Childhood Invasive Bacterial Disease in Kathmandu, Nepal (2005-2013).

BACKGROUND: Invasive bacterial disease (IBD; including pneumonia, meningitis, sepsis) is a major cause of morbidity and mortality in children in low-income countries. METHODS: We analyzed data from a surveillance study of suspected community-acquired IBD in children <15 years of age in Kathmandu, Nepal, from 2005 to 2013 before introduction of pneumococcal conjugate vaccines (PCV). We detailed the serotype-specific distribution of invasive pneumococcal disease (IPD) and incorporated antigen and PCR testing of cerebrospinal fluid (CSF) from children with meningitis. RESULTS: Enhanced surveillance of IBD was undertaken during 2005-2006 and 2010-2013. During enhanced surveillance, a total of 7956 children were recruited of whom 7754 had blood or CSF culture results available for analysis, and 342 (4%) had a pathogen isolated. From 2007 to 2009, all 376 positive culture results were available, with 259 pathogens isolated (and 117 contaminants). Salmonella enterica serovar Typhi was the most prevalent pathogen isolated (167 cases, 28% of pathogens), followed by Streptococcus pneumoniae (98 cases, 16% pathogens). Approximately, 73% and 78% of pneumococcal serotypes were contained in 10-valent and 13-valent PCV, respectively. Most cases of invasive pneumococcal disease (IPD) were among children ≥5 years of age from 2008 onward. Antigen and PCR testing of CSF for pneumococci, Haemophilus influenzae type b and meningococci increased the number of these pathogens identified from 33 (culture) to 68 (culture/antigen/PCR testing). CONCLUSIONS: S. enterica serovar Typhi and S. pneumoniae accounted for 44% of pathogens isolated. Most pneumococcal isolates were of serotypes contained in PCVs. Antigen and PCR testing of CSF improves sensitivity for IBD pathogens.

Alternative Complement Pathway Inhibition Abrogates Pneumococcal Opsonophagocytosis in Vaccine-Naïve, but Not in Vaccinated Individuals.

To assess the relative contribution of opsonisation by antibodies, classical and alternative complement pathways to pneumococcal phagocytosis, we analyzed killing of pneumococci by human blood leukocytes collected from vaccine-naïve and PCV13-vaccinated subjects. With serotype 4 pneumococci as model, two different physiologic opsonophagocytosis assays based on either hirudin-anticoagulated whole blood or on washed cells from EDTA-anticoagulated blood reconstituted with active serum, were compared. Pneumococcal killing was measured in the presence of inhibitors targeting the complement components C3, C5, MASP-2, factor B or factor D. The two assay formats yielded highly consistent and comparable results. They highlighted the importance of alternative complement pathway activation for efficient opsonophagocytic killing in blood of vaccine-naïve subjects. In contrast, alternative complement pathway inhibition did not affect pneumococcal killing in PCV13-vaccinated individuals. Independent of amplification by the alternative pathway, even low capsule-specific antibody concentrations were sufficient to efficiently trigger classical pathway mediated opsonophagocytosis. In heat-inactivated or C3-inhibited serum, high concentrations of capsule-specific antibodies were required to trigger complement-independent opsonophagocytosis. Our findings suggest that treatment with alternative complement pathway inhibitors will increase susceptibility for invasive pneumococcal infection in non-immune subjects, but it will not impede pneumococcal clearance in vaccinated individuals.

Differences in Pneumococcal and Haemophilus influenzae Natural Antibody Development in Papua New Guinean Children in the First Year of Life.

Background: Development of vaccines to prevent disease and death from Streptococcus pneumoniae, and nontypeable Haemophilus influenzae (NTHi), the main pathogens that cause otitis media, pneumonia, meningitis and sepsis, are a global priority. Children living in low and lower-middle income settings are at the highest risk of contracting and dying from these diseases. Improved vaccines with broader coverage are required. Data on the natural development of antibodies to putative vaccine antigens, especially in high-risk settings, can inform the rational selection of the best antigens for vaccine development. Methods: Serum IgG titres to four pneumococcal proteins (PspA1, PspA2, CbpA, and Ply) and five NTHi antigens (P4, P6, OMP26, rsPilA and ChimV4) were measured in sera collected from 101 Papua New Guinean children at 1, 4, 9, 10, 23 and 24 months of age using multiplexed bead-based immunoassays. Carriage density of S. pneumoniae and H. influenzae were assessed by quantitative PCR on genomic DNA extracted from nasopharyngeal swabs using species-specific primers and probes. All data were log-transformed for analysis using Student's unpaired t-tests with geometric mean titre (GMT) or density (GMD) calculated with 95% confidence intervals (CI). Results: Serum -pneumococcal protein-specific IgG titres followed a "U" shaped pattern, with a decrease in presumably maternally-derived IgG titres between 1 and 4 months of age and returning to similar levels as those measured at 1 month of age by 24 months of age. In contrast, NTHi protein-specific IgG titres steadily increased with age. There was no correlation between antibody titres and carriage density for either pathogen. Conclusion: This longitudinal study indicates that the waning of maternally- derived antibodies that is usually observed in infants, after infants does not occur for NTHi antigens in Papua New Guinean infants. Whether NTHi antigen IgG can be transferred maternally remains to be determined. Vaccines that are designed to specifically increase the presence of protective NTHi antibodies in the first few months of life may be most effective in reducing NTHi disease. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT01619462.

The circulatory small non-coding RNA landscape in community-acquired pneumonia on intensive care unit admission.

Community-acquired pneumonia (CAP) is a major cause of sepsis. Despite several clinical trials targeting components of the inflammatory response, no specific treatment other than antimicrobial therapy has been approved. This argued for a deeper understanding of sepsis immunopathology, in particular factors that can modulate the host response. Small non-coding RNA, for example, micro (mi)RNA, have been established as important modifiers of cellular phenotypes. Notably, miRNAs are not exclusive to the intracellular milieu but have also been detected extracellular in the circulation with functional consequences. Here, we sought to determine shifts in circulatory small RNA levels of critically ill patients with CAP-associated sepsis and to determine the influence of clinical severity and causal pathogens on small RNA levels. Blood plasma was collected from 13 critically ill patients with sepsis caused by CAP on intensive care unit admission and from 5 non-infectious control participants. Plasma small RNA-sequencing identified significantly altered levels of primarily mature miRNAs in CAP relative to controls. Pathways analysis of high or low abundance miRNA identified various over-represented cellular biological pathways. Analysis of small RNA levels against common clinical severity and inflammatory parameters indices showed direct and indirect correlations. Additionally, variance of plasma small RNA levels in CAP patients may be explained, at least in part, by differences in causal pathogens. Small nuclear RNA levels were specifically altered in CAP due to Influenza infection in contrast to Streptococcus pneumoniae infection. Pathway analysis of plasma miRNA signatures unique to Influenza or Streptococcus pneumoniae infections showed enrichment for specific proteoglycan, cell cycle, and immunometabolic pathways.

Stable Incidence of Invasive Pneumococcal Disease in Children in Northern France From 2014 Through 2018.

BACKGROUND: The 13-valent pneumococcal conjugate vaccine (PCV13) has been recommended in France since June 2010. The aim of this study was to evaluate the trends in the incidence of invasive pneumococcal disease (IPD) resulting in hospitalization of children younger than 18 years of age, to identify the vaccination status of these patients and to analyze the serotypic evolution of the pneumococci involved in the various types of IPD. METHODS: This multicenter retrospective study reviewed all admissions of children younger than 18 years of age for IPD from 2014 through 2018 in all hospitals with a pediatric or neonatal unit in northern France. Data completeness was obtained by matching 3 independent databases. The incidence of IPD resulting in hospitalization was calculated per age group. The clinical course and the vaccine and nonvaccine types were described overall and by the IPD type. RESULTS: One hundred thirty cases of IPD were identified: 51 with bacteremia, 45 meningitis, 28 pneumonia or pleuropneumonia and 6 arthritis. The IPD incidence ranged from 2.4 to 3.0/100,000 in children under 18 years of age (95% confidence intervals, 1.4-3.3 and 1.9-4.1, respectively), and from 9.5 to 15.9/100,000 in children under 2 years of age, with no significant differences over time. Nonvaccine types were predominant (81%), mainly 24F, 23B and 10A. Vaccine serotype 3 was involved in 10 cases of IPD, 2 of which were in correctly vaccinated children. Two cases of IPD could have been prevented by vaccination. Neurologic sequelae affected 26% of these children (62% of those with meningitis). Six children died from IPD (5%). CONCLUSION: The incidence of IPD resulting in hospitalization remained stable in northern France during the study period, with no significant increase in nonvaccine types. Further surveillance is needed to adjust the vaccination strategy if necessary.

Immunogenicity of alternative ten-valent pneumococcal conjugate vaccine schedules in infants in Ho Chi Minh City, Vietnam: results from a single-blind, parallel-group, open-label, randomised, controlled trial.

BACKGROUND: Data are scarce from low-income and middle-income countries (LMICs) to support the choice of vaccination schedule for the introduction of pneumococcal conjugate vaccines (PCV). We aimed to compare the immunogenicity of four different infant PCV10 schedules in infants in Vietnam. METHODS: In this single-blind, parallel-group, open-label, randomised controlled trial, infants aged 2 months were recruited by community health staff in districts 4 and 7 of Ho Chi Minh City, Vietnam. Eligible infants had no clinically significant maternal or prenatal history and were born at or after 36 weeks' gestation. Participants were randomly assigned (3:3:5:4:5:4) using block randomisation, stratified by district, to one of six PCV10 or PCV13 vaccination schedules. Here we report results for four groups: group A, who were given PCV10 at ages 2, 3, 4, and 9 months (a 3 + 1 schedule); group B, who were vaccinated at ages 2, 3, and 4 months (3 + 0 schedule); group C, who were vaccinated at ages 2, 4, and 9·5 months (2 + 1 schedule); and group D, who were vaccinated at ages 2 and 6 months (two-dose schedule). Laboratory-based assessors were masked to group allocation. Blood samples were collected at different prespecified timepoints between ages 3-18 months depending on group allocation, within 27-43 days after vaccination, and these were analysed for serotype-specific IgG and opsonophagocytic responses. Participants were followed-up until age 24 months. The primary outcome was the proportion of infants with serotype-specific IgG levels of 0·35 µg/mL or higher at age 5 months, analysed as a non-inferiority comparison (10% margin) of the two-dose and three-dose primary series (group C vs groups A and B combined). We also compared responses 4 weeks after two doses administered at either ages 2 and 4 months (group C) or at ages 2 and 6 months (group D). The primary endpoint was analysed in the per-protocol population. Reactogenicity has been reported previously. This study is registered with ClinicalTrials.gov, NCT01953510, and is now closed to accrual. FINDINGS: Between Sept 30, 2013, and Jan 9, 2015, 1201 infants were enrolled and randomly assigned to group A (n=152), group B (n=149), group C (n=250), group D (n=202), or groups E (n=251) and F (n=197). In groups A-D, 388 (52%) of 753 participants were female and 365 (48%) were male. 286 (95%) participants in groups A and B combined (three-dose primary series) and 237 (95%) in group C (two-dose primary series) completed the primary vaccination series and had blood samples taken within the specified time window at age 5 months (per-protocol population). At this timepoint, a two-dose primary series was non-inferior to a three-dose primary series for eight of ten vaccine serotypes; exceptions were 6B (84·6% [95% CI 79·9-88·6] of infants had protective IgG concentrations after three doses [groups A and B combined] vs 76·8% [70·9-82·0] of infants after two doses [group C]; risk difference 7·8% [90% CI 2·1-13·6]) and 23F (90·6% [95% CI 86·6-93·7] vs 77·6% [71·8-82·2]; 12·9% [90% CI 7·7-18·3]). Two doses at ages 2 and 6 months produced higher antibody levels than two doses at ages 2 and 4 months for all serotypes except 5 and 7F. INTERPRETATION: A two-dose primary vaccination series was non-inferior to a three-dose primary vaccination series while two doses given with a wider interval between doses increased immunogenicity. The use of a two-dose primary vaccination schedule using a wider interval could be considered in LMIC settings to extend protection in the second year of life. FUNDING: Australian National Health and Medical Research Council, and The Bill & Melinda Gates Foundation.

Impact of HIV-1 Infection and Antigen Class on T Follicular Helper Cell Responses to Pneumococcal Polysaccharide-Protein Conjugate Vaccine-13.

Pneumococcal infections are common and serious complications of HIV-1 disease. Prevention has been compromised by the limited magnitude and quality of Ab responses to T cell-independent type 2 pneumococcal capsular polysaccharides (PPS). The pneumococcal polysaccharide-protein conjugate vaccine-13 (PCV-13) contains PPS conjugated to the T cell-dependent protein (diphtheria toxoid [DT] [CRM197]). We investigated the differential response to PPS and DT by human Ab-secreting B cells (ASC) after immunization with PCV-13 in newly diagnosed healthy HIV+ and control adults. The numbers of PPS-specific IgG ASC increased significantly and similarly in HIV+ and controls. However, DT-specific IgG ASC increased in controls but not HIV+ subjects. To determine the cellular basis of these disparate responses to DT and PPS, we characterized the frequency and activation of T follicular helper (Tfh) cells, the predominant T cell subset providing B cell help. Expression of inducible T cell costimulator (ICOS), which sustains Tfh function and phenotype, increased significantly among controls, when compared with the HIV+ group. Increases in ICOS+ Tfh correlated with changes in T-dependent, DT-specific IgG ASC in controls but not in HIV+ In contrast, ICOS expression did not correlate with T cell-independent type 2 PPS-specific ASC in either group. Of note, upon optimized ex vivo stimulation, CD4 T cells from HIV+ subjects differentiated into Tfh cells and formed synapses with Raji B cells at frequencies similar to that of controls. In summary, PCV-13-induced increase in ICOS expression on Tfh was associated with responses to DT, which was compromised in recently diagnosed healthy HIV+ adults and can be restored ex vivo by providing effective Tfh-differentiating signals.

23-valent polysaccharide vaccine (PPSV23)-targeted serotype-specific identification of Streptococcus pneumoniae using the loop-mediated isothermal amplification (LAMP) method.

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

Serum levels of anti-PspA and anti-PspC IgG decrease with age and do not correlate with susceptibility to experimental human pneumococcal colonization.

Older adults are at increased risk of pneumococcal disease. This work aims to evaluate whether there is any decrease in serum IgG against variants of the antigens Pneumococcal surface protein A (PspA) and Pneumococcal surface protein C (PspC) in healthy adults with increasing age. Levels of IgG against PspA and PspC variants were determined by ELISA in serum samples comparing volunteers 18-30 years of age with volunteers who were 50-70+ before and after an experimental pneumococcal colonization challenge. The serotype 6B strain used in the challenge belongs to a minor group of pneumococcal isolates expressing two PspC variants. There was a decrease in levels of IgG with increasing age for the most common PspA variants and for all PspC variants analyzed. No correlation was found between basal levels of IgG against these antigens and protection against colonization. There was an increase in levels of IgG against PspA variants that are more cross-reactive with the variant expressed by the challenge strain post challenge in younger individuals who became colonized. Since the challenge strain used in our study expresses two different PspC variants, an increase in serum IgG against all PspC variants tested was observed in younger individuals who became colonized. For some of the antigen variants tested, a decrease in serum IgG was observed in young volunteers who were challenged but did not become colonized. Serum IgG antibodies against PspA and PspC variants thus decrease with age in healthy adults, but there is no correlation between levels of IgG against these antigens and protection against human experimental colonization. Though no correlation between naturally induced serum IgG antibodies against PspA and PspC and protection against colonization was observed, these results do not rule out the protective potential of these antigens as vaccines against pneumococcal infections.

Pneumococcal induced thrombotic thrombocytopenic purpura with features of purpura fulminans.

A 42-year-old woman with a history of acute myeloid leukaemia status postallogeneic stem cell transplant presented with fevers, altered mental status, pulmonary infiltrates and septic shock that further progressed to thrombocytopenia and purpura fulminans. Laboratory studies were consistent with a diagnosis of thrombotic thrombocytopenic purpura (TTP). Blood cultures grew Streptococcus pneumoniae On chart review, our patient had a history of low immunoglobulin levels following stem cell transplant, which may have predisposed her to pneumococcal infection. The patient responded to therapy with ceftriaxone, plasma exchange, rituximab and caplacizumab. This is the fourth-documented case of pneumococcal induced TTP and, to the best of our knowledge, the first-describing pneumococcal induced TTP with purpura fulminans. We conclude that patients with TTP should be evaluated for infectious aetiologies and empiric antibiotics should be considered. Clinicians should be aware of the possibility for TTP to lead to purpura fulminans.

Hemoglobin Induces Early and Robust Biofilm Development in Streptococcus pneumoniae by a Pathway That Involves comC but Not the Cognate comDE Two-Component System.

Streptococcus pneumoniae grows in biofilms during both asymptomatic colonization and infection. Pneumococcal biofilms on abiotic surfaces exhibit delayed growth and lower biomass and lack the structures seen on epithelial cells or during nasopharyngeal carriage. We show here that adding hemoglobin to the medium activated unusually early and vigorous biofilm growth in multiple S. pneumoniae serotypes grown in batch cultures on abiotic surfaces. Human blood (but not serum, heme, or iron) also stimulated biofilms, and the pore-forming pneumolysin, ply, was required for this induction. S. pneumoniae transitioning from planktonic into sessile growth in the presence of hemoglobin displayed an extensive transcriptome remodeling within 1 and 2 h. Differentially expressed genes included those involved in the metabolism of carbohydrates, nucleotides, amino acid, and lipids. The switch into adherent states also influenced the expression of several regulatory systems, including the comCDE genes. Inactivation of comC resulted in 67% reduction in biofilm formation, while the deletion of comD or comE had limited or no effect, respectively. These observations suggest a novel route for CSP-1 signaling independent of the cognate ComDE two-component system. Biofilm induction and the associated transcriptome remodeling suggest hemoglobin serves as a signal for host colonization in pneumococcus.

Systemic cytokines/chemokines associated to radiographic abnormalities in pneumonia in children.

Community-acquired pneumonia (CAP) diagnosis remains a challenge in paediatrics. Chest radiography is considered gold standard for definition of pneumonia, however no previous study assessed the relationship between immune response and radiographic-confirmed-pneumonia. We assessed association between cytokines/chemokines levels and radiographic abnormalities in children with CAP. Children < 5-years-old hospitalized with CAP were investigated in a prospective study at the Federal University of Bahia Hospital, Brazil. On admission, clinical data and biological samples were collected to investigate 20 aetiological agents and determine serum cytokines/chemokines levels; chest radiographs were performed. Among 158 patients, radiographic diagnosis of pneumonia was confirmed in 126(79.7%) and 17(10.8%) had pleural effusion. Viral, bacterial and pneumococcal infection were detected in 80(50.6%), 78(49.4%) and 37(23.4%) cases. By comparing the median concentrations of serum cytokines/chemokines between children with or without pleural effusion, interleukin(IL)-6 was higher (26.6[18.6-103.7] vs 3.0[0.0-19.8]; p < 0.001) among those with pleural effusion; and between children with or without radiographic-confirmed-pneumonia, IL-6 was higher in the first subgroup (4.5[0.0-23.4] vs 0.0[0.0-3.6]; p = 0.02) after having excluded cases with pleural effusion. Stratified analyses according to aetiology showed IL-6 increase in the radiographic-confirmed-pneumonia subgroup inside the pneumococcal infection (28.2[5.9-64.1] vs 0.0[0.0-0.0]; p = 0.03) subgroup. By multivariable analysis, with IL-6 as dependent variable, pneumococcal infection and pleural effusion showed independent association with IL-6 elevation [respective OR: 5.071 (95%CI = 2.226-11.548; p < 0.001) and 13.604 (95%CI = 3.463-53.449; p = 0.0001)]. Considering the cases without pleural effusion, the area under the curve of IL-6 to predict pneumococcal infection was 0.76 (95%CI = 0.66-0.86; p < 0.001). IL-6 increase is a potential biomarker of pneumococcal infection among children with CAP without pleural effusion upon admission.

Opsonization-Enhanced Antigen Presentation by MR1 Activates Rapid Polyfunctional MAIT Cell Responses Acting as an Effector Arm of Humoral Antibacterial Immunity.

Mucosa-associated invariant T (MAIT) cells are innate-like antimicrobial T cells recognizing a breadth of important pathogens via presentation of microbial riboflavin metabolite Ags by MHC class Ib-related (MR1) molecules. However, the interaction of human MAIT cells with adaptive immune responses and the role they may play in settings of vaccinology remain relatively little explored. In this study we investigated the interplay between MAIT cell-mediated antibacterial effector functions and the humoral immune response. IgG opsonization of the model microbe Escherichia coli with pooled human sera markedly enhanced the capacity of monocytic APC to stimulate MAIT cells. This effect included greater sensitivity of recognition and faster response kinetics, as well as a markedly higher polyfunctionality and magnitude of MAIT cell responses involving a range of effector functions. The boost of MAIT cell responses was dependent on strongly enhanced MR1-mediated Ag presentation via increased FcγR-mediated uptake and signaling primarily mediated by FcγRI. To investigate possible translation of this effect to a vaccine setting, sera from human subjects before and after vaccination with the 13-valent-conjugated Streptococcus pneumoniae vaccine were assessed in a MAIT cell activation assay. Interestingly, vaccine-induced Abs enhanced Ag presentation to MAIT cells, resulting in more potent effector responses. These findings indicate that enhancement of Ag presentation by IgG opsonization allows innate-like MAIT cells to mount a faster, stronger, and qualitatively more complex response and to function as an effector arm of vaccine-induced humoral adaptive antibacterial immunity.

Factors influencing PCV13 specific antibody response in Danish children starting in day care.

This study examines different factors influencing the 13-valent pneumococcal conjugate vaccine (PCV13) specific antibody response in 8-13 months old Danish children starting in day care. We present secondary findings to the ProbiComp study, which included nose swabs, buccal swabs and blood samples from the children before entering day care (baseline) and again after 6 months. Pneumococci isolated from nose swabs were identified by latex agglutination kit and Quellung reaction. Luminex-based assay was used for antibody measurements against specific anti-pneumococcal capsular IgG. Buccal gene expression was analyzed by qPCR. Statistical analyses were performed in R and included Pearson's Chi-squared test, Welch two sample t-test and linear regression models. The PCV13 antibody response was unaffected by whether the children were carriers or non-carriers of any pneumococcal serotype. Having siblings increased the risk of carrying serotype 21 before day care (p = 0.020), and having siblings increased the PCV13 antibody response at the end of study (p = 0.0135). Hepatitis B-vaccination increased the PCV13 antibody response before day care attendance (p = 0.005). The expression of IL8 and IL1B was higher in children carrying any pneumococcal serotype at baseline compared to non-carriers (p = 0.0125 and p = 0.0268 respectively).

A Retrospective Study of the Impact of Pneumococcal Conjugate Vaccine-13 Immunization in a Northern Israel Hospital.

BACKGROUND: The introduction of pneumococcal conjugate vaccine-13 (PCV-13) has reduced the burden of invasive pneumococcal disease. OBJECTIVES: To characterize true positive blood cultures of children who presented to our hospital following implementation of the PCV-13 vaccine. METHODS: A retrospective study was conducted on positive blood cultures of children presenting with fever from 2010-2017. Subjects were divided into two age groups: a younger group 3-36 months and an older group 3-18 years. Patients were classified as either having or not having a focus of infection at the time of their bacteremia. Pneumococcal isolates were typed at Israel's Streptococcal Reference Laboratory. RESULTS: The samples included 94 true positive blood cultures. Focal infection with concomitant bacteremia was more common than bacteremia without a focus both overall: 67/94 (71%) vs. 27/94 (28.7%), P <0.001 as well as in the two groups: 32/48 (66%) vs. 16/48 (33%), P = 0.02 in the younger group and 35/46 (76%) vs. 11/46 (24%), P = 0.001 in the older group. Streptococcus pneumoniae was the most common pathogen overall, 27/94 (29%), and in the younger group, 21/48 (44%), but rare in the older group, 6/46 (13%). In the latter, Brucella species predominated, 12/46 (26%), along with Staphylococcus aureus 12/46 (26%). CONCLUSIONS: Our findings are consistent with other studies reporting decreased pneumococcal bacteremia, bacteremia primarily accompanying focal infection, and changing etiological agents among PCV-13-vaccinated children. Brucella species was prominent in older children with osteoarticular infections. Ongoing surveillance is warranted to better understand the implications of PCV-13.

An antifouling peptide-based biosensor for determination of Streptococcus pneumonia markers in human serum.

We report a peptide-based sensor that involves a multivalent interaction with L-ascorbate 6-phosphate lactonase (UlaG), a protein marker of Streptococcus pneumonia. By integrating the antifouling feature of the sensor, we significantly improved the signal-to-noise ratio of UlaG detection. The antifouling surface was fabricated via electrodeposition using an equivalent mixture of 4-amino-N,N,N-trimethylanilinium and 4-aminobenzenesulfonate. This antifouling layer not only effectively reduces the non-specific adsorption on the biosensor but also decreases the charge transfer resistance (Rct) of the screen-printed carbon electrode. The aniline-modified S7 peptide, an UlaG-binding peptide, was pre-synthesized and further electrochemically modified to bind onto the antifouling layer. Bio-electrochemical analysis confirms that the antifouling S7-peptide sensor binds strongly to the UlaG with a dissociation constant (Kd) = 0.5 nM. This strong interaction can be attributed to a multivalent interaction between the biosensor and the heximeric form of UlaG. To demonstrate the potential for clinical application, further detection of Streptococcus pneumonia from 50 to 5×104 CFU/mL were successfully performed in 25% human serum.

Submission for Special Issue: The Role of Platelet Activation in the Pathophysiology of HIV, Tuberculosis, and Pneumococcal Disease. Bedaquiline Suppresses ADP-Mediated Activation of Human Platelets In Vitro via Interference With Phosphatidylinositol 3-Kinase.

Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625-10 µg/ml), followed by activation with adenosine 5'-diphosphate (ADP), thrombin or the thromboxane A2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin- or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 µg/ml and was paralleled by inhibition of aggregation and Ca2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the secondary cationic amphiphilic properties of this agent. If operative in vivo, these anti-platelet effects of bedaquiline may contribute to ameliorating the risk of TB-associated cardiovascular disease, but this remains to be explored in the clinical setting.

Neutrophilic thrombophagocytosis.

Platelets phagocytosed by neutrophils is a rare morphological finding on peripheral blood films. It is rarely an EDTA-dependent artifact but mainly caused by an active vascular inflammation process. Here is reported an unexpected observation of neutrophilic thrombophagocytosis, in the context of a disseminated Streptococcus pneumoniae infection.