Surgical restabilization reduces the progression of post-traumatic osteoarthritis initiated by ACL rupture in mice.

OBJECTIVE: People who sustain joint injuries such as anterior cruciate ligament (ACL) rupture often develop post-traumatic osteoarthritis (PTOA). In human patients, ACL injuries are often treated with ACL reconstruction. However, it is still unclear how effective joint restabilization is for reducing the progression of PTOA. The goal of this study was to determine how surgical restabilization of a mouse knee joint following non-invasive ACL injury affects PTOA progression. DESIGN: In this study, 187 mice were subjected to non-invasive ACL injury or no injury. After injury, mice underwent restabilization surgery, sham surgery, or no surgery. Mice were then euthanized on day 14 or day 49 after injury/surgery. Functional analyses were performed at multiple time points to assess voluntary movement, gait, and pain. Knees were analyzed ex vivo with micro-computed tomography, RT-PCR, and whole-joint histology to assess articular cartilage degeneration, synovitis, and osteophyte formation. RESULTS: Both ACL injury and surgery resulted in loss of epiphyseal trabecular bone (-27-32%) and reduced voluntary movement at early time points. Joint restabilization successfully lowered OA score (-78% relative to injured at day 14, p < 0.0001), and synovitis scores (-37% relative to injured at day 14, p = 0.042), and diminished the formation of chondrophytes/osteophytes (-97% relative to injured at day 14, p < 0.001, -78% at day 49, p < 0.001). CONCLUSIONS: This study confirmed that surgical knee restabilization was effective at reducing articular cartilage degeneration and diminishing chondrophyte/osteophyte formation after ACL injury in mice, suggesting that these processes are largely driven by joint instability in this mouse model. However, restabilization was not able to mitigate the early inflammatory response and the loss of epiphyseal trabecular bone, indicating that these processes are independent of joint instability.

A Phase Three Study of the Safety and Immunogenicity of a Four-dose Series of 20-Valent Pneumococcal Conjugate Vaccine in Healthy Infants.

BACKGROUND: The 20-valent pneumococcal conjugate vaccine (PCV20) was developed to extend pneumococcal disease protection beyond 13-valent PCV (PCV13). METHODS: This phase 3, double-blind study conducted in the United States/Puerto Rico evaluated PCV20 safety and immunogenicity. Healthy infants were randomized to receive a 4-dose series of PCV20 or PCV13 at 2, 4, 6 and 12-15 months old. Objectives included demonstrating noninferiority (NI) of PCV20 to PCV13 immunoglobulin G (IgG) geometric mean concentrations after doses 3 and 4 and percentages of participants with predefined IgG concentrations after dose 3, with 7 additional PCV20 serotypes compared with the lowest result among vaccine serotypes in the PCV13 group. Safety assessments included local reactions, systemic events, adverse events, serious adverse events and newly diagnosed chronic medical conditions. RESULTS: Overall, 1991 participants were vaccinated (PCV20, n = 1001; PCV13, n = 990). For IgG geometric mean concentrations 1 month after both doses 3 and 4, all 20 serotypes met NI criteria (geometric mean ratio lower 2-sided 95% confidence interval > 0.5). For percentages of participants with predefined IgG concentrations after dose 3, NI (percentage differences lower 2-sided 95% confidence interval > -10%) was met for 8/13 matched serotypes and 6/7 additional serotypes; 4 serotypes missed the statistical NI criterion by small margins. PCV20 also elicited functional and boosting responses to all 20 serotypes. The safety profile of PCV20 was similar to PCV13. CONCLUSION: A 4-dose series of PVC20 was well tolerated and elicited robust serotype-specific immune responses expected to help protect infants and young children against pneumococcal disease due to the 20 vaccine serotypes. Clinical trial registration: NCT04382326.

Surveillance of pneumococcal serotypes in adults hospitalised with acute lower respiratory tract infection in Bristol, UK.

INTRODUCTION: Pneumococcus remains a major cause of adult lower respiratory tract infections (LRTI). Few data exist on the relative contribution of serotypes included in pneumococcal vaccines to community-acquired pneumonia (CAP) and non-pneumonic (NP) LRTI. We measured the burden of all and vaccine-serotype pneumococcal respiratory infection following SARS-CoV-2 emergence to inform evidence-based vaccination policy. METHODS: A prospective cohort study at two Bristol hospitals (UK) including all adults age ≥ 18-years hospitalised with acute lower respiratory tract disease (aLRTD) from Nov2021-Nov2022. LRTI patients were classified as: a) radiographically-confirmed CAP (CAP+/RAD+), b) clinically-diagnosed CAP without radiological confirmation (CAP+/RAD-), or c) NP-LRTI. Pneumococcus was identified by blood culture, BinaxNOW™and serotype-specific urine antigen detection assays (UAD). RESULTS: Of 12,083 aLRTD admissions, 10,026 had LRTI and 2,445 provided urine: 1,097 CAP + RAD+; 207 CAP + RAD-; and 1,141 NP-LRTI. Median age was 71.1y (IQR57.9-80.2) and Charlson comorbidity index = 4 (IQR2-5); 2.7 % of patients required intensive care, and 4.4 % died within 30-days of hospitalisation. Pneumococcus was detected in 280/2445 (11.5 %) participants. Among adults aged ≥ 65y and 18-64y, 12.9 % (198/1534) and 9.0 % (82/911), respectively, tested pneumococcus positive. We identified pneumococcus in 165/1097 (15.0 %) CAP + RAD+, 23/207 (11.1 %) CAP + RAD-, and 92/1141 (8.1 %) NP-LRTI cases. Of the 280 pneumococcal cases, 102 (36.4 %) were due to serotypes included in PCV13 + 6C, 115 (41.7 %) in PCV15 + 6C, 210 (75.0 %) in PCV20 + 6C/15C and 228 (81.4 %) in PPV23 + 15C. The most frequently identified serotypes were 8 (n = 78; 27.9 % of all pneumococcus), 7F (n = 25; 8.9 %), and 3 (n = 24; 8.6 %). DISCUSSION: Among adults hospitalised with respiratory infection, pneumococcus is an important pathogen across all subgroups, including CAP+/RAD- and NP-LRTI. Despite 20-years of PPV23 use in adults ≥ 65-years and herd protection due to 17-years of PCV use in infants, vaccine-serotype pneumococcal disease still causes a significant proportion of LRTI adult hospitalizations. Direct adult vaccination with high-valency PCVs may reduce pneumococcal disease burden.

A Phase 2 Extension Study Evaluating the Immunogenicity, Safety, and Tolerability of 3 or 4 Doses of a Clostridioides difficile Vaccine in Healthy US Adults Aged 65 to 85 Years.

BACKGROUND: This phase 2 extension explored the long-term antibody persistence of an investigational Clostridioides difficile vaccine and the safety, tolerability, and immunogenicity of dose 4 approximately 12 months post-dose 3. METHODS: One year post-dose 3, healthy US 65- to 85-year-olds (N = 300) were randomized to dose 4 of vaccine at previously received antigen levels (100 or 200 µg) or placebo. Assessments included safety and percentages of participants achieving neutralizing antibody titers above prespecified thresholds (≥219 and ≥2586 neutralization units/mL for toxins A and B, respectively). RESULTS: In participants previously given three 200-µg doses and placebo in the extension, toxin A and B neutralizing antibodies were above prevaccination levels 48 months post-dose 3 (36 months after placebo); 24.0% and 26.0% had toxin A and B antibodies at or above prespecified thresholds, respectively. Neutralizing antibodies increased post-dose 4 (12 months post-dose 3) and persisted to 36 months post-dose 4. Thirty days post-dose 4, all participants had toxin A and 86.5% to 100% had toxin B titers at or above prespecified thresholds. Local reactions were more frequent in vaccine recipients. Systemic and adverse event frequencies were similar across groups. CONCLUSIONS: C difficile vaccine immune responses persisted 48 months post-dose 3. Dose 4 was immunogenic and well tolerated, supporting continued development. Clinical Trials Registration. ClinicalTrials.gov NCT02561195.

Multicountry Review of Streptococcus pneumoniae Serotype Distribution Among Adults With Community-Acquired Pneumonia.

BACKGROUND: Nonbacteremic community-acquired pneumonia (CAP) is a leading presentation of severe pneumococcal disease in adults. Serotype-specific urinary antigen detection (UAD) assay can detect serotypes causing pneumococcal CAP, including nonbacteremic cases, and guide recommendations for use of higher valency pneumococcal conjugate vaccines (PCVs). METHODS: Adult CAP serotype distribution studies that used both Pfizer UADs (UAD1, detects PCV13 serotypes; UAD2, detects PCV20 non-PCV13 serotypes plus 2, 9N, 17F, and 20) were identified by review of an internal study database and included if results were published. The percentages of all-cause radiologically confirmed CAP (RAD + CAP) due to individual or grouped (PCV13, PCV15, and PCV20) serotypes as detected from culture or UAD were reported. RESULTS: Six studies (n = 2, United States; n = 1 each, Germany, Sweden, Spain, and Greece) were included. The percentage of RAD + CAP among adults ≥18 years with PCV13 serotypes equaled 4.6% to 12.9%, with PCV15 serotypes 5.9% to 14.5%, and with PCV20 serotypes 7.8% to 23.8%. The percentage of RAD + CAP due to PCV15 and PCV20 serotypes was 1.1-1.3 and 1.3-1.8 times higher than PCV13 serotypes, respectively. CONCLUSIONS: PCV13 serotypes remain a cause of RAD + CAP among adults even in settings with pediatric PCV use. Higher valency PCVs among adults could address an important proportion of RAD + CAP in this population.

A phase 3 study evaluating the lot consistency, immunogenicity, safety, and tolerability of a Clostridioides difficile vaccine in healthy adults 65 to 85 years of age.

BACKGROUND: A toxoid-based Clostridioides difficile vaccine is currently in development. Here, we report lot-to-lot consistency, immunogenicity, safety, and tolerability of 3 C difficile vaccine doses in healthy older adults. METHODS: This phase 3, placebo-controlled study randomized (1:1:1:1) healthy adults 65 to 85 years of age to 1 of 3 C difficile vaccine lots or placebo. Participants received C difficile vaccine (200 µg total toxoid) or placebo (Months 0, 1, 6). The primary immunogenicity objective was lot-to-lot consistency (2-sided 95 % CIs within 0.5 and 2 for comparisons of geometric mean concentration [GMC] ratios) for toxins A- and B-specific neutralizing antibody levels 1 month after Dose 3. Safety outcomes included local reactions and systemic events ≤7 days after vaccination, adverse events (AEs), and serious AEs (SAEs). RESULTS: Of 1317 enrolled participants, 1218 completed the study. C difficile vaccine immunogenicity was consistent across lots, with neutralizing antibody responses 1 month after Dose 3 for both toxin A (GMC [95 % CI]: lot 1, 878.8 [786.3, 982.2]; lot 2, 873.0 [779.2, 978.1]; lot 3, 872.9 [782.6, 973.5]) and toxin B (lot 1, 5823.9 [5041.0, 6728.4]; lot 2, 5462.8 [4733.4, 6304.7]; lot 3, 5426.0 [4724.4, 6231.8]). Two-sided 95 % CIs for GMC ratios were within 0.5 and 2 for toxins A and B, indicating lot-to-lot consistency was achieved. C difficile vaccine was well tolerated, with similar rates of local reactions and systemic events among vaccine lots. AE and SAE rates were similar across C difficile vaccine (36.5 % and 4.5 %, respectively) and placebo (35.3 % and 6 %). CONCLUSIONS: Three doses (Months 0,1,6) of toxoid-based C difficile vaccine induced robust neutralizing antibody responses and were well tolerated in healthy participants 65 to 85 years of age. Lot-to-lot consistency was excellent, indicating the manufacturing process for this C difficile vaccine formulation was well controlled. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03579459.

Comparison of pneumococcal immunogenicity elicited by the PCV13 and PCV15 vaccines in adults 18 through 49 years of age.

AIM: Pneumococcal conjugate vaccines (PCV13, PCV15, PCV20) effectively target the capsular polysaccharides of the most common disease-causing Streptococcus pneumoniae serotypes. In this short communication, we analyzed healthy participants who received PCV13 and PCV15 vaccines as part of a recently concluded exploratory clinical trial and report antibody responses to the 13 shared serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) as well as functional OPA responses to serotype 3. METHODS: Sera from 87 adult participants (18 through 49 years of age) randomized to receive either PCV13 or PCV15 were collected (n = 46 or n = 41, respectively), from 17 study centers in the US. IgG concentrations of the 13 shared serotypes and serotype 3-specific OPA titers were analyzed before and 1 month after vaccination using internally validated assays. RESULTS: At 1 month after vaccination, IgG GMCs of the 13 shared serotypes in PCV13 were similar to those for PCV15. Specifically, serotype 3 OPA GMTs and 95% CIs were similar 1 month after vaccination for PCV13 (62.9 [48.9, 80.9]) and PCV15 (71.1 [50.9, 99.2]). CONCLUSION: In healthy participants who received either PCV13 or PCV15, similar serotype-specific responses were observed between all shared serotypes when a uniform validated internal assay was used. Of note, data from this study suggest that both vaccines induce similar functional antibody responses against pneumococcal serotype 3.

Misdiagnosis of Clostridioides difficile Infections by Standard-of-Care Specimen Collection and Testing among Hospitalized Adults, Louisville, Kentucky, USA, 2019-20201.

Although Clostridioides difficile infection (CDI) incidence is high in the United States, standard-of-care (SOC) stool collection and testing practices might result in incidence overestimation or underestimation. We conducted diarrhea surveillance among inpatients >50 years of age in Louisville, Kentucky, USA, during October 14, 2019-October 13, 2020; concurrent SOC stool collection and CDI testing occurred independently. A study CDI case was nucleic acid amplification test‒/cytotoxicity neutralization assay‒positive or nucleic acid amplification test‒positive stool in a patient with pseudomembranous colitis. Study incidence was adjusted for hospitalization share and specimen collection rate and, in a sensitivity analysis, for diarrhea cases without study testing. SOC hospitalized CDI incidence was 121/100,000 population/year; study incidence was 154/100,000 population/year and, in sensitivity analysis, 202/100,000 population/year. Of 75 SOC CDI cases, 12 (16.0%) were not study diagnosed; of 109 study CDI cases, 44 (40.4%) were not SOC diagnosed. CDI incidence estimates based on SOC CDI testing are probably underestimated.

Identification of a Novel Keto Sugar Component in Streptococcus pneumoniae Serotype 12F Capsular Polysaccharide and Impact on Vaccine Immunogenicity.

Implementation of conjugate vaccine technology revolutionized the ability to effectively elicit long-lasting immune responses to bacterial capsular polysaccharides. Although expansion of conjugate vaccine serotype coverage is designed to target residual disease burden to pneumococcal serotypes not contained in earlier vaccine versions, details of polysaccharide Ag structure, heterogeneity, and epitope structure components contributing to vaccine-mediated immunity are not always clear. Analysis of Streptococcus pneumoniae serotype 12F polysaccharide by two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry revealed a partial substitution of N-acetyl-galactosamine by the keto sugar 2-acetamido-2,6-dideoxy-xylo-hexos-4-ulose (Sug) in up to 25% of the repeat units. This substitution was not described in previous published structures for 12F. Screening a series of contemporary 12F strains isolated from humans (n = 17) identified Sug incorporation at varying levels in all strains examined. Thus, partial Sug substitution in S. pneumoniae serotype 12F may have always been present but is now detectable by state-of-the-art analytical techniques. During the steps of conjugation, the serotype 12F Sug epitope is modified by reduction, and both polysaccharide PPSV23 and conjugate PCV20 vaccines contain 12F Ags with little to no Sug epitope. Both PCV20 and PPSV23 vaccines were evaluated for protection against circulating 12F strains with varying amounts of Sug in their repeat unit based on an opsonophagocytic killing assay involving HL-60 cells and rabbit complement. Both vaccines elicited human-derived neutralizing Abs against serotype 12F, independent of Sug level between ∼2 and 25 mol%. These findings suggest that the newly identified serotype 12F Sug epitope is likely not an essential epitope for vaccine-elicited protection.

Population-based incidence of hospitalized Clostridioides difficile infection among older adults in Ota-ku, Japan: A prospective surveillance study.

OBJECTIVES: Clostridioides difficile infection (CDI) burden is not well-characterized in Japan. Therefore, we conducted a population-based, hospitalized CDI incidence study, compared the results with standard-of-care (SOC) CDI testing, and generalized the results for nationwide incidence estimates. METHODS: Surveillance identified inpatients ≥50 years-of-age with diarrhea in nine Tokyo hospitals from December 17, 2018-March 30, 2020. A CDI case was defined as a patient with a PCR-positive/cell cytotoxicity neutralization assay (CCNA)-positive stool or a PCR-positive stool and pseudomembranous colitis (PMC). Incidence estimates were adjusted for the hospitalization share of participating hospitals and, in the sensitivity analysis, for missing CDI test results. SOC specimen collection and CDI testing occurred independently. RESULTS: Surveillance during 318 840 patient-days identified 4633 inpatients with diarrhea. Sixty-three CDI cases were identified; 11 (17·5%) had PMC, eight (12·7%) recurrent CDI, and nine (14·3%) died. The hospitalized CDI incidence was 97/100 000 population per year (PPY) in persons ≥50 years-of-age and, in the sensitivity analysis, 324/100 000 PPY. The incidence was 170 and 481/100 000 PPY in persons ≥65 and ≥85 years-of-age, respectively; these estimates increased to 569 and 1609/100 000 PPY in the sensitivity analysis, respectively. There were 12 primary SOC CDI cases in persons ≥50 years-of-age (18/100 000 PPY). CONCLUSIONS: The CDI incidence was high in older adults, with severe clinical consequences. SOC specimen collection and testing under-estimated CDI burden. There are >57 000 hospitalized CDI cases per year in Japan in persons ≥50 years-of-age. Public health interventions are needed to reduce the CDI burden in Japan.

Streptococcus pneumoniae serotype 15B polysaccharide conjugate elicits a cross-functional immune response against serotype 15C but not 15A.

Protection conferred by pneumococcal polysaccharide conjugate vaccines (PCVs) is associated with PCV-induced antibodies against vaccine-covered serotypes that exhibit functional opsonophagocytic activity (OPA). Structural similarity between capsular polysaccharides of closely related serotypes may result in induction of cross-reactive antibodies with or without a cross-functional activity against a serotype not covered by a PCV, with the former providing an additional protective clinical benefit. Serotypes 15B, 15A, and 15C, in the serogroup 15, are among the most prevalent Streptococcus pneumoniae serotypes associated with invasive pneumococcal disease following the implementation of a 13-valent PCV; in addition, 15B contributes significantly to acute otitis media. Serological discrimination between closely related serotypes such as 15B and 15C is complicated; here, we implemented an algorithm to quickly differentiate 15B from its closely related serotypes 15C and 15A directly from whole-genome sequencing data. In addition, molecular dynamics simulations of serotypes 15A, 15B, and 15C polysaccharides demonstrated that while 15B and 15C polysaccharides assume rigid branched conformation, 15A polysaccharide assumes a flexible linear conformation. A serotype 15B conjugate, included in a 20-valent PCV (PCV20), induced cross-functional OPA serum antibody responses against the structurally similar serotype 15C but not against serotype 15A, both not included in PCV20. In PCV20-vaccinated adults (18-49 years), robust OPA antibody titers were detected against both serotypes 15B (the geometric mean titer [GMT] of 19,334) and 15C (GMTs of 1692 and 2747 for strains PFE344340 and PFE1160, respectively), but were negligible against serotype 15A (GMTs of 10 and 30 for strains PFE593551 and PFE647449, respectively). Cross-functional 15B/C responses were also confirmed using sera from a larger group of older adults (60-64 years).

Pivotal Phase 3 Randomized Clinical Trial of the Safety, Tolerability, and Immunogenicity of 20-Valent Pneumococcal Conjugate Vaccine in Adults Aged ≥18 Years.

BACKGROUND: Pneumococcal conjugate vaccines (PCVs) have significantly reduced pneumococcal disease, but disease from non-PCV serotypes remains. The safety, tolerability, and immunogenicity of a 20-valent PCV (PCV20) were evaluated. METHODS: This pivotal phase 3, randomized, double-blind study enrolled adults into 3 age groups (≥60, 50-59, and 18-49 years) at US and Swedish sites. Participants were randomized to receive 1 PCV20 or 13-valent PCV (PCV13) dose. After 1 month, participants aged ≥60 years also received 1 dose of saline or 23-valent polysaccharide vaccine (PPSV23). Safety assessments included local reactions, systemic events, adverse events, serious adverse events, and newly diagnosed chronic medical conditions. Opsonophagocytic activity geometric mean titers 1 month after PCV20 were compared with 13 matched serotypes after PCV13 and 7 additional serotypes after PPSV23 in participants aged ≥60 years; noninferiority was declared if the lower bound of the 2-sided 95% confidence interval for the opsonophagocytic activity geometric mean titer ratio (ratio of PCV20/saline to PCV13/PPSV23 group) was >0.5. PCV20-elicited immune responses in younger participants were also bridged to those in 60-64-year-olds. RESULTS: The severity and frequency of prompted local reactions and systemic events were similar after PCV20 or PCV13; no safety concerns were identified. Primary immunogenicity objectives were met, with immune responses after PCV20 noninferior to 13 matched serotypes after PCV13 and to 6 additional PPSV23 serotypes in participants aged ≥60 years; serotype 8 missed the statistical noninferiority criterion. PCV20 induced robust responses to all 20 vaccine serotypes across age groups. CONCLUSIONS: PCV20 was safe and well tolerated, with immunogenicity comparable to that of PCV13 or PPSV23. PCV20 is anticipated to expand protection against pneumococcal disease in adults. CLINICAL TRIALS REGISTRATION: NCT03760146.

Safety and immunogenicity of different Clostridioides (Clostridium) difficile vaccine formulations in two early phase randomized studies of healthy adults aged 50-85 years.

BACKGROUND: Two phase 1/phase 2 studies assessed 2 formulations of investigational bivalent Clostridioides (Clostridium) difficile vaccine (QS-21 adjuvanted toxoid and toxoid-alone) in healthy adults 50-85 years of age. METHODS: The QS-21 adjuvanted toxoid vaccine study randomized subjects 3:1 to 100 µg QS-21-containing C difficile vaccine or placebo administered in a shortened-month (Months 0, 1, 3) or day (Days 1, 8, 30) regimen. The toxoid-alone vaccine study randomized subjects 3:3:1 to receive 100 or 200 µg unadjuvanted C difficile vaccine formulation or placebo in Stages 1 and 2 (sentinel cohorts of different age groups), and 3:1 to receive the selected dose of unadjuvanted C difficile vaccine formulation or placebo in Stage 3 (Days 1, 8, 30). Safety was the primary outcome for both studies. Immunogenicity was determined by measuring serum toxin A- and B-specific neutralizing antibodies. RESULTS: In the day regimen, 10 reports across both studies of grade 3 injection site redness postdose 2 triggered predefined stopping rules. Local reactions in both studies were more common among vaccine versus placebo recipients. Injection site pain predominated and was generally mild in severity. Systemic events were infrequent and generally mild-to-moderate in severity. Adverse events were reported by 50.0%-75.0% and 16.7%-50.0% of subjects in the QS-21 and toxoid-alone studies, respectively. Immune responses peaked around Day 37 (shortened-month regimen) or between Day 15 and Month 2 (day regimen) and remained above baseline throughout follow-up. CONCLUSIONS: Both formulations demonstrated robust immunogenicity. Both studies stopped early due to grade 3 injection site redness postdose 2 of the day regimen; neither formulation progressed to later stage development. Instead, an aluminum hydroxide-containing formulation of the vaccine candidate administered at 0, 1, and 6 months, which was safe and immunogenic in phase 1 and 2 studies, advanced to phase 3 studies.

Development and clinical validation of an automated cell cytotoxicity neutralization assay for detecting Clostridioides difficile toxins in clinically relevant stools samples.

OBJECTIVES: To improve the diagnostic accuracy of Clostridioides difficile infection, current U.S. and E.U. guidelines recommend multistep testing that detects the presence of C. difficile and toxin in clinically relevant stool samples to confirm active disease. An accepted gold standard to detect C. difficile toxins is the cell cytotoxicity neutralization assay (CCNA). Although highly sensitive, the traditional CCNA has limitations. One such limitation is the subjective interpretation of an analyst to recognize cytopathic effects in cultured cells exposed to a fecal sample containing toxin. To overcome this limitation, an automated CCNA was developed that replaces most human pipetting steps with robotics and incorporates CellTiterGlo® for a semi-quantitative, non-subjective measure of cell viability instead of microscopy. METHODS: To determine sample positivity and control for non-specific cytopathic effects, two thresholds were defined and validated by evaluating the sample with/without antitoxin antisera (sample-antitoxin/sample + antitoxin): 1) a >70% cell viability threshold was validated with samples containing anti-toxin, and 2) a >1.2-fold difference cut-off where sample results above the cut-off are considered positive. RESULTS: Assay validation demonstrated excellent accuracy, precision, and sample linearity with an LOD of 126.9 pg/mL toxin-B in stool. The positivity cut-offs were clinically validated by comparing 322 diarrheal stool sample results with those run in a predicate, microscopic readout-based CCNA. The automated CCNA demonstrated 96% sensitivity and 100% specificity compared with the predicate CCNA. CONCLUSIONS: Overall, the automated CCNA provides a specific, sensitive, and reproducible tool to support determination of CDI epidemiology or the efficacy of interventions such as vaccines.

Expanded Analysis of 20 Pneumococcal Serotypes Associated With Radiographically Confirmed Community-acquired Pneumonia in Hospitalized US Adults.

BACKGROUND: Streptococcus pneumoniae is a causative agent of community-acquired pneumonia (CAP). The 13-valent pneumococcal conjugate vaccine (PCV13) has significantly decreased the burden of PCV13-serotype pneumococcal disease; however, disease from nonvaccine serotypes remains substantial. A recent study documented the persistence of PCV13 serotypes among US adults hospitalized with radiographically confirmed CAP. The current analysis used a recently developed urinary antigen detection (UAD) assay (UAD2) to extend these results to additional serotypes included in an investigational PCV20 vaccine. METHODS: This prospective study enrolled adults aged ≥18 years hospitalized with radiographically confirmed CAP between October 2013 and September 2016. Presence of S pneumoniae was determined by blood and respiratory sample culture, BinaxNOW urine testing, and UAD. In addition to Quellung on cultured isolates when available, serotypes were identified from urine specimens using UAD1 for PCV13 serotypes and UAD2 for 7 PCV20-unique serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) and 4 additional serotypes (2, 9N, 17F, and 20). RESULTS: Among 12 055 subjects with radiographically confirmed CAP, 1482 were positive for S pneumoniae. PCV13- and PCV20-unique serotypes were associated with 37.7% (n = 559) and 27.0% (n = 400) of cases, respectively; 288 subjects were exclusively diagnosed as positive for S pneumoniae by UAD2. Demographic and clinical disease characteristics were similar between subjects with CAP caused by PCV13 and PCV20-unique serotypes. CONCLUSIONS: The current analysis using UAD2 identified a sizeable proportion of hospitalized adult CAP associated with PCV20-unique serotypes. PCV20 may therefore address the burden of CAP caused by the additional serotypes present in the vaccine.

Application of a Pneumococcal Serotype-specific Urinary Antigen Detection Test for Identification of Pediatric Pneumonia in Burkina Faso.

BACKGROUND: Serotype-specific diagnosis of pneumococcal community-acquired pneumonia in children under age 5 years would mark a major advancement for understanding pneumococcal epidemiology and supporting vaccine decision-making. METHODS: A Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and subsequently validated in adults, but its applicability to children is unknown. This study aimed to set appropriate cutoffs for use of the UAD in a healthy pediatric population and apply these cutoffs in children with pneumonia in sub-Saharan Africa. The cutoffs were determined by assessing 379 urines obtained from healthy children under age 5 years from the Bobo-Dioulasso area for serotypes included in 13-valent pneumococcal conjugate vaccine (UAD-1) and the 11 other serotypes unique to 23-valent pneumococcal polysaccharide vaccine (UAD-2). RESULTS: Based on the assigned cutoff values, among 108 children who met the World Health Organization consolidation endpoint criteria, UAD-1 and UAD-2 were positive in 23.3% and 8.3%, respectively; among 364 children with clinically suspected pneumonia who did not meet the World Health Organization criteria, UAD-1 and UAD-2 were positive for 6.6% and 3.6%, respectively. Pneumococcal carriage prevalence was similar among pneumonia cases (30%) versus controls (35%) as was semiquantitative carriage density. CONCLUSIONS: UAD-1 and UAD-2 were able to distinguish community controls from children with pneumonia, particularly pneumonia with consolidation. Future studies are needed to confirm these results and more fully assess the contribution of pneumococcal carriage and concurrent viral infection.

Safety, Tolerability, and Immunogenicity of a 20-Valent Pneumococcal Conjugate Vaccine (PCV20) in Adults 60 to 64 Years of Age.

BACKGROUND: Pneumococcal conjugate vaccines (PCVs) have significantly decreased pneumococcal disease worldwide; however, expanding serotype coverage may further reduce disease burden. A 20-valent PCV (PCV20) containing capsular polysaccharide conjugates of serotypes present in the 13-valent PCV (PCV13) and 7 new serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) is currently in development. This phase 2 study evaluated safety, tolerability, and immunogenicity of PCV20 in adults without prior pneumococcal vaccination. METHODS: In this randomized, active-controlled, double-blinded trial, 444 adults 60 through 64 years of age were randomized to receive either a single dose of PCV20 followed 1 month later by saline placebo or a single dose of PCV13 followed 1 month later by 23-valent polysaccharide vaccine. Local injection site reactions, select systemic symptoms, and adverse events (AEs) were recorded. Immunogenicity was assessed by measuring serotype-specific opsonophagocytic activity (OPA) titers before and approximately 1 month after each vaccination. RESULTS: Local reaction and systemic event rates were similar after vaccination with PCV20 or PCV13; no serious vaccine-related AEs were reported. In the PCV20 group, functional immune responses as measured by OPA were robust for all 20 serotypes included in the vaccine, with geometric mean fold rises from baseline ranging from 6.0 to 113.4. CONCLUSIONS: PCV20 was well tolerated in adults 60 to 64 years of age, with a safety profile consistent with historical experience of PCVs in this age group. Substantial OPA responses were elicited against all serotypes. Results demonstrate the potential for PCV20 to expand pneumococcal disease protection. CLINICAL TRIALS REGISTRATION: NCT03313037.

Developmental exposure to near roadway pollution produces behavioral phenotypes relevant to neurodevelopmental disorders in juvenile rats.

Epidemiological studies consistently implicate traffic-related air pollution (TRAP) and/or proximity to heavily trafficked roads as risk factors for developmental delays and neurodevelopmental disorders (NDDs); however, there are limited preclinical data demonstrating a causal relationship. To test the effects of TRAP, pregnant rat dams were transported to a vivarium adjacent to a major freeway tunnel system in northern California where they were exposed to TRAP drawn directly from the face of the tunnel or filtered air (FA). Offspring remained housed under the exposure condition into which they were born and were tested in a variety of behavioral assays between postnatal day 4 and 50. To assess the effects of near roadway exposure, offspring of dams housed in a standard research vivarium were tested at the laboratory. An additional group of dams was transported halfway to the facility and then back to the laboratory to control for the effect of potential transport stress. Near roadway exposure delayed growth and development of psychomotor reflexes and elicited abnormal activity in open field locomotion. Near roadway exposure also reduced isolation-induced 40-kHz pup ultrasonic vocalizations, with the TRAP group having the lowest number of call emissions. TRAP affected some components of social communication, evidenced by reduced neonatal pup ultrasonic calling and altered juvenile reciprocal social interactions. These findings confirm that living in close proximity to highly trafficked roadways during early life alters neurodevelopment.

Cyclin D2-knock-out mice with attenuated dentate gyrus neurogenesis have robust deficits in long-term memory formation.

Neurobehavioral studies have produced contradictory findings concerning the function of neurogenesis in the adult dentate gyrus. Previous studies have proved inconsistent across several behavioral endpoints thought to be dependent on dentate neurogenesis, including memory acquisition, short-term and long-term retention of memory, pattern separation, and reversal learning. We hypothesized that the main function of dentate neurogenesis is long-term memory formation because we assumed that a newly formed and integrated neuron would have a long-term impact on the local neural network. We used a cyclin D2-knock-out (cyclin D2-/-) mouse model of endogenously deficient dentate neurogenesis to test this hypothesis. We found that cyclin D2-/- mice had robust and sustained loss of long-term memory in two separate behavioral tasks, Morris water maze (MWM) and touchscreen intermediate pattern separation. Moreover, after adjusting for differences in brain volumes determined by magnetic resonance (MR) imaging, reduced dentate neurogenesis moderately correlated with deficits in memory retention after 24 hours. Importantly, cyclin D2-/- mice did not show deficits in learning acquisition in a touchscreen paradigm of intermediate pattern separation or MWM platform location, indicating intact short-term memory. Further evaluation of cyclin D2-/- mice is necessary to confirm that deficits are specifically linked to dentate gyrus neurogenesis since cyclin D2-/- mice also have a reduced size of the olfactory bulb, hippocampus, cerebellum and cortex besides reduced dentate gyrus neurogenesis.