Multiple Pneumococcal Serotypes Research Articles

Association between nasopharyngeal colonization with multiple pneumococcal serotypes and total pneumococcal colonization density in young Peruvian children

ObjectivesWe examined the association of nasopharyngeal (NP) pneumococcal co-colonization (>1 pneumococcal serotype) and pneumococcal density in young Peruvian children enrolled in a prospective cohort study. MethodsNP swabs collected monthly from children aged <3 years during both asymptomatic and acute respiratory illness (ARI) periods underwent culture-enriched microarray for pneumococcal detection and serotyping and lytA polymerase chain reaction for density assessment. We examined the serotypes commonly associated with co-colonization and the distribution of densities by co-colonization, age, current ARI, and other covariates. The association of co-colonization and pneumococcal density was assessed using a multivariable mixed-effects linear regression model, accounting for repeated measures and relevant covariates. ResultsA total of 27 children contributed 575 monthly NP samples. Pneumococcus was detected in 302 of 575 (53%) samples, and co-colonization was detected in 61 of these 302 (20%). The total densities were higher during ARI than non-ARI periods and lowest among the youngest children, increasing with age. In the multivariable analysis, there was no significant association between pneumococcal density and co-colonization (coefficient estimate 0.22, 95% confidence interval 0.11-0.55; reference: single-serotype detections). Serotypes 23B and 19F were detected significantly more frequently as single isolates. ConclusionPneumococcal co-colonization was common and not associated with increased pneumococcal density. Differential propensity for co-colonization was observed among individual serotypes.

Comparison of traditional culture and molecular qPCR for detection of simultaneous carriage of multiple pneumococcal serotypes in African children

S. pneumoniae is a common colonizer of the human nasopharynx in high income and low-middle income countries. Due to limitations of standard culture methods, the prevalence of concurrent colonization with multiple serotypes is unclear. We evaluated the use of multiplex quantitative PCR (qPCR) to detect multiple pneumococcal serotypes/group colonization in archived nasopharyngeal swabs of pneumococcal conjugate vaccine naive children who had previously been investigated by traditional culture methods. Overall the detection of pneumococcal colonization was higher by qPCR (82%) compared to standard culture (71%; p < 0.001), with a high concordance (kappa = 0.73) of serotypes/groups identified by culture also being identified by qPCR. Also, qPCR was more sensitive in detecting multiple serotype/groups among colonized cases (28.7%) compared to culture (4.5%; p < 0.001). Of the additional serotypes detected only by qPCR, the majority were of lower density (<104 CFU/ml) than the dominant colonizing serotype, with serotype/group 6A/B, 19B/F and 23F being the highest density colonizers, followed by serotype 5 and serogroup 9A/L/N/V being the most common second and third colonizers respectively. The ability of qPCR to detect multiple pneumococcal serotypes at a low carriage density might provide better insight into underlying mechanism for changes in serotype colonization in PCV vaccinated children.

High multiple carriage and emergence of Streptococcus pneumoniae vaccine serotype variants in Malawian children.

BackgroundCarriage of either single or multiple pneumococcal serotypes (multiple carriage) is a prerequisite for developing invasive pneumococcal disease. However, despite the reported high rates of pneumococcal carriage in Malawi, no data on carriage of multiple serotypes has been reported previously. Our study provides the first description of the prevalence of multiple pneumococcal carriage in Malawi.MethodsThe study was conducted in Blantyre and Karonga districts in Malawi, from 2008 to 2012. We recruited 116 children aged 0–13 years. These children were either HIV-infected (N = 44) or uninfected (N = 72). Nasopharyngeal samples were collected using sterile swabs. Pneumococcal serotypes in the samples were identified by microarray. Strains that could not be typed by microarray were sequenced to characterise possible genetic alterations within the capsular polysaccharide (CPS) locus.ResultsThe microarray identified 179 pneumococcal strains (from 116 subjects), encompassing 43 distinct serotypes and non-typeable (NT) strains. Forty per cent (46/116) of children carried multiple serotypes. Carriage of vaccine type (VT) strains was higher (p = 0.028) in younger (0–2 years) children (71 %, 40/56) compared to older (3–13 years) children (50 %, 30/60). Genetic variations within the CPS locus of known serotypes were observed in 19 % (34/179) of the strains identified. The variants included 13-valent pneumococcal conjugate vaccine (PCV13) serotypes 6B and 19A, and the polysaccharide vaccine serotype 20. Serotype 6B variants were the most frequently isolated (47 %, 16/34). Unlike the wild type, the CPS locus of the 6B variants contained an insertion of the licD-family phosphotransferase gene. The CPS locus of 19A- and 20-variants contained an inversion in the sugar-biosynthesis (rmlD) gene and a 717 bp deletion within the transferase (whaF) gene, respectively.ConclusionsThe high multiple carriage in Malawian children provides opportunities for genetic exchange through horizontal gene transfer. This may potentially lead to CPS locus variants and vaccine escape. Variants reported here occurred naturally, however, PCV13 introduction could exacerbate the CPS genetic variations. Further studies are therefore recommended to assess the invasive potential of these variants and establish whether PCV13 would offer cross-protection. We have shown that younger children (0–2 years) are a reservoir of VT serotypes, which makes them an ideal target for vaccination.

Multi-serotype pneumococcal nasopharyngeal carriage prevalence in vaccine naïve Nepalese children, assessed using molecular serotyping.

Invasive pneumococcal disease is one of the major causes of death in young children in resource poor countries. Nasopharyngeal carriage studies provide insight into the local prevalence of circulating pneumococcal serotypes. There are very few data on the concurrent carriage of multiple pneumococcal serotypes. This study aimed to identify the prevalence and serotype distribution of pneumococci carried in the nasopharynx of young healthy Nepalese children prior to the introduction of a pneumococcal conjugate vaccine using a microarray-based molecular serotyping method capable of detecting multi-serotype carriage. We conducted a cross-sectional study of healthy children aged 6 weeks to 24 months from the Kathmandu Valley, Nepal between May and October 2012. Nasopharyngeal swabs were frozen and subsequently plated on selective culture media. DNA extracts of plate sweeps of pneumococcal colonies from these cultures were analysed using a molecular serotyping microarray capable of detecting relative abundance of multiple pneumococcal serotypes. 600 children were enrolled into the study: 199 aged 6 weeks to <6 months, 202 aged 6 months to < 12 months, and 199 aged 12 month to 24 months. Typeable pneumococci were identified in 297/600 (49·5%) of samples with more than one serotype being found in 67/297 (20·2%) of these samples. The serotypes covered by the thirteen-valent pneumococcal conjugate vaccine were identified in 44·4% of samples containing typeable pneumococci. Application of a molecular serotyping approach to identification of multiple pneumococcal carriage demonstrates a substantial prevalence of co-colonisation. Continued surveillance utilising this approach following the introduction of routine use of pneumococcal conjugate vaccinates in infants will provide a more accurate understanding of vaccine efficacy against carriage and a better understanding of the dynamics of subsequent serotype and genotype replacement.

New Pneumococcal Diagnostics---further ahead or more confused?

Invited Lecture AbstractsISPPD-0205 New Pneumococcal Diagnostics---further ahead or more confused?: USEFULNESS OF SEROTYPE SPECIFIC URINARY ANTIGEN DETECTION (UAD) ASSAY TO DETECT PNEUMOCOCCAL PNEUMONIA IN ADULTSW. Albrich1, M.W. Pride2, S.A. Madhi3, J. Callahan4, P.V. Adrian3, R. French5, N. van Niekerk6, S. Sebastian2, V. Souza2, K.U. Jansen2, K.P. Klugman31Division of Infectious Diseases and Hospital Epidemiology, Cantonal Hospital St. Gallen, St. Gallen, Switzerland; 2Pfizer Vaccine Research and Early Development, Pfizer Inc., Pearl River, USA; 3Respiratory and Meningeal Pathogens Research Unit/Medical Research Council, National Institute for Communicable Diseases, Johannesburg, South Africa; 4Callahan Associates Inc, Callahan Associates Inc, La Jolla, USA; 5Biotechnology Clinical Development Statistics, Pfizer Inc., Pearl River, USA; 6Respiratory and Meningeal Pathogens Research Unit/Medical Research Council, Respiratory and Meningeal Pathogens Research Unit/Medical Research Council, Johannesburg, South AfricaBackground and Aims: A urinary antigen detection assay (UAD) for 13 pneumococcal serotypes was recently reported as a useful diagnostic tool for pneumococcal pneumonia. We tested this assay in a well-defined cohort of HIV-infected South African adults with X-ray confirmed pneumonia.Methods: A Luminex xMAP bead-based multiplex UAD detecting the PCV13 serotypes was tested retrospectively in HIV-infected South African adults with pneumonia. UAD positivity cut-offs were established in HIV-infected South Africans without respiratory disease. Pneumonia was considered pneumococcal if a composite diagnostic including any of sputum Gram stain, sputum culture, blood culture or urinary Binax was positive.Results: The UAD identified 6/7 (85.7%) patients with bacteremia due to a PCV13 serotype and did not misclassify any of 3 patients with bacteremia due to a non-PCV13 serotype. Even though the UAD detects only 13 serotypes, it detected more cases of presumed pneumococcal pneumonia among X — ray confirmed cases than the serotype-independent urine Binax (106/235 (45.1%) vs. 52/235 (22.1%); p < 0.001). The UAD was positive in 52/71 (73.2%) patients with a positive composite diagnostic suggesting that 73% of disease was due to PCV13 vaccine types and overall detected significantly more cases than the composite (106/235 (45.1%) vs 71/235 (30.2%); p < 0.001). No correlation was detected between nasopharyngeal colonization density measured by lytA rtPCR and quantitative UAD values.Conclusions: The UAD compared more favourably with other diagnostic tests for pneumococcal pneumonia in adults and may become a new standard for detection of pneumococcal pneumonia in adults.Conflict of interestISPPD-0569 New Pneumococcal Diagnostics---further ahead or more confused?: DISSECTING THE COMPLEXITY OF CARRIAGE: MOLECULAR MINING BY MICROARRAYJ. Hinds11Bacterial Microarray Group, St George’s University of London, London, United KingdomNasopharyngeal carriage represents the natural host reservoir from which Streptococcus pneumoniae may achieve transmission to a new host or cause invasive disease. The biology of the pneumococcus has evolved for successful colonization of this complex niche, including strategies to avoid host defences and co-exist with other microbes. The dynamic microbial community of the nasopharynx is in constant flux and presents opportunities for genetic exchange, both impacting on the effectiveness of pneumococcal vaccines and antimicrobial resistance. Diagnostic methods have revealed key insights into carriage of co-colonizing species and pneumococcal serotypes. As nasopharyngeal carriage is a precursor to invasive pneumococcal disease and has a role in herd immunity, monitoring the effect of vaccines on carriage is especially important. In the era of polysaccharide conjugate vaccines, serotyping has been a key technique to study the impact upon circulating vaccine and non-vaccine serotypes. The microarray-based molecular serotyping method has shown high specificity and sensitivity for detection of multiple pneumococcal serotypes in carriage samples. Enhanced detection of additional serotypes at low abundance may affect metrics used in assessing vaccine efficacy, such as acquisition and duration of carriage, and also case:carrier ratios. Furthermore, the ability to detect serotype variants and non-typeables along with closely related species, antimicrobial resistance determinants and pneumococcal genotype provide valuable supplementary information. This combines to help dissect the complex nasopharyngeal niche and also contextualise an individual sample within other local or global studies.Conflict of interest

A survey of maternal pertussis vaccine uptake in England

Pneumococcal surface protein A (PspA) Protective aganist multiple pneumococcal serotypes in mice models of invasive disease pneumonia, and colonization. Vaccination of healthy volunteers with PspA generated antibodies aganist multiple strains that protected mice aganist invasive disease. Human studies in progess. Pneumolysins.Pneumolysoids Protection aganist Pneuminia and increased survival after intraperitoneal challenge. human studies in progress Neuraminidases (Nan) Protection aganist colonization and otitis media Pneumococcal surface protein C (PspC) Protection aganist colonization. Protection aganist sepsis Pneumococcal protective protein A (PppA) Protection aganist lung infection. Protective aganist nasopharyngeal colonization. Pneumococcal Histidine Trial Protein D Protective aganist animal models of invasive disease and pneumonia phase II trial reports awaited. IC4 antigens Protection aganist sepsis, pneumonia, nasopharyngeal carriage.

Field Evaluation of Culture plus Latex Sweep Serotyping for Detection of Multiple Pneumococcal Serotype Colonisation in Infants and Young Children

BackgroundNasopharyngeal swab (NPS) culture by World Health Organisation (WHO) methodology underestimates multiple pneumococcal serotype colonisation compared to a simple culture and latex sweep method. The impacts of this on descriptions of pneumococcal serotype distributions and colonisation dynamics in infancy are not clear.Methods8,736 NPS collected from infants enrolled into a longitudinal study were processed to evaluate the field utility of the latex sweep method. 1,107 had previously been cultured by WHO methodology. Additionally, colonisation results were compared in 100 matched pairs of infants, where swabs from an individual were cultured either by WHO or latex sweep method.ResultsIn 1,107 swabs cultured by both methods, the latex sweep method was three times more likely to detect colonisation with multiple pneumococcal serotypes than the WHO method (p<0.001). At least one common serotype was identified in 91.2% of swabs from which typeable pneumococci were detected by both methods. Agreement improved with increasing colonisation density (p = 0.03). Estimates of age at first pneumococcal acquisition and colonisation duration were not affected by culture/serotyping method. However, a greater number of serotype carriage episodes were detected in infants cultured by latex sweep (p = 0.03). The overall rate of non-vaccine type pneumococcal acquisition was also greater in infants cultured by latex sweep (p = 0.04).ConclusionsLatex sweep serotyping was feasible to perform on a large specimen collection. Multiple serotype colonisation detection was significantly improved compared with WHO methodology. However, use of the latex sweep method is unlikely to significantly alter colonisation study serotype distribution or colonisation dynamics results.

Pneumococcal Transmission and Disease In Silico: A Microsimulation Model of the Indirect Effects of Vaccination

BackgroundThe degree and time frame of indirect effects of vaccination (serotype replacement and herd immunity) are key determinants in assessing the net effectiveness of vaccination with pneumococcal conjugate vaccines (PCV) in control of pneumococcal disease. Using modelling, we aimed to quantify these effects and their dependence on coverage of vaccination and the vaccine's efficacy against susceptibility to pneumococcal carriage.Methods and FindingsWe constructed an individual-based simulation model that explores the effects of large-scale PCV programmes and applied it in a developed country setting (Finland). A population structure with transmission of carriage taking place within relevant mixing groups (families, day care groups, schools and neighbourhoods) was considered in order to properly assess the dependency of herd immunity on coverage of vaccination and vaccine efficacy against carriage. Issues regarding potential serotype replacement were addressed by employing a novel competition structure between multiple pneumococcal serotypes. Model parameters were calibrated from pre-vaccination data about the age-specific carriage prevalence and serotype distribution. The model predicts that elimination of vaccine-type carriage and disease among those vaccinated and, due to a substantial herd effect, also among the general population takes place within 5–10 years since the onset of a PCV programme with high (90%) coverage of vaccination and moderate (50%) vaccine efficacy against acquisition of carriage. A near-complete replacement of vaccine-type carriage by non-vaccine-type carriage occurs within the same time frame.ConclusionsThe changed patterns in pneumococcal carriage after PCV vaccination predicted by the model are unequivocal. The overall effect on disease incidence depends crucially on the magnitude of age- and serotype-specific case-to-carrier ratios of the remaining serotypes relative to those of the vaccine types. Thus the availability of reliable data on the incidence of both pneumococcal carriage and disease is essential in assessing the net effectiveness of PCV vaccination in a given epidemiological setting.

Development and Characterization of a Multiplex Bead-Based Immunoassay To Quantify Pneumococcal Capsular Polysaccharide-Specific Antibodies

Enzyme-linked immunosorbent assay (ELISA), the traditional antibody quantification technique, has several limitations, especially when used to evaluate multivalent and/or infant vaccines. We have developed a multiplex bead-based antibody quantification assay (MBIA) to measure antibody response to multiple pneumococcal (Pn) serotypes (St) in a single assay. MBIA was compared with the WHO ELISA using a WHO panel of 12 international calibration sera for 7 Pn Sts. An agreement of 75 to 92% was obtained for all 7 Sts. MBIA exhibited good robustness, with the assay variability at ≤ 16%. A major contributor to MBIA variability was the cell wall polysaccharide (CWPs) content in Pn St-specific capsular Ps. This necessitated careful CWPs (20 μg/ml) preadsorption of sera. MBIA is specific, robust, and reproducible and offers high throughput. The use of MBIA will greatly reduce the cost and time required to evaluate the immune response to multiple Pn Sts and could help promote the licensure of future Pn and other multivalent vaccines.

Contribution of vaccines to our understanding of pneumococcal disease

Pneumonia is the leading cause of mortality in children in developing countries and is also the leading infectious cause of death in adults. The most important cause of pneumonia is the Gram-positive bacterial pathogen, Streptococcus pneumoniae, also known as the pneumococcus. It has thus become the leading vaccine-preventable cause of death and is a successful and diverse human pathogen. The development of conjugate pneumococcal vaccines has made possible the prevention of pneumococcal disease in infants, but has also elucidated aspects of pneumococcal biology in a number of ways. Use of the vaccine as a probe has increased our understanding of the burden of pneumococcal disease in children globally. Vaccination has also elucidated the clinical spectrum of vaccine-preventable pneumococcal infections; the identification of a biological niche for multiple pneumococcal serotypes in carriage and the differential invasiveness of pneumococcal serotypes; the impact of pneumococcal transmission among children on disease burden in adults; the role of carriage as a precursor to pneumonia; the plasticity of a naturally transformable pathogen to respond to selective pressure through capsular switching and the accumulation of antibiotic-resistance determinants; and the role of pneumococcal infections in hospitalization and mortality associated with respiratory viral infections, including both seasonal and pandemic influenza. Finally, there has been a recent demonstration that pneumococcal pneumonia in children may be an important cause of hospitalization for those with underlying tuberculosis.

Improved Detection of Nasopharyngeal Cocolonization by Multiple Pneumococcal Serotypes by Use of Latex Agglutination or Molecular Serotyping by Microarray

Identification of Streptococcus pneumoniae in the nasopharynx is critical for an understanding of transmission, estimates of vaccine efficacy, and possible replacement disease. Conventional nasopharyngeal swab (NPS) culture and serotyping (the WHO protocol) is likely to underestimate multiple-serotype carriage. We compared the WHO protocol with methods aimed at improving cocolonization detection. One hundred twenty-five NPSs from an infant pneumococcal-carriage study, containing ≥ 1 serotype by WHO culture, were recultured in duplicate. A sweep of colonies from one plate culture was serotyped by latex agglutination. DNA extracted from the second plate was analyzed by S. pneumoniae molecular-serotyping microarray. Multiple serotypes were detected in 11.2% of the swabs by WHO culture, 43.2% by sweep serotyping, and 48.8% by microarray. Sweep and microarray were more likely to detect multiple serotypes than WHO culture (P < 0.0001). Cocolonization detection rates were similar between microarray and sweep, but the microarray identified the greatest number of serotypes. A common serogroup type was identified in 95.2% of swabs by all methods. WHO methodology significantly underestimates multiple-serotype carriage compared to these alternate methods. Sweep serotyping is cost-effective and field deployable but may fail to detect serotypes at low abundance, whereas microarray serotyping is more costly and technology dependent but may detect these additional minor carried serotypes.

Efficacy of Whole‐Cell Killed Bacterial Vaccines in Preventing Pneumonia and Death during the 1918 Influenza Pandemic

Most deaths in the 1918 influenza pandemic were caused by secondary bacterial pneumonia. We performed a systematic review and reanalysis of studies of bacterial vaccine efficacy (VE) in preventing pneumonia and mortality among patients with influenza during the 1918 pandemic. A meta-analysis of 6 civilian studies of mixed killed bacterial vaccines containing pneumococci identified significant heterogeneity among studies and estimated VE at 34% (95% confidence interval [CI], 19%-47%) in preventing pneumonia and 42% (95% CI, 18%-59%) in reducing case fatality rates among patients with influenza, using random-effects models. Using fixed-effect models, the pooled VE from 3 military studies was 59% (95% CI, 43%-70%) for pneumonia and 70% (95% CI, 50%-82%) for case fatality. Military studies showed less heterogeneity and may provide more accurate results than civilian studies, given the potential biases in the included studies. Findings of 1 military study using hemolytic streptococci also suggested that there was significant protection. Despite significant methodological problems, the systematic biases in these studies do not exclude the possibilities that whole-cell inactivated pneumococcal vaccines may confer cross-protection to multiple pneumococcal serotypes and that bacterial vaccines may play a role in preventing influenza-associated pneumonia.

Multiplex PCR reveals a high rate of nasopharyngeal pneumococcal 7-valent conjugate vaccine serotypes co-colonizing indigenous Warao children in Venezuela

Knowledge of co-colonization with multiple pneumococcal serotypes is becoming very important in the light of both serotype replacement and switching as a result of vaccination. Co-colonization has been reported to occur in up to 30% of carriers, especially in populations with high Streptococcus pneumoniae carriage rates. For the determination of co-colonization, single colonies of nasopharyngeal specimens are serotyped with the Quellung method, a costly method with a low sensitivity. Here we explore the use of a multiplex PCR to identify simultaneous carriage of the capsular serotypes targeted by the 7-valent conjugate vaccine. We applied this multiplex PCR to 50 primary cultures from the nasopharyngeal swabs of healthy Warao Amerindian children, a population with a high pneumococcal carriage rate, most of them with vaccine serotypes, and we identified a second serotype in 20% (n=10) of the pneumococci carriers. These results were confirmed by detailed serotyping of multiple colonies isolated from the primary culture with the Quellung method. We conclude that the multiplex PCR is a sensitive, simple and cost-effective method for detecting multiple serotypes in nasopharyngeal cultures, and thus might be useful for the monitoring of pneumococcal colonization over time, especially in the surveillance of nasopharyngeal colonization after conjugate vaccination.

Mucosal Immunization with Polyamine Transport Protein D (PotD) Protects Mice Against Nasopharyngeal Colonization with Streptococcus pneumoniae

Streptococcus pneumoniae is an encapsulated pathogen that can cause invasive disease following colonization of the nasopharynx. Targeting colonization of mucosal surfaces may, therefore, be the best approach for vaccination to prevent pneumococcal invasive disease. Previous studies in our laboratory have shown that immunization with recombinant polyamine transport protein D (PotD) protects mice against systemic pneumococcal infections. In this study we investigated the efficacy of mucosal immunization with rPotD to protect against pneumococcal carriage and invasion in a murine model. Mice were intranasally immunized with either rPotD and cholera toxin B subunit (CTB) or CTB alone. Significantly less pneumococci were recovered from the nasopharynx of immunized mice compared to the control animals following intranasal challenge with either EF3030 (serotype 19F) (P < 0.05) or an invasive serotype 4 isolate (TIGR4) (P < 0.05). PotD immunized mice also had lesser bacteria in their sinus tissues (P < 0.05), brains (P < 0.05), lungs and olfactory bulbs following intranasal challenge with TIGR4. ELISA analysis demonstrated the presence of IgG antibodies to PotD in the serum and IgA antibodies in the saliva. These results indicate that mucosal immunization with PotD generates both mucosal and systemic immune responses and prevents establishment of nasopharyngeal carriage by multiple pneumococcal serotypes. Thus, PotD is a potentially important antigen for development of a pneumococcal protein vaccine.

A minimally hypomorphic mutation in Btk resulting in reduced B cell numbers but no clinical disease

Reduced B cell numbers and a mutation in Btk are considered sufficient to make the diagnosis of X-linked agammaglobulinaemia. In the process of conducting family studies, we identified a 58-year-old healthy man with an amino acid substitution, Y418H, in the adenosine-5'-triphosphate binding site of Btk. Immunofluorescence studies showed that this man had 0.85% CD19+ B cells (normal 4-18%) in the peripheral circulation and his monocytes were positive for Btk. He had borderline low serum immunoglobulins but normal titres to tetanus toxoid and multiple pneumococcal serotypes. To determine the functional consequences of the amino acid substitution, a Btk- chicken B cell line, DT40, was transfected with expression vectors producing wild-type Btk or Y418H Btk. The transfected cells were stimulated with anti-IgM and calcium flux and inositol triphosphate (IP3) production were measured. Cells bearing the mutant protein demonstrated consistently a 15-20% decrease in both calcium flux and IP3 production. These findings indicate that even a modest decrease in Btk function can impair B cell proliferation or survival. However, a mutation in Btk and reduced numbers of B cells are not always associated with clinical disease.

Emergence of Streptococcus pneumoniae with very-high-level resistance to penicillin.

Penicillin resistance threatens the treatment of pneumococcal infections. We used sentinel hospital surveillance (1978 to 2001) and population-based surveillance (1995 to 2001) in seven states in the Active Bacterial Core surveillance of the Emerging Infections Program Network to document the emergence in the United States of invasive pneumococcal isolates with very-high-level penicillin resistance (MIC > or = 8 microg/ml). Very-high-level penicillin resistance was first detected in 1995 in multiple pneumococcal serotypes in three regions of the United States. The prevalence increased from 0.56% (14 of 2,507) of isolates in 1995 to 0.87% in 2001 (P = 0.03), with peaks in 1996 and 2000 associated with epidemics in Georgia and Maryland. For a majority of the strains the MICs of amoxicillin (91%), cefuroxime (100%), and cefotaxime (68%), were > or =8 microg/ml and all were resistant to at least one other drug class. Pneumonia (50%) and bacteremia (36%) were the most common clinical presentations. Factors associated with very highly resistant infections included residence in Tennessee, age of <5 or > or =65 years, and resistance to at least three drug classes. Hospitalization and case fatality rates were not higher than those of other pneumococcal infection patients; length of hospital stay was longer, controlling for age. Among the strains from 2000 and 2001, 39% were related to Tennessee(23F)-4 and 35% were related to England(14-)9. After the introduction of the pneumococcal conjugate vaccine, the incidence of highly penicillin resistant infections decreased by 50% among children <5 years of age. The emergence, clonality, and association of very-high-level penicillin resistance with multiple drug resistance requires further monitoring and highlights the need for novel agents active against the pneumococcus.

Colony blot assay: a useful method to detect multiple pneumococcal serotypes within clinical specimens

The efficacy of pneumococal conjugate vaccines in young children may be complicated by serotype replacement. We developed a colony blot assay which enables the identification of re-colonization with novel serotypes (replacement), overgrowth by minor co-colonizing serotypes or suppression of previously predominant vaccine serotype strains as a result of vaccination. This method allows the identification of multiple serotypes in a single specimen in a ratio of 1:1000. In order to demonstrate the potential of our method, we investigated the consecutive nasopharyngeal samples of 26 children who had shown a shift in pneumococcal colonization after conjugate vaccination. Mixed colonization was found once in 15 pre-vaccination samples and four times in 26 post-vaccination samples. In the remaining children `true replacement' had presumably occurred. Hence, we conclude that the colony blot assay is an easy to apply method, which allows the identification of different pneumococcal serotypes within single clinical specimens.

Clinical correlates of response to pneumococcal immunization.

The inability to form antibodies to polysaccharide antigens may occur as a part of a more significant immunodeficiency or as an isolated defect. The latter has been reported in some children with recurrent upper and lower respiratory tract infections and evaluation of the responsiveness of such patients to polysaccharide antigens is indicated as part of their assessment. The present study evaluated the pattern of antibody responses of patients immunized with pneumococcal vaccine as part of the investigation of recurrent upper and lower respiratory tract infections to determine if any correlation exists between these responses and clinical presentation. An analysis was performed of antibody responses to pneumococcal serotypes 3, 4 and 6 following immunization with a 23-valent vaccine in 42 children with normal IgG levels who were evaluated for recurrent infections. Antibody responses were assessed in relation to clinical features and the results of other immunological investigations. Of the 42 patients evaluated, 25 (59%) were responders to all serotypes tested. Failure to respond to serotype 3 alone was the least common pattern of non-response. Recurrent pneumonia, but not otitis media with discharge or chronic productive cough, was significantly associated with a lack of response to two or three serotypes. Failure to respond to serotype 3 alone or in combination with other serotypes was associated with more significant immune abnormalities. In a selected population of children with recurrent bacterial infections, pneumococcal serotype 3 is a strong immunogen. In this clinical group recurrent pneumonia is associated with a defect in response to multiple pneumococcal serotypes.

A latex bead-based flow cytometric immunoassay capable of simultaneous typing of multiple pneumococcal serotypes (Multibead assay).

A simple and rapid method of simultaneously determining 15 Streptococcus pneumoniae serotypes was developed. Fifteen latex beads of different sizes and different red fluorescence levels were coated with 1 of 15 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, and 23F) of pneumococcal capsular polysaccharide (PS). The bead mixture was incubated with individual pneumococcal lysate, a pool of rabbit antisera capable of binding the 15 serotypes, and fluorescein (green fluorescence)-conjugated anti-rabbit antibody. Bead size, red fluorescence, and green fluorescence were measured in a single flow cytometer run. The green fluorescence of the beads was inhibited only when there was a serotypic match between PS on the bead and PS in the pneumococcal lysate. This method distinguished cross-reactive serotypes and correctly identified the serotypes in 100% of 86 pneumococcal isolates tested.

Therapy for pneumococcal infection at the millennium: doubts and certainties

Rapidly burgeoning worldwide multiple drug-resistant pneumococcal serotypes pose an urgent demand for new management approaches. Perhaps modern intensive care methods may have alternatives to offer. Indeed, standard assessments such as the admission APACHE II score may overestimate individual risk of death in severe CAP, and mortality can be reduced. However, among those at highest risk for mortality in the early phase of invasive disease, the conclusions reached 2-3 decades ago, that it is questionable whether a more effective drug than penicillin can be developed, and that a reduction in the number of deaths consequent to this infection can be accomplished only by widespread immunoprophylactic measures, remain inescapable. Clearly, as discussed elsewhere in this supplement, the continuing validity of these 20-year-old conclusions and the global prevalence of DRSP demand the development and marketing of new conjugate vaccines, although more widespread use of the existing 23-valent polysaccharide vaccine among high-risk populations is essential in the interim. With respect to resistance selection pressures, antibiotic prescription control may provide the answer. However, patient expectations of antibiotic therapy for trivial respiratory infection is high and, in the United Kingdom, 75% of previously healthy adults will receive it; those who do not will usually consult another physician in an effort to secure such therapy. Thus, without the intervention of government or managed care organizations, self-regulation in prescribing is unlikely. The evidence for beta-lactam treatment failure in meningitis has led to alternative approaches, with vancomycin as the primary agent. Penicillins may remain effective for otitis media, but oral cephalosporins are suspect. Data on pediatric pneumococcal pneumonia continue to suggest use of beta-lactams, at least for disease caused by strains with intermediate penicillin sensitivity. Pallares et al concluded that penicillins and cephalosporins remain the drugs of choice for severe pneumococcal pneumonia in adults. Others who share this conclusion often cite that study as evidence. However, in the case of penicillins, the mortality rate was 6% higher in a subgroup selected for monomicrobial infection and reduced risk factors for mortality when penicillin-resistant infection was present, and the overall mortality was 14% higher with penicillin-resistant strains (taking into account "all comers"). Those who depend on the findings of evidence-based medicine may accept the premise that penicillins and cephalosporins remain the drugs of choice, and agree with Goldstein and Garau that it would indeed be a mistake to adopt alternative therapies. Others may consider the deaths of 6 of 100 patients who were not in the highest-risk group too high a price to pay for statistical significance and may be skeptical of the continued use of beta-lactam therapy on higher-risk patients. In addition, the persistent selection pressure applied by continued use of beta-lactams offers a powerful population-based argument for alternatives. As DRSP continues to spread and resistant strains with penicillin MIC >2 mg/L become more prevalent, new agents such as the azabicyclo-methoxyquinolone, moxifloxacin, and perhaps grepafloxacin, but not the more toxic sparfloxacin and trovafloxacin, will undoubtedly flourish as treatments for CAP. By that time, the results of clinical studies on ketolides and oxazolidinones could offer further choices.