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Streptococcus pneumoniae is a colonizer of the human nasopharynx, which accounts for most of the community-acquired pneumonia cases and can cause non-invasive and invasive diseases. Current available vaccines are serotype-specific and the use of recombinant proteins associated with virulence is an alternative to compose vaccines and to overcome these problems. In a previous work, we describe the identification of proteins in S. pneumoniae by reverse vaccinology and the genetic diversity of these proteins in clinical isolates. It was possible to purify a half of 20 selected proteins in soluble form. The expression of these proteins on the pneumococcal cells surface was confirmed by flow cytometry. We demonstrated that some of these proteins were able to bind to extracellular matrix proteins and were recognized by sera from patients with pneumococcal meningitis infection caused by several pneumococcal serotypes. In this context, our results suggest that these proteins may play a role in pneumococcal pathogenesis and might be considered as potential vaccine candidates.

In 2010, the 10-valent pneumococcal conjugate vaccine (PCV10) was introduced into the Brazilian childhood vaccination programme. Concerns have been raised that non-vaccine serotypes could increase in prevalence and reduce the benefits of vaccination; therefore, we examined non-PCV10 isolates recovered from meningitis during pre- (January 2008-May 2010) and post-vaccine (June 2010-December 2012) periods. Surveillance for pneumococcal meningitis was established at the Reference Hospital of Infectious Diseases in Salvador, Brazil. Serotypes were determined by multiplex PCR and/or Quellung reaction. Antimicrobial susceptibility testing was conducted by E-test and broth microdilution. Genotyping used PFGE and multi-locus sequence typing. A total of 148 cases of meningitis were identified from January 2008 to December 2012, 77 (52 %) of which were due to non-PCV10 isolates, with 50 (52.1 %) from pre-vaccine and 27 (52 %) from post-vaccine periods. In the post-vaccine period, the non-PCV10 serotypes 12F (n=6; 22.2 %), 10A (n=3; 11.1 %), 15B (n=2; 7.4 %) and 18B (n=2; 7.4 %) were the most prevalent. Forty-three isolates (55.8 %) were non-susceptible to one or more antibiotics. Non-susceptibility to penicillin was observed among serotypes 19A (three isolates), 9N (one isolate) and 12F (one isolate). PFGE and multi-locus sequence typing results demonstrated a wide genetic diversity among the isolates. During the early period following PCV10 introduction, no obvious emergence of a particular serotype was evident among non-PCV10 strains. This study underscores the importance of monitoring any changes among non-PCV10 cases after the introduction of PCV10.

Information on pneumococcal carriage in the pre-vaccine period is essential to predict and assess the impact of PCV in settings where disease surveillance is particularly difficult. Therefore, we present data on pneumococcal carriage before the introduction of the 10-valent-pneumococcal conjugate vaccine (PCV10) in Brazil. We conducted a prospective study on a cohort of 203 children aged <5 years old, randomly selected in an urban community located in the periphery of the city of Salvador, Brazil and followed them from January/2008 to January/2009. Nasopharyngeal swabs were collected from each child at four times. In total, 721 swabs were collected, yielding a pneumococcal carriage prevalence of 55% (n=398). In multivariate analyses, the variables associated with carriage were having contact with three or more children <2 years old (OR, 2.00; 95% CI 1.33-2.89) and living in a house with an average of 3 residents per room (OR, 1.77; 95% CI 1.05-3.10). Also, white participants were more likely to be protected from colonization (OR, 0.52; 95% CI 0.29-0.93), and prevalence of carriage varied over time, with lower prevalence occurring from February to June (OR, 0.53; 95% CI 0.37-0.78) compared to July to January. Contact with children under 2 years of age and living in crowded housing also were associated with colonization by highly invasive serotypes, although this relationship was not significant. The most prevalent vaccine serotypes were 6A/B (25.4%), 19F (10.1%) and 14 (9.0%), while the most prevalent non-vaccine serotypes were 16F (4.8%), 15B/C (4.5%) and 6C/D (3.5%). Overall, 38.4% (153/398) of the isolates were non-susceptible to penicillin, and of those, 73.8% (113/153) were non-susceptible to trimethoprim/sulfamethoxazole. Colonization rate by PCV10 serotypes was 52.2%. Routine PCV10 vaccination can lead to significant changes in pneumococcal serotypes found in NP colonization, indicating a need for continued monitoring, especially in crowded settings, as occurs in Brazil's slums., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Invasive pneumococcal disease is a relevant public health problem in Brazil, especially among children and the elderly. In July/2010 a 10-valent pneumococcal conjugate vaccine was introduced to the immunization schedule of Brazilian children under two years of age. Between July/2010 and December/2013 we conducted a case-series study on invasive pneumococcal disease in Salvador, Brazil to describe the clinical and bacteriological profile of invasive pneumococcal disease cases during the post-implementation period. Eighty-two cases were eligible. Mean age was 31 years (interquartile range, 3-42); 17.1% and 30.5% were under 2 years and 5 years, respectively. Pneumococcal meningitis (n=64, 78.1%), bacteraemic pneumococcal pneumonia (n=12, 14.6%) and bacteraemia (n=6, 7.3%) were the clinical syndromes identified. Thirty-three different serotypes were found. Of these, serotype 14 (n=12, 14.6%) was the most common, followed by 23F (n=10, 12.2%), 12F (n=8, 9.8%), 18C (n=5, 6.1%) and 6B (n=5, 6.1%). Investigations conducted in Salvador in the pre-vaccine period did not identify serotype 12F as one of the most prevalent serotypes. Increase of serotype 12F was observed in different regions of Brazil, in the post-vaccine period. Among children under two years of age, the target group for 10-valent pneumococcal conjugate vaccine, 11 (78.6%) of the 14 isolated strains of Streptococcus pneumoniae belonged to vaccine serotypes; at least 50% of these children were not vaccinated. The relatively recent implementation of 10-valent pneumococcal conjugate vaccine in Brazil reinforces the need to maintain an active surveillance of invasive pneumococcal disease cases, considering the possible increase of invasive pneumococcal disease cases related to non-vaccine serotypes and the changes on the clinical presentation of the disease., (Copyright © 2015 Elsevier Editora Ltda. All rights reserved.)

Background: Hospital-based surveillance for pneumococcal meningitis has been conducted since January 1996 in the city of Salvador, Brazil. The purpose of this study was to describe the temporal evolution of Penicillin Non-Susceptible Streptococcus pneumoniae (PNSSP) in regards to serotype distributions and clonal diversity recovered from meningitis cases over 17 years., Methods: Broth microdilution was used to identify pneumococcal isolates that were PNSSP (Minimum Inhibitory Concentration > 0.12 μg/ml). The annual incidence rate of meningitis cases was calculated. Serotyping was defined using multiplex polymerase chain reaction assays and quellung reaction. Genetic diversity of PNSSP isolates was assessed using both pulsed-field gel electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) analyses., Results: A total of 854 cerebrospinal fluid (CSF) culture pneumococcal isolates were tested by broth microdilution method and serotyped. A total of 173 (20.3%) were penicillin non-susceptible (PNSSP) (Minimum Inhibitory concentration ≥ 0.12 μg/ml). The annual incidence of meningitis cases declined from 1.65/100,000 population (1996) to 0.2/100,000 population in 2012 and the rate due to PNSSP declined 82% over the 17-years of surveillance. PNSSP isolates were restricted to 13 serotypes, being the most common ones serotypes 14 (45.1%; 78/173), 23 F (19.1%; 33/173), 6B (14.4%; 25/173), 19 F (9.2%; 16/173) and 19A (5.2%; 9/173). Among the PNSSP isolates, 94% had serotypes represented in the 10-valent conjugate vaccine (PCV10). The predominant serotype 14 clonal groups were identified as PFGE group A/multilocus sequence type 66 (ST66) [35.3% (61/173)] and PFGE group GK/ST156 [4.6% (8/173)], the latter one associated with high level resistance to penicillin and ceftriaxone., Conclusions: Our results show sustained reductions in pneumococcal meningitis cases in the Metropolitan region of Salvador from 1996 to 2012. This might reflect a beneficial impact of conjugate vaccines. Continued surveillance and further studies need to be conducted to better understanding on PCV10 vaccine impact.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. Our aim was to identify conserved targets in pneumococci that showed positive prediction for lipoprotein and extracellular subcellular location using bioinformatics programs and verify the distribution and the degree of conservation of these targets in pneumococci. These targets can be considered potential vaccine candidate to be evaluated in the future. A set of 13 targets were analyzed and confirmed the presence in all pneumococci tested. These 13 genes were highly conserved showing around >96 % of amino acid and nucleotide identity, but they were also present and show high identity in the closely related species Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. S. oralis clusters away from S. pneumoniae, while S. pseudopneumoniae and S. mitis cluster closer. The divergence between the selected targets was too small to be observed consistently in phylogenetic groups between the analyzed genomes of S. pneumoniae. The proteins analyzed fulfill two of the initial criteria of a vaccine candidate: targets are present in a variety of different pneumococci strains including different serotypes and are conserved among the samples evaluated.

Background: Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard., Methods and Findings: CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously., Results: Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF., Conclusion: Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.

Background: Prior to the availability of generic third-generation cephalosporins, penicillins were widely used for treatment of pneumococcal meningitis in developing countries despite concerns about rising levels of penicillin resistance among pneumococcal isolates. We examined the impact of penicillin resistance on outcomes of pneumococcal meningitis over a ten year period in an infectious diseases hospital in Brazil., Methods: Clinical presentation, antimicrobial therapy and outcomes were reviewed for 548 patients with culture-confirmed pneumococcal meningitis from December, 1995, to November, 2005. Pneumococcal isolates from meningitis patients were defined as penicillin-resistant if Minimum Inhibitory Concentrations for penicillin were greater than 0.06 μg/ml. Proportional hazards regression was used to identify risk factors for fatal outcomes., Results: During the ten-year period, ceftriaxone replaced ampicillin as first-line therapy for suspected bacterial meningitis. In hospital case-fatality for pneumococcal meningitis was 37%. Of 548 pneumococcal isolates from meningitis cases, 92 (17%) were resistant to penicillin. After controlling for age and severity of disease at admission, penicillin resistance was associated with higher case-fatality (Hazard Ratio [HR], 1.62; 95% Confidence Interval [CI], 1.08-2.43). Penicillin-resistance remained associated with higher case-fatality when initial therapy included ceftriaxone (HR, 1.68; 95% CI 1.02-2.76)., Conclusions: Findings support the use of third generation cephalosporin antibiotics for treatment of suspected pneumococcal meningitis even at low prevalence of pneumococcal resistance to penicillins.

This study describes the serotype distribution and antibiotic resistance patterns among 397 S. pneumoniae meningitis case isolates recovered in Salvador, Brazil, during the period of 2000-2007, before introduction of the 10-valent pneumococcal conjugate vaccine. The active hospital-based surveillance showed a decline in the annual incidence rates of pneumococcal meningitis during the period of study, from 1.12 cases to 0.83 cases/100,000 persons for all age groups (P<0.001), with an overall case-fatality rate of 28.6% (113 of 395) for all patients and 41.9% (57 of 136) for those <5 years of age. Serotypes 14 (n=55; 13.9%), 3 (n=32; 8.1%), 23F (n=32; 8.1%), 19F (n=31; 7.8%), 6B (n=30; 7.6%), 18C (n=28; 7.1%), and 6A (n=20; 5%) were the most prevalent serotypes. In patients <5 years the estimated projected coverage of 7-, 10- and 13-valent conjugate vaccines was 74.3%, 75.7% and 83.1%, respectively. Antimicrobial susceptibility testing revealed that 22.1% (n=88) of isolates were non-susceptible to penicillin, 56% were non-susceptible to trimethoprim/sulphamethoxazole, and 29.6% were non-susceptible to tetracycline. Nonsusceptibility to penicillin and cefotaxime was detected solely among serotype 14 isolates (n=4; 1%). This study provides an important baseline to assess the impact of conjugate vaccine implantation on the epidemiology of meningitis due to Streptococcus pneumoniae in Salvador, Brazil., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The newly described Streptococcus pneumoniae serotype 6C accounted for 2.3% (16/709) of meningitis cases and 3.2% (3/95) of nasopharyngeal isolates from healthy individuals in Brazil. The strains were multidrug resistant (18.8%) and genetically diverse. Despite low serotype 6C prevalence, continuous surveillance is necessary to guide vaccine strategies.

Background: Inhabitants of slum settlements represent a significant proportion of the population at risk for pneumococcal disease in developing countries., Methods: We conducted a household survey of pneumococcal carriage among residents of a slum community in the city of Salvador, Brazil., Results: Among 262 subjects, 95 (36%) were colonized with Streptococcus pneumoniae. Children <5 years of age (OR, 8.0; 95% CI, 3.5-18.6) and those who attended schools (OR, 2.7, 95% CI, 1.2-6.0) had significantly higher risk of being colonized. Of 94 isolates obtained from colonized individuals, 51% had serotypes included in the seven-valent pneumococcal conjugate vaccine. Overall, 10% (9 of 94 isolates) were nonsusceptible to penicillin and 28% (27 of 94 isolates) were resistant to cotrimoxazole. BOX-PCR, PFGE and MLST analyses found that 44% of the carriage isolates belonged to 14 distinct clonal groups. Strains of the same clonal group were isolated from multiple members of 9 out of the 39 study households. Nineteen carriage isolates had genotypes that were the same as those identified among 362 strains obtained from active surveillance for meningitis., Conclusions: The study's findings indicate that there is significant intra- and inter-household spread of S. pneumoniae in the slum community setting. However, a limited number of clones encountered during carriage among slum residents were found to cause invasive disease.

Background: Pilus components of Streptococcus pneumoniae encoded by rlrA were recently shown to elicit protection in an animal model of infection. Limited data are available on the prevalence of the rlrA operon in pneumococci; therefore, we investigated its distribution and its antigenic variation among disease-causing strains., Methods: The prevalence of rlrA and its association with serotype and genotype were evaluated in a global panel of 424 pneumococci isolates (including the 26 drug-resistant clones described by the Pneumococcal Molecular Epidemiology Network)., Results: The rlrA islet was found in 130 isolates (30.6%) of the defined collection. Sequence alignment of 15 rlrA islets defined the presence of 3 clade types, with an overall homology of 88%-92%. The presence or absence of a pilus-encoding operon correlated with S. pneumoniae genotype (P < .001), as determined by multilocus sequence typing, and not with serotype. Further investigation identified a positive trend of rlrA occurrence among antimicrobial-resistant pneumococci., Conclusions: On the basis of S. pneumoniae genotype, it is possible to predict the incidence of the rlrA pilus operon in a collection of pneumococcal isolates. This will facilitate the development of a protein vaccine.

Penicillin-nonsusceptible strains were isolated from 15% of 303 individuals with pneumococcal meningitis identified during a 4-year surveillance study in Salvador, Brazil. The estimated rate of coverage of the seven-valent conjugate vaccine was 74% among patients <5 years of age and 94% among those infected with nonsusceptible isolates, indicating that the use of conjugate vaccines may be an approach to the control of emerging penicillin resistance in Brazil.

Active hospital-based surveillance in the city of Salvador, Brazil, from December 1995 through October 1998, identified 221 patients with confirmed pneumococcal meningitis. Of these 221 patients, 29 (13%) had isolates with intermediate-level resistance to penicillin. Infection with these penicillin-nonsusceptible isolates was significantly associated with age of <2 years (P<.0019), previous antibiotic use (P<.0006), and coresistance to trimethoprim-sulfamethoxazole (P<.0000). Serotype 14 was the most prevalent serotype (55.2%) of penicillin-nonsusceptible isolates. Strain typing by repetitive element BOX polymerase chain reaction (PCR) analysis showed that penicillin-nonsusceptible serotype 14 isolates had closely related BOX PCR patterns, whereas penicillin-susceptible serotype 14 isolates each had distinct, unrelated patterns. Penicillin-nonsusceptible serotype 14 isolates from Salvador and other Brazilian cities had similar BOX PCR patterns. These observations indicate that in Brazil a large proportion of cases of penicillin-nonsusceptible pneumococcal meningitis appear to be caused by a closely related group of serotype 14 strains that may have disseminated to widely separate geographic areas.