Evolución de los serotipos causantes de enfermedad neumocócica invasiva aislados durante 2008-2022 en un Hospital Público Madrileño de nivel dos, en relación con su inclusión en diferentes vacunas conjugadas / Evolution of the serotypes causing invasive pneumococcal disease along 2008-2022 in a Level 2 Public Hospital of the Madrid Region, in relation to their inclusion in different conjugate vaccines

Introducción. El uso de vacunas conjugadas frente a Streptococcus pneumoniae ocasiona cambios en la epidemio logía de la Enfermedad Neumocócica Invasiva (ENI). El objetivo de este estudio fue analizar la evolución de los serotipos de S. pneumoniae aislados en el Hospital Universitario de Getafe entre 2008 y 2022. Material y métodos. Se estudiaron 313 cepas de S. pneu moniae. El serotipado se realizó mediante el test de aglutina ción por látex (Pneumotest-latex) y la reacción de Quellung. Además, se determinó la concentración mínima inhibitoria (CMI) frente a penicilina, eritromicina y levofloxacino por el método de gradiente de concentración (E-test) según los cri terios de corte EUCAST. Resultados. Los serotipos más frecuentes en todo el pe riodo de estudio fueron 8, 3, 19A, 1, 11A y 22F correspondien do con el 46,6 % de los aislados. Durante los años 2008-2012, los serotipos 3, 1, 19A, 7F, 6C y 11A supusieron en conjunto el 53,6% de los aislamientos. Entre 2013 y 2017 los serotipos 3, 8, 12F, 19A, 22F y 19F representaron el 51% de los aislados. Entre 2018-2022 los serotipos 8, 3, 11A, 15A, 4 y 6C incluyeron al 55,5% de los casos. En total, 5 cepas (1,6%) se mostraron resistentes a penicilina, 64 (20,4%) resistentes a eritromicina y 11 (3,5%) resistentes a levofloxacino. Los niveles de CMI50 y CMI90 frente a los tres antibióticos se mantuvieron estables a lo largo del tiempo. Conclusiones. El uso de vacunas conjugadas condicionó un descenso de los serotipos cubiertos junto con un aumento de los no vacunales. Los patrones de sensibilidad a eritromicina y levofloxacino se mantuvieron relativamente estables. La re sistencia a penicilina fue muy baja, no encontrándose este tipo de cepas resistentes en el último periodo de estudio (AU)

Introduction. The use of conjugate vaccines against Streptococcus pneumoniae originates changes in the invasive pneumococcal disease (IPD). The aim of this study was to in vestigate the evolution of S. pneumoniae serotypes isolated in the Hospital Universitario de Getafe between 2008 and 2022. Material and Methods. 313 of S. pneumoniae strains were studied. Serotyping was carried out by latex agglutina tion (Pneumotest-latex) and the Quellung reaction. In addi tion, the minimal inhibitory concentration (MIC) was deter mined against penicillin, erythromycin and levofloxacin by the concentration gradient method (E-test) according the EUCAST breakpoints. Results. The most frequent serotypes throughout the study period were 8, 3, 19A, 1, 11A and 22F corresponding to 46.6% of the isolates. Along 2008-2012 the serotypes 3, 1, 19A, 7F, 6C and 11A represented altogether 53.6% of the isolates. Between 2013 and 2017 the serotypes 3, 8, 12F, 19A, 22F and 19F grouped 51% of the isolates. During 2018-2022 the serotypes 8, 3, 11A, 15A, 4 and 6C included the 55.5% of the cases. In total 5 strains (1.6%) were penicillin resistant, 64 (20.4%) erythromycin resistant and 11 (3.5%) levofloxacin re sistant. The MIC50 and MIC90 levels maintained stables along the time. Conclusion. The conjugate vaccines use with different se rotype coverage conditioned a decrease of the vaccine-includ ed and an increase of non-covered. Despite these changes, the global antimicrobial susceptibility patterns to erythromycin and levofloxacin maintained relatively stables. The resistance a penicillin was low, not finding this type of resistant strains in the last study period (AU)

Capsular typing of Streptococcus pneumoniae isolated from clinical specimens in Gauhati Medical College and hospital, Assam, India.

PURPOSE: Streptococcus pneumoniae is an important human respiratory tract pathogen causing pneumococcal diseases in majority of children and adults. The capsule is a significant virulence factor of Pneumococci which determines the bacterial serotype and is the component used for synthesis of pneumococcal vaccines. This cross-sectional study aimed to isolate Streptococcus pneumoniae from clinical samples and determine the occurrence of its circulating serotypes in Assam, North East India. MATERIALS AND METHODS: A total of 80 clinical samples were collected from June 2019 to May 2020 from patients clinically suspected from pneumococcal infection and also included samples routinely sent to bacteriology laboratory. Isolation and identification of S. pneumoniae was performed using conventional culture and molecular methods. Antibiotic susceptibility patterns were monitored. Capsular serotyping was performed using PCR of cpsA gene followed by DNA sequencing. RESULTS: Majority of the cases suspected of pneumococcal infection belong to the paediatric group aged less than 5 years. Out of 80 samples, 10 (12.50%) were found to be positive by PCR of recP gene. Culture was positive in 80% (8/10) of the total positives. Co-trimoxazole resistance was seen in 33.33% of the isolate from sputum. Serotypes 6A, 6B, 6C and 19F were detected in our region, out of which 6C is a non-vaccine serotype. CONCLUSION: Continued surveillance is needed to monitor trends in non-vaccine serotypes that may emerge as highly associated with antibiotic resistance. Also, the need to continuous monitoring of the antibiotic susceptibility of S. pneumoniae in North eastern parts of India is of outmost importance.

SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by Streptococcus pneumoniae and Staphylococcus aureus.

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike-biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients.

Widespread sharing of pneumococcal strains in a rural African setting: proximate villages are more likely to share similar strains that are carried at multiple timepoints.

The transmission dynamics of Streptococcus pneumoniae in sub-Saharan Africa are poorly understood due to a lack of adequate epidemiological and genomic data. Here we leverage a longitudinal cohort from 21 neighbouring villages in rural Africa to study how closely related strains of S. pneumoniae are shared among infants. We analysed 1074 pneumococcal genomes isolated from 102 infants from 21 villages. Strains were designated for unique serotype and sequence-type combinations, and we arbitrarily defined strain sharing where the pairwise genetic distance between strains could be accounted for by the mean within host intra-strain diversity. We used non-parametric statistical tests to assess the role of spatial distance and prolonged carriage on strain sharing using a logistic regression model. We recorded 458 carriage episodes including 318 (69.4 %) where the carried strain was shared with at least one other infant. The odds of strain sharing varied significantly across villages (χ2=47.5, df=21, P-value <0.001). Infants in close proximity to each other were more likely to be involved in strain sharing, but we also show a considerable amount of strain sharing across longer distances. Close geographic proximity (<5 km) between shared strains was associated with a significantly lower pairwise SNP distance compared to strains shared over longer distances (P-value <0.005). Sustained carriage of a shared strain among the infants was significantly more likely to occur if they resided in villages within a 5 km radius of each other (P-value <0.005, OR 3.7). Conversely, where both infants were transiently colonized by the shared strain, they were more likely to reside in villages separated by over 15 km (P-value <0.05, OR 1.5). PCV7 serotypes were rare (13.5 %) and were significantly less likely to be shared (P-value <0.001, OR -1.07). Strain sharing was more likely to occur over short geographical distances, especially where accompanied by sustained colonization. Our results show that strain sharing is a useful proxy for studying transmission dynamics in an under-sampled population with limited genomic data. This article contains data hosted by Microreact.

Evolution of pneumococcal serotype epidemiology in Botswana following introduction of 13-valent pneumococcal conjugate vaccine.

Pneumococcal conjugate vaccines reduce the burden of invasive pneumococcal disease, but the sustained effect of these vaccines can be diminished by an increase in disease caused by non-vaccine serotypes. To describe pneumococcal serotype epidemiology in Botswana following introduction of 13-valent pneumococcal conjugate vaccine (PCV-13) in July 2012, we performed molecular serotyping of 268 pneumococcal strains isolated from 221 children between 2012 and 2017. The median (interquartile range) age of the children included in this analysis was 6 (3,12) months. Fifty-nine percent of the children had received at least one dose of PCV-13 and 35% were fully vaccinated with PCV-13. While colonization by vaccine serotypes steadily declined following PCV-13 introduction, 25% of strains isolated more than 3 years after vaccine introduction were PCV-13 serotypes. We also observed an increase in colonization by non-vaccine serotypes 21 and 23B, which have been associated with invasive pneumococcal disease and antibiotic resistance in other settings.

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

Current real-time high-throughput Polymerase Chain Reaction (qPCR) methods do not distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-2011), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92-101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/mL of sample. There was high concordance (Kappa = 0.73-0.92); sensitivity (85-100%) and specificity (96-100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

Serotypes and Clonal Composition of Streptococcus pneumoniae Isolates Causing IPD in Children and Adults in Catalonia before 2013 to 2015 and after 2017 to 2019 Systematic Introduction of PCV13.

The goal of this study was to investigate the distribution of serotypes and clonal composition of Streptococcus pneumoniae isolates causing invasive pneumococcal disease (IPD) in Catalonia, before and after systematic introduction of PCV13. Pneumococcal strains isolated from normally sterile sites obtained from patients of all ages with IPD received between 2013 and 2019 from 25 health centers of Catalonia were included. Two study periods were defined: presystematic vaccination period (2013 and 2015) and systematic vaccination period (SVP) (2017 to 2019). A total of 2,303 isolates were analyzed. In the SVP, there was a significant decrease in the incidence of IPD cases in children 5 to 17 years old (relative risk [RR] 0.61; 95% confidence interval [CI] 0.38 to 0.99), while there was a significant increase in the incidence of IPD cases in 18- to 64-year-old adults (RR 1.33; 95% CI 1.16 to 1.52) and adults over 65 years old (RR 1.23; 95% CI 1.09 to 1.38). Serotype 8 was the major emerging serotype in all age groups except in 5- to 17-year-old children. In children younger than 5 years old, the main serotypes in SVP were 24F, 15A, and 3, while in adults older than 65 years they were serotypes 3, 8, and 12F. A significant decrease in the proportions of clonal complexes CC156, CC191, and ST306 and an increase in those of CC180, CC53, and CC404 were observed. A steady decrease in the incidence of IPD caused by PCV13 serotypes indicates the importance and impact of systematic vaccination. The increase of non-PCV13 serotypes highlights the need to expand serotype coverage in future vaccines and rethink vaccination programs for older adults. IMPORTANCE We found that with the incorporation of the PCV13 vaccine, the numbers of IPD cases caused by serotypes included in this vaccine decreased in all of the age groups. Still, there was an unforeseen increase of the serotypes not included in this vaccine causing IPD, especially in the >65-year-old group. Moreover, a significant increase of serotype 3 included in the vaccine has been observed; this event has been reported by other researchers. These facts call for the incorporation of more serotypes in future vaccines and a more thorough surveillance of the dynamics of this microorganism.

Re-emergence of invasive pneumococcal disease (IPD) and increase of serotype 23B after easing of COVID-19 measures, Switzerland, 2021.

Incidence of invasive pneumococcal disease (IPD) has been low during the peak of the COVID-19 pandemic. In this study, we found that the IPD numbers again increased in Switzerland during the first six months of 2021 and that this coincides with the loosening of COVID-19 measures. Vaccine pneumococcal serotypes have continued to decrease and non-vaccine type serotype 23B has emerged (8% of the isolates in 2021). Worryingly, serotype 23B is associated with reduced susceptibility to penicillin.

Isolation and Characterization of Human Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide 3.

The current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human ST3 pneumococcal capsular polysaccharide (PPS3) antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven PPS3-specific human monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro, and four mediated both activities. Two humAbs, namely, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03) both altered ST3 gene expression in vitro; however, C10 had fewer VL somatic mutations, higher PPS3 affinity, and promoted in vitro ST3 opsonophagocytic and agglutinating activity, whereas C27 did not. In C57BL/6 mice, both humAbs reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. After performing VL swaps, C10VH/C27VL exhibited reduced ST3 binding and agglutination, but C27VH/C10VL binding was unchanged. However, both humAbs lost the ability to reduce colonization in vivo when their light chains were replaced. Our findings associate the ability of PPS3-specific humAbs to reduce colonization with ST3 agglutination and opsonophagocytic activity, and reveal an unexpected role for the VL in their functional activity in vitro and in vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3. IMPORTANCE Despite the global success of vaccination with pneumococcal conjugate vaccines, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice; however, neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analyzed the functional and in vivo efficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protection in vivo, and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next-generation vaccines for ST3.

Cytokine response in cerebrospinal fluid of meningitis patients and outcome associated with pneumococcal serotype.

Streptococcus pneumoniae causes life-threatening meningitis. Its capsular polysaccharide determines the serotype and influences disease severity but the mechanism is largely unknown. Due to evidence of elevated cytokines levels in the meningeal inflammatory response, we measured 41 cytokines/chemokines and growth factors in cerebrospinal fluid (CSF) samples from 57 South African meningitis patients (collected in the period 2018-2019), with confirmed S. pneumoniae serotypes, using a multiplexed bead-based immunoassay. Based on multivariable Bayesian regression, using serotype 10A as a reference and after adjusting for HIV and age, we found IL-6 concentrations significantly lower in patients infected with serotypes 6D (undetectable) and 23A (1601 pg/ml), IL-8 concentrations significantly higher in those infected with 22A (40,459 pg/ml), 7F (32,400 pg/ml) and 15B/C (6845 pg/ml), and TNFα concentration significantly higher in those infected with serotype 18A (33,097 pg/ml). Although a relatively small number of clinical samples were available for this study and 28% of samples could not be assigned to a definitive serotype, our data suggests 15B/C worthy of monitoring during surveillance as it is associated with in-hospital case fatality and not included in the 13-valent polysaccharide conjugate vaccine, PCV13. Our data provides average CSF concentrations of a range of cytokines and growth factors for 18 different serotypes (14, 19F, 3, 6A, 7F, 19A, 8, 9N, 10A, 12F, 15B/C, 22F, 16F, 23A, 31, 18A, 6D, 22A) to serve as a basis for future studies investigating host-pathogen interaction during pneumococcal meningitis. We note that differences in induction of IL-8 between serotypes may be particularly worthy of future study.

Pneumococcal Carriage Among Indigenous Kichwa Children From the Ecuadorian Andes After the 10-Valent Pneumococcal Vaccine Introduction.

BACKGROUND: We assessed nasopharyngeal pneumococcal carriage in Andean Kichwa children, the largest Amerindian indigenous population in the Ecuadorian Andes. All children in our study had been vaccinated with the 10-valent pneumococcal vaccine (PCV10). METHODS: Nasopharyngeal swabs from 63 families, 100 children <10 years old including 38 children under 5 years and 63 adult caregivers, from 5 different communities, were cultivated for Streptococcus pneumoniae and isolates were serotyped and antibiotic susceptibility testing was performed. RESULTS: Respectively, 67% of the 38 children under 5 years old, 49% of the 62 children between 6 and 10 years old and 16% of the 100 adults were colonized with S. pneumoniae. Of these, 30.9% carried a vaccine serotype, 5.4% a serotype shared by the PCV10/13-valent pneumococcal vaccine (PCV13) vaccine and 25.5% a PCV13 serotype or PCV13 vaccine-related serotype, with 19A (10.9%) and 6C (10.9%) as the most prominent. Drug susceptibility testing revealed that 46% of the S. pneumoniae strains were susceptible to 6 tested antibiotics. However, 20.3% of the strains were multidrug-resistant or extensively drug-resistant strains, including 82% of the vaccine (-related) serotype 19A and 6C strains. CONCLUSIONS: Kichwa children, vaccinated with PCV10, were highly colonized with pneumococci and should be considered a high-risk group for pneumococcal disease. Twenty-five percent of the colonizing S. pneumoniae strains were PCV13-only vaccine-targeted serotypes, and in addition to that, most were multidrug-resistant or extensively drug-resistant strains. The vaccine benefits for this population possibly will significantly increase with the introduction of PCV13.

Population genetic structure, serotype distribution and antibiotic resistance of Streptococcus pneumoniae causing invasive disease in children in Argentina.

Invasive disease caused by Streptococcus pneumoniae (IPD) is one of the leading causes of morbidity and mortality in young children worldwide. In Argentina, PCV13 was introduced into the childhood immunization programme nationwide in 2012 and PCV7 was available from 2000, but only in the private market. Since 1993 the National IPD Surveillance Programme, consisting of 150 hospitals, has conducted nationwide pneumococcal surveillance in Argentina in children under 6 years of age, as part of the SIREVA II-OPS network. A total of 1713 pneumococcal isolates characterized by serotype (Quellung) and antimicrobial resistance (agar dilution) to ten antibiotics, belonging to three study periods: pre-PCV7 era 1998-1999 (pre-PCV), before the introduction of PCV13 2010-2011 (PCV7) and after the introduction of PCV13 2012-2013 (PCV13), were available for inclusion. Fifty-four serotypes were identified in the entire collection and serotypes 14, 5 and 1 represented 50 % of the isolates. Resistance to penicillin was 34.9 %, cefotaxime 10.6 %, meropenem 4.9 %, cotrimoxazole 45 %, erythromycin 21.5 %, tetracycline 15.4 % and chloramphenicol 0.4 %. All the isolates were susceptible to levofloxacin, rifampin and vancomycin. Of 1713 isolates, 1061 (61.9 %) were non-susceptible to at least one antibiotic and 235(13.7 %) were multidrug resistant. A subset of 413 isolates was randomly selected and whole-genome sequenced as part of Global Pneumococcal Sequencing Project (GPS). The genome data was used to investigate the population structure of S. pneumoniae defining pneumococcal lineages using Global Pneumococcal Sequence Clusters (GPSCs), sequence types (STs) and clonal complexes (CCs), prevalent serotypes and their associated pneumococcal lineages and genomic inference of antimicrobial resistance. The collection showed a great diversity of strains. Among the 413 isolates, 73 known and 36 new STs were identified belonging to 38 CCs and 25 singletons, grouped into 52 GPSCs. Important changes were observed among vaccine types when pre-PCV and PCV13 periods were compared; a significant decrease in serotypes 14, 6B and 19F and a significant increase in 7F and 3. Among non-PCV13 types, serogroup 24 increased from 0 % in pre-PCV to 3.2 % in the PCV13 period. Our analysis showed that 66.1 % (273/413) of the isolates were predicted to be non-susceptible to at least one antibiotic and 11.9 % (49/413) were multidrug resistant. We found an agreement of 100 % when comparing the serotype determined by Quellung and WGS-based serotyping and 98.4 % of agreement in antimicrobial resistance. Continued surveillance of the pneumococcal population is needed to reveal the dynamics of pneumococcal isolates in Argentina in post-PCV13. This article contains data hosted by Microreact.

Safety and Immunogenicity of a 20-valent Pneumococcal Conjugate Vaccine in Healthy Infants in the United States.

BACKGROUND: The development and widespread use of pneumococcal conjugate vaccines (PCVs) substantially reduced the global burden of pneumococcal disease. Expanding the serotypes covered by PCVs may further reduce disease burden. A 20-valent PCV (PCV20) has been developed to add coverage for 7 additional serotypes (8, 10A, 11A, 12F, 15B, 22F and 33F) to those in the existing 13-valent PCV (PCV13). This phase 2 study evaluated the safety, tolerability and immunogenicity of PCV20 in healthy US infants. METHODS: In this randomized, active-controlled, double-blind study, 460 infants were randomized 1:1 to receive a 4-dose series of either PCV20 or PCV13 at 2, 4, 6 and 12 months of age. Solicited local reactions and systemic events, adverse events (AEs) and serious AEs were recorded. Immunogenicity was assessed by measuring serotype-specific IgG concentrations and opsonophagocytic activity titers at 1 month after Dose 3, before Dose 4 and 1 month after Dose 4. RESULTS: Of 460 infants, 82.8% completed the 1-month visit after Dose 4. Local reactions and systemic events were mostly mild to moderate in severity and similar between the PCV20 and PCV13 groups. Treatment-related AEs were uncommon, with no related serious AEs or deaths reported. IgG and opsonophagocytic activity responses elicited by PCV20 were robust and demonstrated a booster response after Dose 4. CONCLUSIONS: Administration of PCV20 in US infants was well tolerated, with a safety profile similar to PCV13, and induced robust serotype-specific immune responses. These findings support continued development of PCV20 in the pediatric population.

A LAMP-based system for rapid detection of eight common pathogens causing lower respiratory tract infections.

Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality worldwide and lack a rapid diagnostic method. To improve the diagnosis of LRTIs, we established an available loop-mediated isothermal amplification (LAMP) assay for the detection of eight common lower respiratory pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. The whole process can be achieved within 1 h (sample to results read out). We established an extraction free isothermal system. 528 sputum samples collected from patients suspected to have LRTIs were analyzed by the system (8 tests in each sample, a total of 4224 tests) and compared with the standard culture method (SCM). The samples with inconsistent results were further verified by Sanger sequencing and High-throughput sequencing (NGS). The detection limits of the LAMP assay for the 8 pathogens ranged from 103 to 104 CFU/mL. Upon testing 528 samples, the Kappa coefficients of all pathogens ranged between 0.5 and 0.7 indicated a moderate agreement between the LAMP assay and the SCM. All inconsistent samples were further verified by Sanger sequencing, we found that the developed LAMP assay had a higher consistency level with Sanger sequencing than the SCM for all pathogens. Additionally, when the NGS was set to a diagnostic gold standard, the specificity and sensitivity of the LAMP assay for LRTIs were 94.49% and 75.00%. The present study demonstrated that the developed LAMP has high consistency with the sequencing methods. Meanwhile, the LAMP assay has a higher detection rate compared to the SCM. It may be a powerful tool for rapid and reliable clinical diagnosis of LRTIs in primary hospitals.

Streptococcus pneumoniae serotypes that frequently colonise the human nasopharynx are common recipients of penicillin-binding protein gene fragments from Streptococcus mitis.

Streptococcus pneumoniae is an important global pathogen that causes bacterial pneumonia, sepsis and meningitis. Beta-lactam antibiotics are the first-line treatment for pneumococcal disease, however, their effectiveness is hampered by beta-lactam resistance facilitated by horizontal genetic transfer (HGT) with closely related species. Although interspecies HGT is known to occur among the species of the genus Streptococcus, the rates and effects of HGT between Streptococcus pneumoniae and its close relatives involving the penicillin binding protein (pbp) genes remain poorly understood. Here we applied the fastGEAR tool to investigate interspecies HGT in pbp genes using a global collection of whole-genome sequences of Streptococcus mitis, Streptococcus oralis and S. pneumoniae. With these data, we established that pneumococcal serotypes 6A, 13, 14, 16F, 19A, 19F, 23F and 35B were the highest-ranking serotypes with acquired pbp fragments. S. mitis was a more frequent pneumococcal donor of pbp fragments and a source of higher pbp nucleotide diversity when compared with S. oralis. Pneumococci that acquired pbp fragments were associated with a higher minimum inhibitory concentration (MIC) for penicillin compared with pneumococci without acquired fragments. Together these data indicate that S. mitis contributes to reduced ß-lactam susceptibility among commonly carried pneumococcal serotypes that are associated with long carriage duration and high recombination frequencies. As pneumococcal vaccine programmes mature, placing increasing pressure on the pneumococcal population structure, it will be important to monitor the influence of antimicrobial resistance HGT from commensal streptococci such as S. mitis.

Persistence of Immunity Following 2-Dose Priming with a 10-Valent Pneumococcal Conjugate Vaccine at 6 and 10 Weeks or 6 and 14 Weeks of Age in Nepalese Toddlers.

BACKGROUND: The pneumococcal conjugate vaccine has had a substantial impact on invasive pneumococcal disease. Previously, we compared immunity following vaccination with the 10-valent pneumococcal conjugate vaccine (PCV10) administered at 2 slightly different schedules: at 6 and 10 weeks of age, and at 6 and 14 weeks of age, both followed by a 9-month booster. In this study, we followed up those participants to evaluate the medium-term persistence of serotype-specific pneumococcal immunity at 2-3 years of age. METHOD: Children from the previous studies were contacted and after taking informed consent from their parents, blood samples and nasopharyngeal swabs were collected. Serotype-specific IgG antibody concentrations were determined by enzyme-linked immunosorbent assay, for the 10 vaccine serotypes, at a WHO pneumococcal serology reference laboratory. FINDINGS: Two hundred twenty of the 287 children who completed the primary study returned at 2-3 years of age to provide a blood sample and nasopharyngeal swab. The nasopharyngeal carriage rate of PCV10 serotypes in the 6 + 14 group was higher than the 6 + 10 group (13.4% vs. 1.9%). Nevertheless, the proportion of toddlers with serum pneumococcal serotype-specific IgG greater than or equal to 0.35 µg/mL was comparable for all PCV10 serotypes between the 6 + 10 week and 6 + 14 week groups. Similarly, the geometric mean concentrations of serum pneumococcal serotype-specific IgG levels were similar in the 2 groups for all serotypes, except for serotype 19F which was 32% lower in the 6 + 10 group than the 6 + 14 group. CONCLUSION: Immunization with PCV10 at 6 + 10 weeks or 6 + 14 weeks, with a booster at 9 months in each case, results in similar persistence of serotype-specific antibody at 2-3 years of age. Thus, protection from pneumococcal disease is expected to be similar when either schedule is used.

Examining the Distribution and Impact of Single-Nucleotide Polymorphisms in the Capsular Locus of Streptococcus pneumoniae Serotype 19A.

Streptococcus pneumoniae serotype 19A prevalence has increased after the implementation of the PCV7 and PCV10 vaccines. In this study, we have provided, with high accuracy, the genetic diversity of the 19A serotype in a cohort of Dutch invasive pneumococcal disease patients and asymptomatic carriers obtained in the period from 2004 to 2016. The whole genomes of the 338 pneumococcal isolates in this cohort were sequenced and their capsule (cps) loci compared to examine their diversity and determine the impact on the production of capsular polysaccharide (CPS) sugar precursors and CPS shedding. We discovered 79 types with a unique cps locus sequence. Most variation was observed in the rmlB and rmlD genes of the TDP-Rha synthesis pathway and in the wzg gene, which is of unknown function. Interestingly, gene variation in the cps locus was conserved in multiple alleles. Using RmlB and RmlD protein models, we predict that enzymatic function is not affected by the single-nucleotide polymorphisms as identified. To determine if RmlB and RmlD function was affected, we analyzed nucleotide sugar levels using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). CPS precursors differed between 19A cps locus subtypes, including TDP-Rha, but no clear correlation was observed. Also, significant differences in multiple nucleotide sugar levels were observed between phylogenetically branched groups. Because of indications of a role for Wzg in capsule shedding, we analyzed if this was affected. No clear indication of a direct role in shedding was found. We thus describe genotypic variety in rmlB, rmlD, and wzg in serotype 19A in the Netherlands, for which we have not discovered an associated phenotype.

The 13-Valent Pneumococcal Conjugate Vaccine Elicits Serological Response and Lasting Protection in Selected Patients With Primary Humoral Immunodeficiency.

Background: Patients with primary humoral immunodeficiency are more prone to invasive as well as recurrent pneumococcal infections. Therefore, anti-pneumococcal vaccination including the 13-valent conjugate vaccine is recommended. Nevertheless, to date, no data is available on immunogenicity of this vaccine in this population. Objective: To assess the immunogenicity and the persistence of protection up to one year after a 13-valent pneumococcal conjugate vaccine in patients with primary humoral immunodeficiency. Methods: Twenty-nine patients with common variable immunodeficiency or IgG subclass deficiency were vaccinated. Immune response and immune protection at baseline as well as at one, six and twelve months after vaccination were evaluated by measuring specific IgG serum concentrations (ELISA), and opsonophagocytic activities directed against selected pneumococcal (MOPA). Results: By ELISA, half of the patients had protective IgG concentrations before vaccination, 35.7% showed an immune response one month after vaccination, 71.4%, 66.7% and 56.0% of the patients were protected at one, six and twelve months respectively. Conversely, by MOPA, 3.4% of the patients were protected at baseline, 10.7% showed an immune response and 28.6%, 48.2% and 33.3% were protected at one, six and twelve months respectively. IgG subclass deficiency, Ig replacement therapy and higher IgG2 concentrations at diagnosis were associated with long-term protection. Conclusion: Pneumococcal conjugate vaccine improves immune protection and antibodies' functionality in a subset of patients with primary immunodeficiency. Prime-boost vaccine strategy needs to be better and individually adapted.

Streptococcus Pneumoniae septic arthritis in adults in Bristol and Bath, United Kingdom, 2006-2018: a 13-year retrospective observational cohort study.

Few studies on adult pneumococcal septic arthritis are sufficiently large enough to assess both epidemiological trends following routine pneumococcal immunization and clinical disease. With major shifts in serotypes causing invasive pneumococcal disease (IPD), we wanted to determine the clinical phenotype of adult septic arthritis caused by Streptococcus pneumoniae. We conducted a retrospective cohort study of pneumococcal infections in Bristol and Bath, UK, 2006-2018. We defined pneumococcal septic arthritis as adults with clinically-confirmed septic arthritis, with pneumococcus isolated from sterile-site culture or urinary antigen test positivity. Clinical records were reviewed for each patient in the cohort. Septic arthritis accounted for 1.7% of all IPD cases. 45 cases of adult pneumococcal septic arthritis occurred, with disease typically affecting older adults and those with underlying comorbidity. 67% patients had another focus of infection during their illness. 66% patients required increased care on discharge and 43% had reduced range of movement. In-hospital case fatality rate was 6.7%. One-year patient mortality was 31%. Currently most cases of adult pneumococcal septic arthritis are due to non-PCV13 serotypes which are associated with more severe disease. Non-PCV-13 serotypes had higher prevalence of concomitant pneumococcal infection at another site (73.7% versus 36.6%), increased intensive care or high-dependency unit requirement (32.4% versus 0%), and increased inpatient and 1-year case fatality rate (8.8% versus 0%, and 32.4% versus 27.4% respectively) compared to PCV-13 serotypes. Pneumococcal septic arthritis remains a small proportion of IPD. However, there is significant associated morbidity and mortality, and pneumococcal septic arthritis requires monitoring in coming years.

A Structural Model for the Ligand Binding of Pneumococcal Serotype 3 Capsular Polysaccharide-Specific Protective Antibodies.

Capsular polysaccharides (CPSs) are major virulence factors that decorate the surfaces of many human bacterial pathogens. In their pure form or as glycoconjugate vaccines, CPSs are extensively used in vaccines deployed in clinical practice worldwide. However, our understanding of the structural requirements for interactions between CPSs and antibodies is limited. A longstanding model based on comprehensive observations of antibody repertoires binding to CPSs is that antibodies expressing heavy chain variable gene family 3 (VH3) predominate in these binding interactions in humans and VH3 homologs in mice. Toward understanding this highly conserved interaction, we generated a panel of mouse monoclonal antibodies (MAb) against Streptococcus pneumoniae serotype 3 CPS, determined an X-ray crystal structure of a protective MAb in complex with a hexasaccharide derived from enzymatic hydrolysis of the polysaccharide, and elucidated the structural requirements for this binding interaction. The crystal structure revealed a binding pocket containing aromatic side chains, suggesting the importance of hydrophobicity in the interaction. Through mutational analysis, we determined the amino acids that are critical in carbohydrate binding. Through elucidating the structural and functional properties of a panel of murine MAbs, we offer an explanation for the predominant use of the human VH3 gene family in antibodies against CPSs with implications in knowledge-based vaccine design. IMPORTANCE Infectious diseases caused by pathogenic bacteria are a major threat to human health. Capsular polysaccharides (CPSs) of many pathogenic bacteria have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide, with various degrees of success. Immunization with a glycoconjugate vaccine elicits T cell help for B cells that produce IgG antibodies to the CPS. Thus, it is important to develop an in-depth understanding of the interactions of carbohydrate epitopes with the antibodies. Structural characterization of the ligand binding of polysaccharide-specific antibodies laid out in this study may have fundamental biological implications for our comprehension of how the humoral immune system recognizes polysaccharide antigens, and in future knowledge-based vaccine design.