal clones in paediatric carriage as part of an ongoing longitudinal study of pneumococcal carriage in children less than 5 years of age. Across three years, 87 different sequence types (STs) were found amongst 310 pneumococci. A decline in PCV-7 related STs was observed during the study period. STs 62, 199, 433 and 1692 increased after the implementation of PCV-7 and were related to increases in serotypes 11A, 19A, 22F, and to serotype 6C, respectively. Overall, a strong correlation was observed between ST and serotype. Thirteen STs contained multiple serotypes and 74 STs were associated with only one serotype. On-going molecular epidemiological surveillance of pneumococcal carriage is warranted during the implementation of pneumococcal conjugate vaccines.

Clonal expansion within pneumococcal serotype 6C after use of seven-valent vaccine.

Streptococcus pneumoniae causes invasive infections, primarily at the extremes of life. A seven-valent conjugate vaccine (PCV7) is used to protect against invasive pneumococcal disease in children. Within three years of PCV7 introduction, we observed a fourfold increase in serotype 6C carriage, predominantly due to a single clone. We determined the whole-genome sequences of nineteen S. pneumoniae serotype 6C isolates, from both carriage (n = 15) and disease (n = 4) states, to investigate the emergence of serotype 6C in our population, focusing on a single multi-locus sequence type (MLST) clonal complex 395 (CC395). A phylogenetic network was constructed to identify different lineages, followed by analysis of variability in gene sets and sequences. Serotype 6C isolates from this single geographical site fell into four broad phylogenetically distinct lineages. Variation was seen in the 6C capsular locus and in sequences of genes encoding surface proteins. The largest clonal complex was characterised by the presence of lantibiotic synthesis locus. In our population, the 6C capsular locus has been introduced into multiple lineages by independent capsular switching events. However, rapid clonal expansion has occurred within a single MLST clonal complex. Worryingly, plasticity exists within current and potential vaccine-associated loci, a consideration for future vaccine use, target selection and design.

Risk of red queen dynamics in pneumococcal vaccine strategy.

Pathogens increasingly evade current vaccines, and new strategies to control them are needed. There is mounting evidence that replacement of vaccine serotypes of Streptococcus pneumoniae with non-vaccine serotypes has taken place following widespread use of limited-serotype conjugate vaccines. New strategies to control vaccine evasion are needed and understanding evolutionary theory is important for the development of such approaches. Hosts are under selection pressure to evolve resistance against pathogens whereas pathogens are under selection pressure to evolve counter-resistance against the resistance mechanism of their host. Evolutionary changes in both host and pathogen lead to a continuous turnover of host and pathogen genotypes; this is known as Red Queen dynamics. We argue that integrating evolutionary thinking into pneumococcal vaccine design will lead to the avoidance of Red Queen dynamics and improved interventions against pneumococci.

Declining serotype coverage of new pneumococcal conjugate vaccines relating to the carriage of Streptococcus pneumoniae in young children.

BACKGROUND: Asymptomatic carriage of the opportunistic pathogen Streptococcus pneumoniae is known to precede the development of invasive disease. Young children are one of the major reservoirs for pneumococci and worldwide over 700,000 children under two years old die due to invasive pneumococcal disease each year. Heptavalent conjugate vaccine (PCV-7) was introduced into the UK childhood immunisation schedule in September 2006. Our objective was to assess the emergence of colonising serotypes in young children in the three years following PCV-7 implementation. METHODS: Time-series prevalence survey set in the paediatric outpatients department of a large UK teaching hospital. Participants were children aged four years and under attending the outpatients department during PCV-7 introduction (October 2006-February 2007) and in the same months of the two subsequent years. The main outcome measure was prevalence of pneumococcal carriage by serotype. RESULTS: The rate of pneumococcal nasopharyngeal carriage remained stable during the three year period. We observed a significant 69% (95% CI, -40% to -118%, p<0.0001) decrease in carriage of PCV-7 serotypes during PCV-7 implementation and a concomitant increase in the proportion of non PCV-7 serotypes. The most prevalent emerging non-vaccine serotypes were 6C, 11A, 19A and 22F. By March 2009, PCV-13 was predicted to cover only 33.3% (95% CI, 24.2-42.5%) of strains carried in the study population. CONCLUSIONS: Although the overall pneumococcal carriage rate remained stable between 2006 and 2009, we observed a significant decrease in the serotype coverage of PCV-7 and PCV-13. PCV-7 was highly successful in reducing carriage of vaccine serotypes. However, the increase in the proportion of non-vaccine serotypes found both in our study and causing invasive disease currently in the UK, underlines the importance of continued surveillance of carriage and disease.

Death or survival from invasive pneumococcal disease in Scotland: associations with serogroups and multilocus sequence types.

We describe associations between death from invasive pneumococcal disease (IPD) and particular serogroups and sequence types (STs) determined by multilocus sequence typing (MLST) using data from Scotland. All IPD episodes where blood or cerebrospinal fluid (CSF) culture isolates were referred to the Scottish Haemophilus, Legionella, Meningococcal and Pneumococcal Reference Laboratory (SHLMPRL) from January 1992 to February 2007 were matched to death certification records by the General Register Office for Scotland. This represented 5959 patients. The median number of IPD cases in Scotland each year was 292. Deaths, from any cause, within 30 days of pneumococcal culture from blood or CSF were considered to have IPD as a contributing factor. Eight hundred and thirty-three patients died within 30 days of culture of Streptococcus pneumoniae from blood or CSF [13.95 %; 95 % confidence interval (13.10, 14.80)]. The highest death rates were in patients over the age of 75. Serotyping data exist for all years but MLST data were only available from 2001 onward. The risk ratio of dying from infection due to particular serogroups or STs compared to dying from IPD due to all other serogroups or STs was calculated. Fisher's exact test with Bonferroni adjustment for multiple testing was used. Age adjustment was accomplished using the Cochran-Mantel-Haenszel test and 95 % confidence intervals were reported. Serogroups 3, 11 and 16 have increased probability of causing fatal IPD in Scotland while serogroup 1 IPD has a reduced probability of causing death. None of the 20 most common STs were significantly associated with death within 30 days of pneumococcal culture, after age adjustment. We conclude that there is a stronger association between a fatal outcome and pneumococcal capsular serogroup than there is between a fatal outcome and ST.

Presence of nonhemolytic pneumolysin in serotypes of Streptococcus pneumoniae associated with disease outbreaks.

Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumoniae.

Identification of invasive serotype 1 pneumococcal isolates that express nonhemolytic pneumolysin.

Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of S. pneumoniae, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing "wild-type" pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.

Genomic diversity between strains of the same serotype and multilocus sequence type among pneumococcal clinical isolates.

The important human pathogen Streptococcus pneumoniae is known to be a genetically diverse species. We have used comparative genome hybridization (CGH) microarray analysis to investigate this diversity in a collection of clinical isolates including several capsule serotype 14 pneumococci, a dominant serotype among disease isolates. We have identified three new regions of diversity among pneumococcal isolates and, importantly, clearly demonstrate genetic differences between strains of the same multilocus sequence type (ST) and capsule serotype. CGH may therefore, under certain circumstances, prove to be a valuable tool to supplement current typing methods. Finally, we show that these clonal strains with the same serotype and ST behave differently in an animal model. Strains of the same ST and serotype therefore have important genetic and phenotypic differences.

Genetic analysis of diverse disease-causing pneumococci indicates high levels of diversity within serotypes and capsule switching.

We have used multilocus sequence typing (MLST) and serotyping to build a phylogenetic framework for pneumococcal disease isolates in Scotland that provides a snapshot of the relationships between capsular type and genotype. The results show that while the MLST type correlates with the serotype, isolates within a serotype can belong to a number of individual clonal complexes or sequence types (STs). We also show that isolates of the same ST can express different capsular polysaccharides, i.e., display capsular switching, and that this phenomenon is observed both for capsular types commonly isolated from patients with invasive disease and for serogroups less commonly isolated from patients with invasive disease but which may commonly be carried asymptomatically in the human nasopharynx.