Surveillance of pneumococcal colonization and invasive pneumococcal disease reveals shift in prevalent carriage serotypes in Massachusetts' children to relatively low invasiveness.

BACKGROUND: Following the introduction of pneumococcal conjugate vaccines (PCV), overall nasopharyngeal colonization rates have not changed significantly, however a dramatic and sustained decline in invasive pneumococcal disease (IPD) in children was observed in every setting where the PCVs were implemented. We aimed to describe the differences in invasive disease potential of serotypes that are common colonizers in pre- and post-vaccine eras in order to provide further insight in our understanding of dynamic epidemiology of pneumococcal diseases. METHODS: Using data from surveillance of nasopharyngeal carriage and enhanced surveillance for IPD, a serotype specific "invasive capacity (IC)" was computed by dividing the incidence of IPD due to serotype x by the carriage prevalence of that same serotype in children <7years of age in Massachusetts. We have evaluated the serotype specific invasive capacity in two periods; pre-PCV13 (2001/02, 2003/04, 2006/07, 2008/09) and post-PCV13 (2010/11 and 2013/14), and by age groups; <24monthsvs. ≥24months. RESULTS: An approximate 50-fold variation in the point estimate was observed between the serotypes having the highest (7F, 38, 19A, 3, 33F) and the lowest (6C, 35B, 21, 11A, 23B and 23A) computed serotype specific invasive disease potential. In the post-PCV13 era (6C, 35B, 11A, 23B and 23A), 5 of the 7 most common serotypes colonizing the nasopharynx were serotypes with the lowest invasive capacity. Serotype specific invasive capacity trended down in older children for majority of the serotypes, and serotypes 3, 10A and 19A had significantly lower invasive capacity in children older than 24months of age compared to younger children. CONCLUSION: Invasive capacity differs among serotypes and likely by age. Point estimates of IC for most of the common serotypes colonizing children in Massachusetts in post-PCV13 era were low and likely explain the continued reduction in IPD from the pre-PCV era in the absence of specific protection against these serotypes.

Efficacy of Solithromycin (CEM-101) for Experimental Otitis Media Caused by Nontypeable Haemophilus influenzae and Streptococcus pneumoniae.

Solithromycin (CEM-101) is a "fourth-generation" macrolide, as it has three binding site and is acid stable. The three binding sites confer activity against bacteria resistant to the older macrolides and ketolides, including multidrug-resistant Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi). The objective of this study was to evaluate solithromycin pharmacokinetics (PK), middle ear fluid (MEF) concentrations, and microbiologic efficacy in a chinchilla model of experimental otitis media (EOM) due to strains of S. pneumoniae or NTHi. Plasma PK (maximum concentration of drug in serum [Cmax] and area under the concentration-time curve from 0 to 24 h [AUC0-24]) and middle ear fluid (MEF) concentrations were determined. Isolates with specified antimicrobial susceptibility patterns were inoculated directly into the middle ear (ME). Plasma and MEF were collected for PK and MEF cultures performed to determine efficacy. Solithromycin administered at 150 mg/kg of body weight/day resulted in Cmax and AUC0-24 values of 2.2 µg/ml and 27.4 µg · h/ml in plasma and 1.7 µg/ml and 28.2 µg · h/ml in extracellular MEF on day 1. By day 3, Cmax and AUC0-24 values had increased to 4.5 µg/ml and 54 µg · h/ml in plasma and 4.8 µg/ml and 98.6 µg · h/ml in extracellular MEF. For NTHi EOM, three isolates with MIC/minimal bactericidal concentration (MBC) ratios of 0.5/1 µg/ml (isolate BCH1), 2/2 µg/ml (isolate BMC1247C), and 4/4 µg/ml (isolate BMC1213C) were selected. The MEF of >85% of animals infected with BCH1 and BMC1247C was sterilized. For NTHi BMC1213, >85% of MEF cultures remained positive. For S. pneumoniae EOM, 3 isolates with MIC/MBC ratios of 0.06/0.125 µg/ml (S. pneumoniae 331), 0.125/1 µg/ml (S. pneumoniae CP-645 [MLSB phenotype]), and 0.5/2 µg/ml (CP-712 [mefA subclass mefA resistance]) were selected. Solithromycin sterilized MEF in 100% of animals infected with S. pneumoniae 331 and S. pneumoniae CP-645. ME infection persisted in 60% of animals infected with CP-712. In a model of EOM, solithromycin sterilized MEF in >85% of animals challenged with NTHi with an MIC of ≤2 µg/ml and 100% of ME infected with S. pneumoniae with an MIC of ≤0.125 µg/ml.

Do community-level predictors of pneumococcal carriage continue to play a role in the conjugate vaccine era?

This paper examined whether previously identified community-level factors (high proportion of crowded households and/or persons below the poverty level) remained associated with childhood pneumococcal carriage in the heptavalent pneumococcal conjugate vaccine (PCV7) era. Using logistic regression, individual factors were used to develop base models to which community-level factors were added to evaluate impact on pneumococcal carriage within two paediatric study cohorts from Massachusetts (urban Boston, outside Boston). Six years after introduction of universal childhood PCV7 vaccination, we found no consistent evidence that census tract characteristics (e.g. population size and density, age and race distribution, percent participating in group childcare, parental education, percent lacking in-unit plumbing, poverty, and community stability) affected odds of pneumococcal carriage when added to individual predictors (e.g. younger age, current respiratory tract infections, and attendance in group childcare). How community-level factors influence pneumococcal carriage continues to change in the era of increasing immunization coverage.

Clonal replacement among 19A Streptococcus pneumoniae in Massachusetts, prior to 13 valent conjugate vaccination.

As part of an ongoing study of the response of the Streptococcus pneumoniae population to conjugate vaccination, we applied multi-locus sequence typing (MLST) to 291 isolates sampled from nasopharyngeal carriage in Massachusetts children. We found 94 distinct sequence types (STs), including 19 that had not been previously recorded, and a xpt allele containing a large insertion. Comparison with a similar sample collected in 2007 revealed no significant overall difference in the ST composition (p=0.51) suggesting that the population has reached a new equilibrium following the introduction of 7 valent vaccination in 2000. Within serotypes, a large and statistically significant increase (p=0.014 Fisher's Exact test) was noted in the prevalence of the major multiresistant clone ST 320, which is apparently outcompeting ST 199 among serotype 19A strains. This sample will be used as a baseline to study the future evolution of the pneumococcal population in Massachusetts following introduction of vaccines with higher valency.

Modeling the decline in pneumococcal acute otitis media following the introduction of pneumococcal conjugate vaccines in the US.

We hypothesized that following the introduction of PCV7, the exchange of vaccine serotypes (VST) for non-vaccine serotypes (NVST) in the nasopharynx has resulted in fewer episodes of pneumococcal acute otitis media (AOM) due to the reduced capacity for common NVST strains to cause disease. We modeled the change in the proportion of children colonized with S. pneumoniae who would develop pneumococcal AOM that would occur due to serotype replacement, and projected the future impact of PCV13. Our model is based on observed changes in the nasopharyngeal pneumococcal serotype distribution from the pre- to post-PCV7 era, and an estimated capacity of each serotype to produce pneumococcal AOM given colonization; the latter was derived by dividing serotype-specific disease prevalence by serotype-specific carriage prevalence in the same population. Our results indicate a 12% (95% CI 0.5-26) decline in the number of AOM episodes attributable to S. pneumoniae in children less than 3 years of age between 2000 and 2007 due to the combined effects of PCV7 vaccine efficacy and vaccine-induced serotype replacement, and predicts that PCV13 will further decrease pneumococcal AOM an additional 27% (95% CI 13-40) from 2007 to 2013. Evaluation of changes in VST disease revealed a 91% (95% CI 83-97) decrease in PCV7-VST AOM from 2000 to 2007, and predicted an additional 65% (95% CI 57-74) decrease in PCV13-VST AOM from 2007 to 2013. Our model indicates that following vaccination, nasopharyngeal replacement of VST by NVST has led to a decrease in the amount of pneumococcal AOM despite a consistent rate of S. pneumoniae colonization, and that pneumococcal AOM may continue to decrease as pneumococcal serotypes with greater capacity to cause disease are replaced by less locally invasive serotypes.

Streptococcus pneumoniae serotype 6C: an intra- and interclonal complex comparison.

We report the annotated draft genome sequences of four serotype 6C Streptococcus pneumoniae isolates of differing genetic backgrounds. Serotype 6C isolates are increasing in prevalence and becoming progressively more resistant to antibiotics. As a result, these strains are likely to become more important in the near future.

Serotype specific invasive capacity and persistent reduction in invasive pneumococcal disease.

Defining the propensity of Streptococcus pneumoniae (SP) serotypes to invade sterile body sites following nasopharyngeal (NP) acquisition has the potential to inform about how much invasive pneumococcal disease (IPD) may occur in a typical population with a given distribution of carriage serotypes. Data from enhanced surveillance for IPD in Massachusetts children ≤7 years in 2003/04, 2006/07 and 2008/09 seasons and surveillance of SP NP carriage during the corresponding respiratory seasons in 16 Massachusetts communities in 2003/04 and 8 of the 16 communities in both 2006/07 and 2008/09 were used to compute a serotype specific "invasive capacity (IC)" by dividing the incidence of IPD due to serotype x by the carriage prevalence of that same serotype in children of the same age. A total of 206 IPD and 806 NP isolates of SP were collected during the study period. An approximate 50-fold variation in the point estimates between the serotypes having the highest (18C, 33F, 7F, 19A, 3 and 22F) and lowest (6C, 23A, 35F, 11A, 35B, 19F, 15A, and 15BC) IC was observed. Point estimates of IC for most of the common serotypes currently colonizing children in Massachusetts were low and likely explain the continued reduction in IPD from the pre-PCV era in the absence of specific protection against these serotypes. Invasive capacity differs among serotypes and as new pneumococcal conjugate vaccines are introduced, ongoing surveillance will be essential to monitor whether serotypes with high invasive capacity emerge (e.g. 33F, 22F) as successful colonizers resulting in increased IPD incidence due to replacement serotypes.

Otitis media across nine countries: disease burden and management.

OBJECTIVE: To assess the perceived disease burden and management of otitis media (OM) among an international cohort of experienced physicians. METHODS: A cross-sectional survey conducted in France, Germany, Spain, Poland, Argentina, Mexico, South Korea, Thailand and Saudi Arabia. Face-to-face interviews conducted with 1800 physicians (95% paediatricians, 5% family practitioners).Main outcome measures were the perceived burden on clinical practice (number of cases, complications and referrals) and first- and second-line management strategies for OM. Results are expressed as mean and range across the nine countries over three continents. RESULTS: Respondents estimated an average annual caseload of 375 (range 128-1003) children under 5 years of age with OM; 54% (range 44-71%) with an initial episode and 38% (range 27-54%) with recurrent OM (ROM). OM with complications was estimated to be approximately 20 (range 7-49) cases per year and an estimated 15% (8-41%) of children with OM was recalled as needing specialist referral. There was high awareness of Streptococcus pneumoniae and Haemophilus influenzae as causative bacterial pathogens: 77% (range 65-91%) and 74% (range 68-83%), respectively, but less recognition of non-typeable H. influenzae (NTHi); 59% (range 45-67%). Although concern over antimicrobial resistance was widespread, empirical treatment with antibiotics was the most common first-line treatment (mean 81%, range 40-96%). The burden of disease is substantial enough that many physicians would consider vaccination to prevent OM (mean score 5.1, range 4.3-6.2 on 1-7 scale). CONCLUSIONS: This large, multinational survey shows that OM remains a significant burden for clinical practice. Despite awareness of shortcomings, antimicrobial therapy remains the most frequent treatment for OM.

Evolution and transmission of stable CTL escape mutations in HIV infection.

Increasing evidence indicates that potent anti-HIV-1 activity is mediated by cytotoxic T lymphocytes (CTLs); however, the effects of this immune pressure on viral transmission and evolution have not been determined. Here we investigate mother-child transmission in the setting of human leukocyte antigen (HLA)-B27 expression, selected for analysis because it is associated with prolonged immune containment in adult infection. In adults, mutations in a dominant and highly conserved B27-restricted Gag CTL epitope lead to loss of recognition and disease progression. In mothers expressing HLA-B27 who transmit HIV-1 perinatally, we document transmission of viruses encoding CTL escape variants in this dominant Gag epitope that no longer bind to B27. Their infected infants target an otherwise subdominant B27-restricted epitope and fail to contain HIV replication. These CTL escape variants remain stable without reversion in the absence of the evolutionary pressure that originally selected the mutation. These data suggest that CTL escape mutations in epitopes associated with suppression of viraemia will accumulate as the epidemic progresses, and therefore have important implications for vaccine design.

Radiographic resolution of lymphocytic interstitial pneumonitis (LIP) in children with human immunodeficiency virus (HIV): not a sign of clinical deterioration.

BACKGROUND: The literature and anecdotal evidence associate the resolution of radiographic findings of lymphocytic interstitial pneumonitis (LIP) with a decline in immune and clinical status of human immunodeficiency virus (HIV) infected children. OBJECTIVE: As our clinical impression was the opposite, we sought to elucidate this contradiction. MATERIALS AND METHODS: Of 52 pediatric patients infected with the HIV currently being followed at our institution, 20 (38.5%) carried the diagnosis of LIP and 13 (65%) of these have had complete resolution of radiographic findings of LIP. We retrospectively reviewed the chest radiographs, CD4 counts, and clinical history of these 13 patients. RESULTS: Of the 13 patients who had resolution of radiographic findings, 11 (84.6%) had no significant change in CD4 count at the time of resolution and remained clinically stable during a mean follow-up period of 32 months. Two patients (15.3%) developed severe CD4 lymphocytopenia at the time of resolution of LIP, but clinically remained stable. None of these 13 patients had a recurrence of LIP, even with subsequent increases in CD4 count. CONCLUSION: We suggest that in contradiction to previously published data, resolution of LIP on chest radiographs is not an indicator for poor prognosis for the HIV-infected pediatric patient.

Constancy of distribution of serogroups of invasive pneumococcal isolates among children: experience during 4 decades.

Serogroups of pneumococci that caused bacteremia or meningitis in children were examined from 1981 through 1998 at Boston City Hospital/Boston Medical Center. There were 410 episodes of pneumococcal bacteremia (13--36 cases per year), of which 14 occurred in human immunodeficiency virus (HIV)--infected children and 9 occurred in children with sickle-cell disease. The 7 most common serogroups were 14 (30.7% of isolates), 19 (11.7%), 6 (11%), 18 (10.7%), 9 (7.6%), 23 (7.3%), and 4 (5.6%). The rate of episodes due to serogroups 4, 6, 9, 14, 18, 19, and 23 ranged from 80% to 91.9% during the study period. The rate of episodes due to serogroups 4, 6, 14, 18, 19, and 23 was 84.6% among patients with HIV infection, 100% among patients with sickle-cell disease, and 94.1% among the 18 patients for whom cultures of CSF specimens revealed pneumococcal meningitis. The results demonstrate that type 14 was the dominant pneumococcal serogroup responsible for invasive disease throughout the 18-year study period and that serogroup distribution overall remained constant. A comparison of these findings with historical pediatric data from our institution showed serogroup stability dating back to 1957.

Functionally inert HIV-specific cytotoxic T lymphocytes do not play a major role in chronically infected adults and children.

The highly sensitive quantitation of virus-specific CD8(+) T cells using major histocompatibility complex-peptide tetramer assays has revealed higher levels of cytotoxic T lymphocytes (CTLs) in acute and chronic virus infections than were recognized previously. However, studies in lymphocytic choriomeningitis virus infection have shown that tetramer assays may include measurement of a substantial number of tetramer-binding cells that are functionally inert. Such phenotypically silent CTLs, which lack cytolytic function and do not produce interferon (IFN)-gamma, have been hypothesized to explain the persistence of virus in the face of a quantitatively large immune response, particularly when CD4 help is impaired. In this study, we examined the role of functionally inert CTLs in chronic HIV infection. Subjects studied included children and adults (n = 42) whose viral loads ranged from <50 to >100,000 RNA copies/ml plasma. Tetramer assays were compared with three functional assays: enzyme-linked immunospot (Elispot), intracellular cytokine staining, and precursor frequency (limiting dilution assay [LDA]) cytotoxicity assays. Strong positive associations were observed between cell numbers derived by the Elispot and the tetramer assay (r = 0.90). An even stronger association between tetramer-derived numbers and intracellular cytokine staining for IFN-gamma was present (r = 0.97). The majority (median 76%) of tetramer-binding cells were consistently detectable via intracellular IFN-gamma cytokine staining. Furthermore, modifications to the LDA, using a low input cell number into each well, enabled LDAs to reach equivalence with the other methods of CTL enumeration. These data together show that functionally inert CTLs do not play a significant role in chronic pediatric or adult HIV infection.

Variability of outer membrane protein P1 and its evaluation as a vaccine candidate against experimental otitis media due to nontypeable Haemophilus influenzae: an unambiguous, multifaceted approach.

Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (

HLA-B57-restricted cytotoxic T-lymphocyte activity in a single infected subject toward two optimal epitopes, one of which is entirely contained within the other.

Viral peptides are recognized by cytotoxic T lymphocytes (CTL) as a complex with major histocompatibility complex (MHC) class I molecules, but the extent to which a single HLA allele can accommodate epitope peptides of different length and sequence is not well characterized. Here we report the identification of clonal CTL responses from the same donor that independently recognize one of two HLA-B57-restricted epitopes, KAFSPEVIPMF (KF11; p24(Gag) residues 30 to 40) and KAFSPEVI (KF8; p24(Gag) residues 30 to 37). Although lysis studies indicated that the KF11 peptide stabilized the HLA-B57-peptide complex more efficiently than the KI8 peptide, strong clonal responses were directed at each epitope. In samples from a second donor, the same phenomenon was observed, in which distinct CTL clones recognized peptide epitopes presented by the same HLA class I allele (in this case, HLA-A3) which were entirely overlapping. These data are relevant to the accurate characterization of CTL responses, which is fundamental to a detailed understanding of MHC class I-restricted immunity. In addition, these studies demonstrate marked differences in the length of peptides presented by HLA-B57, an allele which is associated with nonprogressive human immunodeficiency virus infection.

Differential narrow focusing of immunodominant human immunodeficiency virus gag-specific cytotoxic T-lymphocyte responses in infected African and caucasoid adults and children.

Cytotoxic T-lymphocyte (CTL) activity plays a central role in control of viral replication and in determining outcome in cases of human immunodeficiency virus type 1 (HIV-1) infection. Incorporation of important CTL epitope sequences into candidate vaccines is, therefore, vital. Most CTL studies have focused upon small numbers of adult Caucasoid subjects infected with clade-B virus, whereas the global epidemic is most severe in sub-Saharan African populations and predominantly involves clade-C infection in both adults and children. In this study, sensitive enzyme-linked immunospot (elispot) assays have been utilized to identify the dominant Gag-specific CTL epitopes targeted by adults and children infected with clade-B or -C virus. Cohorts evaluated included 44 B-clade-infected Caucasoid American and African American adults and children and 37 C-clade-infected African adults and children from Durban, South Africa. The results show that 3 out of 46 peptides spanning p17(Gag) and p24(Gag) sequences tested contain two-thirds of the dominant Gag-specific epitopes, irrespective of the clade, ethnicity, or age group studied. However, there were distinctive differences between the dominant responses made by Caucasoids and Africans. Dominant responses in Caucasoids were more often within p17(Gag) peptide residues 16 to 30 (38 versus 12%; P < 0.01), while p24(Gag) peptide residues 41 to 60 contained the dominant Gag epitope more often in the African subjects tested (39 versus 4%; P < 0.005). Within this 20-mer p24(Gag), an epitope presented by both B42 and B81 is defined which represents the dominant Gag response in >30% of the total infected population in Durban. This epitope is closely homologous with dominant HIV-2 and simian immunodeficiency virus Gag-specific CTL epitopes. The fine focusing of dominant CTL responses to these few regions of high immunogenicity is of significance to vaccine design.

Efficacy of linezolid in experimental otitis media.

Therapy for otitis media (OM) due to resistant Streptococcus pneumoniae (MIC of penicillin, >/=2.0 microgram/ml) is challenging. Linezolid, an oxazolidinone, represent a new class of antimicrobial agents with excellent in vitro activity against penicillin-resistant S. pneumoniae; however, in vitro activity against nontypeable Haemophilus influenzae (NTHI) is limited. We evaluated its efficacy against experimental acute OM due to a multidrug-resistant S. pneumoniae isolate and two isolates of NTHI. The chinchilla model was utilized to evaluate the efficacy of linezolid against experimental infection due to S. pneumoniae or NTHI. Serum and middle ear antibiotic concentrations were determined, and sterilization of experimental OM was evaluated. Chinchillas were inoculated directly with S. pneumoniae into the superior bulla. Twenty-four hours after inoculation, all animals had positive middle ear and nasopharyngeal cultures. Animals were given linezolid at 25 mg/kg/dose twice a day (b.i.d.) by orogastric feeding tube or amoxicillin at 40 mg/kg/dose b.i.d. intramuscularly for 5 days. By day 5, all animals in the linezolid group had sterile middle ear cultures and eradication of S. pneumoniae from the nasopharynx. In the amoxicillin group, all nine animals remained middle ear and nasopharynx positive (P < 0.01). In animals inoculated with NTHI, 25 and 37.5 mg/kg b.i.d. failed to sterilize middle ear infection or eradicate colonization. Mean levels in middle ear fluid measured during experimental infection were 12.8 microgram/ml at 2 to 6 h and 4. 1 mirogram/ml at 16 to 17 h after orogastric dosing at 25 mg/kg. Linezolid achieved a high concentration in the middle ear during experimental OM. Linezolid eradicated multidrug-resistant S. pneumoniae from the middle ear and nasopharynx. Experimental infection and nasopharyngeal colonization due to NTHI persisted despite achievement of concentrations in the middle ear that were above the MIC (for NTHI).

Acute otitis media in the era of effective pneumococcal conjugate vaccine: will new pathogens emerge?

The immunogenicity of pneumococcal conjugate vaccine (PCV) in young infants and its serotype-specific efficacy in otitis media (OM) results in a modest reduction in total episodes of OM and a more substantial reduction in disease due to the most frequent pneumococcal serotypes. Since PCV will only prevent disease due to the most common serotypes, concerns about potential changes in the microbiology of OM have emerged. Insight into potential changes can be obtained from reviewing middle ear and nasopharyngeal isolates from studies of antimicrobial prophylaxis and bacterial polysaccharide immune globulin for prevention of OM and PCV for prevention of invasive pneumococcal disease, respectively. In children receiving PCV, a shift in serotypes of SP colonizing the nasopharynx has been observed. Since non-vaccine serotypes are already present in the community as the etiology of acute purulent OM, it is predictable that these non-vaccine serotypes will become more common especially in children less than two years of age.