Primary Exposure to SARS-CoV-2 via Infection or Vaccination Determines Mucosal Antibody-Dependent ACE2 Binding Inhibition.

BACKGROUND: Mucosal antibodies play a critical role in preventing SARS-CoV-2 infections or reinfections by blocking the interaction of the receptor-binding domain (RBD) with the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface. In this study, we investigated the difference between the mucosal antibody response after primary infection and vaccination. METHODS: We assessed longitudinal changes in the quantity and capacity of nasal antibodies to neutralize the interaction of RBD with the ACE2 receptor using the spike protein and RBD from ancestral SARS-CoV-2 (Wuhan-Hu-1), as well as the RBD from the Delta and Omicron variants. RESULTS: Significantly higher mucosal IgA concentrations were detected postinfection vs postvaccination, while vaccination induced higher IgG concentrations. However, ACE2-inhibiting activity did not differ between the cohorts. Regarding whether IgA or IgG drove ACE2 inhibition, infection-induced binding inhibition was driven by both isotypes, while postvaccination binding inhibition was mainly driven by IgG. CONCLUSIONS: Our study provides new insights into the relationship between antibody isotypes and neutralization by using a sensitive and high-throughput ACE2 binding inhibition assay. Key differences are highlighted between vaccination and infection at the mucosal level, showing that despite differences in the response quantity, postinfection and postvaccination ACE2 binding inhibition capacity did not differ.

Nasopharyngeal colonisation dynamics of bacterial pathogens in patients with fever in rural Burkina Faso: an observational study.

BACKGROUND: Nasopharyngeal colonisation with clinically relevant bacterial pathogens is a risk factor for severe infections, such as pneumonia and bacteraemia. In this study, we investigated the determinants of nasopharyngeal carriage in febrile patients in rural Burkina Faso. METHODS: From March 2016 to June 2017, we recruited 924 paediatric and adult patients presenting with fever, hypothermia or suspicion of severe infection to the Centre Medical avec Antenne Chirurgicale Saint Camille de Nanoro, Burkina Faso. We recorded a broad range of clinical data, collected nasopharyngeal swabs and tested them for the presence of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Klebsiella pneumoniae by quantitative polymerase chain reaction. Using logistic regression, we investigated the determinants of carriage and aimed to find correlations with clinical outcome. RESULTS: Nasopharyngeal colonisation with S. pneumoniae, H. influenzae and M. catarrhalis was highly prevalent and strongly dependent on age and season. Females were less likely to be colonised with S. pneumoniae (OR 0.71, p = 0.022, 95% CI 0.53-0.95) and M. catarrhalis (OR 0.73, p = 0.044, 95% CI 0.54-0.99) than males. Colonisation rates were highest in the age groups < 1 year and 1-2 years of age and declined with increasing age. Colonisation also declined towards the end of the rainy season and rose again during the beginning of the dry season. K. pneumoniae prevalence was low and not significantly correlated with age or season. For S. pneumoniae and H. influenzae, we found a positive association between nasopharyngeal carriage and clinical pneumonia [OR 1.75, p = 0.008, 95% CI 1.16-2.63 (S. pneumoniae) and OR 1.90, p = 0.004, 95% CI 1.23-2.92 (H. influenzae)]. S. aureus carriage was correlated with mortality (OR 4.01, p < 0.001, 95% CI 2.06-7.83), independent of bacteraemia caused by this bacterium. CONCLUSIONS: Age, sex and season are important determinants of nasopharyngeal colonisation with S. pneumoniae, H. influenzae and M. catarrhalis in patients with fever in Burkina Faso. S. pneumoniae and H. influenzae carriage is associated with clinical pneumonia and S. aureus carriage is associated with mortality in patients with fever. These findings may help to understand the dynamics of colonisation and the associated transmission of these pathogens. Furthermore, understanding the determinants of nasopharyngeal colonisation and the association with disease could potentially improve the diagnosis of febrile patients.

Broad range detection of viral and bacterial pathogens in bronchoalveolar lavage fluid of children to identify the cause of lower respiratory tract infections.

BACKGROUND: Knowledge on the etiology of LRTIs is essential for improvement of the clinical diagnosis and accurate treatment. Molecular detection methods were applied to identify a broad range of bacterial and viral pathogens in a large set of bronchial alveolar lavage (BAL) fluid samples. The patterns of detected pathogens were correlated to the clinical symptoms. METHODS: BAL fluid samples and clinical data were collected from 573 hospitalized children between 1 month and 14 years of age with LRTIs, enrolled from January to December 2018. Pathogens were detected using standardized clinical diagnostics, with a sensitive, high-throughput GeXP-based multiplex PCR and with multiplex qPCR. Data were analyzed to describe the correlation between the severity of respiratory tract disease and the pathogens identified. RESULTS: The pathogen detection rate with GeXP-based PCR and multiplex qPCR was significantly higher than by clinical routine diagnostics (76.09% VS 36.13%,χ2 = 8.191, P = 0.004). The most frequently detected pathogens in the BAL fluid were human adenovirus (HADV)(21.82%), Mycoplasma pneumoniae (20.24%), human rhinovirus (13.96%), Streptococcus pneumoniae (8.90%) and Haemophilus influenzae (8.90%). In 16.4% of the cases co-detection with two or three different pathogens was found. Viral detection rates declined with age, while atypical pathogen detection rates increased with age. Oxygen supply in the HADV and Influenza H1N1 infected patients was more frequent (49.43%) than in patients infected with other pathogens. CONCLUSION: Broad range detection of viral and bacterial pathogens using molecular methods is a promising and implementable approach to improve clinical diagnosis and accurate treatment of LRTI in children.

Desialylation of Platelets by Pneumococcal Neuraminidase A Induces ADP-Dependent Platelet Hyperreactivity.

Platelets are increasingly recognized to play a role in the complications of Streptococcus pneumoniae infections. S. pneumoniae expresses neuraminidases, which may alter glycans on the platelet surface. In the present study, we investigated the capability of pneumococcal neuraminidase A (NanA) to remove sialic acid (desialylation) from the platelet surface, the consequences for the platelet activation status and reactivity, and the ability of neuraminidase inhibitors to prevent these effects. Our results show that soluble NanA induces platelet desialylation. Whereas desialylation itself did not induce platelet activation (P-selectin expression and platelet fibrinogen binding), platelets became hyperreactive to ex vivo stimulation by ADP and cross-linked collagen-related peptide (CRP-XL). Platelet aggregation with leukocytes also increased. These processes were dependent on the ADP pathway, as inhibitors of the pathway (apyrase and ticagrelor) abrogated platelet hyperreactivity. Inhibition of NanA-induced platelet desialylation by neuraminidase inhibitors (e.g., oseltamivir acid) also prevented the platelet effects of NanA. Collectively, our findings show that soluble NanA can desialylate platelets, leading to platelet hyperreactivity, which can be prevented by neuraminidase inhibitors.

Th17-Mediated Cross Protection against Pneumococcal Carriage by Vaccination with a Variable Antigen.

Serotype-specific protection against Streptococcus pneumoniae is an important limitation of the current polysaccharide-based vaccines. To prevent serotype replacement, reduce transmission, and limit the emergence of new variants, it is essential to induce broad protection and restrict pneumococcal colonization. In this study, we used a prototype vaccine formulation consisting of lipopolysaccharide (LPS)-detoxified outer membrane vesicles (OMVs) from Salmonella enterica serovar Typhimurium displaying the variable N terminus of PspA (α1α2) for intranasal vaccination, which induced strong Th17 immunity associated with a substantial reduction of pneumococcal colonization. Despite the variable nature of this protein, a common major histocompatibility complex class (MHC-II) epitope was identified, based on in silico prediction combined with ex vivo screening, and was essential for interleukin-17 A (IL-17A)-mediated cross-reactivity and associated with cross protection. Based on 1,352 PspA sequences derived from a pneumococcal carriage cohort, this OMV-based vaccine formulation containing a single α1α2 type was estimated to cover 19.1% of strains, illustrating the potential of Th17-mediated cross protection.

High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infections in infants. A small percentage of the infected infants develops a severe infection, while most of these severely ill patients were previously healthy. It remains unclear why these children develop severe RSV infections. In this study, we investigate whether pneumococcal nasopharyngeal carriage patterns correlate with mucosal inflammation and severity of disease. METHODS: In total, 105 infants hospitalized with RSV infection were included and recovery samples were taken from 42 patients. The presence and density of Streptococcus pneumoniae was determined by RT qPCR to study its relation to viral load, inflammation (MMP-9 and IL-6) and severity of RSV disease. RESULTS: We show that pneumococcal presence or absence in the nasopharynx does not correlate with viral load, inflammation or severity of disease. However, when pneumococcus is present in patients, a higher nasopharyngeal pneumococcal density was correlated with a higher RSV load, higher MMP-9 levels and a less severe course of disease. CONCLUSIONS: Our results show correlations between S. pneumoniae density and viral load, inflammation and disease severity, suggesting that pneumococcal density may be an indicator for severity in paediatric RSV disease.

Deciphering the genetic basis of Moraxella catarrhalis complement resistance: a critical role for the disulphide bond formation system.

The complement system is an important innate defence mechanism, and the ability to resist complement-mediated killing is considered a key virulence trait of the respiratory tract pathogen M. catarrhalis. We studied the molecular basis of complement resistance by transcriptional profiling and Tn-seq, a genome-wide negative-selection screenings technology. Exposure of M. catarrhalis to human serum resulted in increased expression of 84 genes and reduced expression of 134 genes, among which genes encoding ABC transporter systems and surface proteins UspA1 and McaP. By subjecting a ∼ 15 800 transposon mutant library to serum, mutants of 53 genes were negatively selected, including the key complement-resistance factor uspA2H. Validation with directed mutants confirmed Tn-seq phenotypes of uspA2H and 11 newly identified genes, with mutants of MCR_0424, olpA, MCR_1483, and dsbB most severely attenuated. Detailed analysis showed that both components of the disulphide bond formation (DSB) system, DsbB and DsbA, were required for complement-resistance in multiple isolates, and fulfil a critical role in evasion of IgG-dependent classical pathway-mediated killing. Lipooligosaccharide (LOS) structure and membrane stability were severely affected in ΔdsbA strains, suggesting a pivotal role for the DSB system in LOS structure safeguarding and membrane stability maintenance.

From microbial gene essentiality to novel antimicrobial drug targets.

BACKGROUND: Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. RESULT: Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept. CONCLUSION: Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Bacterial colonisation of surface and core of palatine tonsils among Tanzanian children with recurrent chronic tonsillitis and obstructive sleep apnoea who underwent (adeno)tonsillectomy.

OBJECTIVE: Acute and chronic tonsillitis are frequently treated with antibiotics. This study aimed to understand the presence of pathogenic micro-organisms on the surface and core of chronically infected tonsils among Tanzanian children. METHODS: The study enrolled children undergoing adenotonsillectomy. Surface and core tonsillar swabs were taken. Quantitative polymerase chain reaction was performed for Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Neisseria meningitidis and Pseudomonas aeruginosa. RESULTS: Surface and core combined, isolated N meningitidis (86.1 per cent) was found the most, followed by H influenzae (74.9 per cent), S pneumoniae (42.6 per cent) and S aureus (28.7 per cent). M catarrhalis and P aeruginosa were only found in a few patients, 5.6 per cent and 0.8 per cent respectively. CONCLUSION: Colonisation of the tonsillar surface and core has been found. Potentially pathogenic micro-organisms are likely to be missed based on a throat swab. Hence, the practice of surface tonsillar swabbing may be misleading or insufficient.

Streptococcus pneumoniae folate biosynthesis responds to environmental CO2 levels.

Although carbon dioxide (CO2) is known to be essential for Streptococcus pneumoniae growth, it is poorly understood how this respiratory tract pathogen adapts to the large changes in environmental CO2 levels it encounters during transmission, host colonization, and disease. To identify the molecular mechanisms that facilitate pneumococcal growth under CO2-poor conditions, we generated a random S. pneumoniae R6 mariner transposon mutant library representing mutations in 1,538 different genes and exposed it to CO2-poor ambient air. With Tn-seq, we found mutations in two genes that were involved in S. pneumoniae adaptation to changes in CO2 availability. The gene pca, encoding pneumococcal carbonic anhydrase (PCA), was absolutely essential for S. pneumoniae growth under CO2-poor conditions. PCA catalyzes the reversible hydration of endogenous CO2 to bicarbonate (HCO3(-)) and was previously demonstrated to facilitate HCO3(-)-dependent fatty acid biosynthesis. The gene folC that encodes the dihydrofolate/folylpolyglutamate synthase was required at the initial phase of bacterial growth under CO2-poor culture conditions. FolC compensated for the growth-phase-dependent decrease in S. pneumoniae intracellular long-chain (n > 3) polyglutamyl folate levels, which was most pronounced under CO2-poor growth conditions. In conclusion, S. pneumoniae adaptation to changes in CO2 availability involves the retention of endogenous CO2 and the preservation of intracellular long-chain polyglutamyl folate pools.

Effect of prophylactic amoxicillin on tonsillar bacterial pathogens after (adeno)tonsillectomy in children.

OBJECTIVES: Unnecessary and inappropriate antibiotic use is an increasing global health challenge. In limited resource settings, prophylactic antibiotics are still often used in (adeno)tonsillectomy (AT), despite evidence against their effectiveness. This study aimed to investigate the effect of prophylactic amoxicillin, given after AT in children. METHODS: This is a secondary analysis from a two-center, double-blinded, randomized controlled, non-inferiority trial to study the effect of prophylactic amoxicillin on post-AT morbidity. Children aged 2-14 years with recurrent chronic tonsillitis and/or obstructive sleep apnea were randomly assigned to receive either placebo or amoxicillin for 5 days after the operation. Pre- and postoperative samples were collected for polymerase chain reaction (PCR) analyses to detect the five most important pathogens known to be common causes of tonsillitis. PCR results were compared before and after surgery as well as between placebo and amoxicillin. RESULTS: PCR results were obtained, 109 in the amoxicillin group and 115 in the placebo group. In the amoxicillin group, 91% of patients had at least one positive PCR test before surgery and 87% after surgery. In the placebo group, the respective percentages were 92% and 90%. In both groups, a decrease in the total number of pathogens was found after surgery. CONCLUSION: Prophylactic amoxicillin given after AT in children did not show a clinically relevant effect with respect to the number of oropharyngeal microorganisms as compared to placebo.

Timing and sequence of vaccination against COVID-19 and influenza (TACTIC): a single-blind, placebo-controlled randomized clinical trial.

Background: Novel mRNA-based vaccines have been used to protect against SARS-CoV-2, especially in vulnerable populations who also receive an annual influenza vaccination. The TACTIC study investigated potential immune interference between the mRNA COVID-19 booster vaccine and the quadrivalent influenza vaccine, and determined if concurrent administration would have effects on safety or immunogenicity. Methods: TACTIC was a single-blind, placebo-controlled randomized clinical trial conducted at the Radboud University Medical Centre, the Netherlands. Individuals ≥60 years, fully vaccinated against COVID-19 were eligible for participation and randomized into one of four study groups: 1) 0.5 ml influenza vaccination Vaxigrip Tetra followed by 0.3 ml BNT162b2 COVID-19 booster vaccination 21 days later, (2) COVID-19 booster vaccination followed by influenza vaccination, (3) influenza vaccination concurrent with the COVID-19 booster vaccination, and (4) COVID-19 booster vaccination only (reference group). Primary outcome was the geometric mean concentration (GMC) of IgG against the spike (S)-protein of the SARS-CoV-2 virus, 21 days after booster vaccination. We performed a non-inferiority analysis of concurrent administration compared to booster vaccines alone with a predefined non-inferiority margin of -0.3 on the log10-scale. Findings: 154 individuals participated from October, 4, 2021, until November, 5, 2021. Anti-S IgG GMCs for the co-administration and reference group were 1684 BAU/ml and 2435 BAU/ml, respectively. Concurrent vaccination did not meet the criteria for non-inferiority (estimate -0.1791, 95% CI -0.3680 to -0.009831) and antibodies showed significantly lower neutralization capacity compared to the reference group. Reported side-effects were mild and did not differ between study groups. Interpretation: Concurrent administration of both vaccines is safe, but the quantitative and functional antibody responses were marginally lower compared to booster vaccination alone. Lower protection against COVID-19 with concurrent administration of COVID-19 and influenza vaccination cannot be excluded, although additional larger studies would be required to confirm this. Trial registration number: EudraCT: 2021-002186-17. Funding: The study was supported by the ZonMw COVID-19 Programme.

The impact of Bacillus Calmette-Guérin vaccination on antibody response after COVID-19 vaccination.

Earlier studies showed that BCG vaccination improves antibody responses of subsequent vaccinations. Similarly, in older volunteers we found an increased IgG receptor-binding domain (RBD) concentration after SARS-CoV-2 infection if they were recently vaccinated with BCG. This study aims to assess the effect of BCG on the serum antibody concentrations induced by COVID-19 vaccination in a population of adults older than 60 years. Serum was collected from 1,555 participants of the BCG-CORONA-ELDERLY trial a year after BCG or placebo, and we analyzed the anti-SARS-CoV-2 antibody concentrations using a fluorescent-microsphere-based multiplex immunoassay. Individuals who received the full primary COVID-19 vaccination series before serum collection and did not test positive for SARS-CoV-2 between inclusion and serum collection were included in analyses (n = 945). We found that BCG vaccination before first COVID-19 vaccine (median 347 days [IQR 329-359]) did not significantly impact the IgG RBD concentration after COVID-19 vaccination in an older European population.

Differential Pneumococcal Growth Features in Severe Invasive Disease Manifestations.

The nasopharyngeal commensal Streptococcus pneumoniae can become invasive and cause metastatic infection. This requires the pneumococcus to have the ability to adapt, grow, and reside in diverse host environments. Therefore, we studied whether the likelihood of severe disease manifestations was related to pneumococcal growth kinetics. For 383 S. pneumoniae blood isolates and 25 experimental mutants, we observed highly reproducible growth curves in nutrient-rich medium. The derived growth features were lag time, maximum growth rate, maximum density, and stationary-phase time before lysis. First, the pathogenicity of each growth feature was probed by comparing isolates from patients with and without marked preexisting comorbidity. Then, growth features were related to the propensity of causing severe manifestations of invasive pneumococcal disease (IPD). A high maximum bacterial density was the most pronounced pathogenic growth feature, which was also an independent predictor of 30-day mortality (P = 0.03). Serotypes with an epidemiologically higher propensity for causing meningitis displayed a relatively high maximum density (P < 0.005) and a short stationary phase (P < 0.005). Correspondingly, isolates from patients diagnosed with meningitis showed an especially high maximum density and short stationary phase compared to isolates from the same serotype that had caused uncomplicated bacteremic pneumonia. In contrast, empyema-associated strains were characterized by a relatively long lag phase (P < 0.0005), and slower growth (P < 0.005). The course and dissemination of IPD may partly be attributable to the pneumococcal growth features involved. If confirmed, we should tailor the prevention and treatment strategies for the different infection sites that can complicate IPD. IMPORTANCE Streptococcus pneumoniae is a leading infectious cause of deaths worldwide. To understand the course and outcome of pneumococcal infection, most research has focused on the host and its response to contain bacterial growth. However, bacterial epidemiology suggest that certain pneumococcal serotypes are particularly prone to causing complicated infections. Therefore, we took the bacterial point of view, simply examining in vitro growth features for hundreds of pneumococcal blood isolates. Their growth curves were very reproducible. Certain poles of pneumococcal growth features were indeed associated with specific clinical manifestations like meningitis or pleural empyema. This indicates that bacterial growth style potentially affects the progression of infection. Further research on bacterial growth and adaptation to different host environments may therefore provide key insight into pathogenesis of complicated invasive disease. Such knowledge could lead to more tailored vaccine targets or therapeutic approaches to reduce the million deaths that are caused by pneumococcal disease every year.

Genome-wide identification of Streptococcus pneumoniae genes essential for bacterial replication during experimental meningitis.

Meningitis is the most serious of invasive infections caused by the Gram-positive bacterium Streptococcus pneumoniae. Vaccines protect only against a limited number of serotypes, and evolving bacterial resistance to antimicrobials impedes treatment. Further insight into the molecular pathogenesis of invasive pneumococcal disease is required in order to enable the development of new or adjunctive treatments and/or pneumococcal vaccines that are efficient across serotypes. We applied genomic array footprinting (GAF) in the search for S. pneumoniae genes that are essential during experimental meningitis. A total of 6,000 independent TIGR4 marinerT7 transposon mutants distributed over four libraries were injected intracisternally into rabbits, and cerebrospinal fluid (CSF) was collected after 3, 9, and 15 h. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 and 11 genes mutants of which had become attenuated or enriched, respectively, during infection. The results point to essential roles for capsular polysaccharides, nutrient uptake, and amino acid biosynthesis in bacterial replication during experimental meningitis. The GAF phenotype of a subset of identified targets was followed up by detailed studies of directed mutants in competitive and noncompetitive infection models of experimental rat meningitis. It appeared that adenylosuccinate synthetase, flavodoxin, and LivJ, the substrate binding protein of a branched-chain amino acid ABC transporter, are relevant as targets for future therapy and prevention of pneumococcal meningitis, since their mutants were attenuated in both models of infection as well as in competitive growth in human cerebrospinal fluid in vitro.

Polymorphisms in genes controlling inflammation and tissue repair in rheumatoid arthritis: a case control study.

BACKGROUND: Various cytokines and inflammatory mediators are known to be involved in the pathogenesis of rheumatoid arthritis (RA). We hypothesized that polymorphisms in selected inflammatory response and tissue repair genes contribute to the susceptibility to and severity of RA. METHODS: Polymorphisms in TNFA, IL1B, IL4, IL6, IL8, IL10, PAI1, NOS2a, C1INH, PARP, TLR2 and TLR4 were genotyped in 376 Caucasian RA patients and 463 healthy Caucasian controls using single base extension. Genotype distributions in patients were compared with those in controls. In addition, the association of polymorphisms with the need for anti-TNF-α treatment as a marker of RA severity was assessed. RESULTS: The IL8 781 CC genotype was associated with early onset of disease. The TNFA -238 G/A polymorphism was differentially distributed between RA patients and controls, but only when not corrected for age and gender. None of the polymorphisms was associated with disease severity. CONCLUSIONS: We here report an association between IL8 781 C/T polymorphism and age of onset of RA. Our findings indicate that there might be a role for variations in genes involved in the immune response and in tissue repair in RA pathogenesis. Nevertheless, additional larger genomic and functional studies are required to further define their role in RA.

SARS-CoV-2 mucosal antibody development and persistence and their relation to viral load and COVID-19 symptoms.

Although serological studies have shown that antibodies against SARS-CoV-2 play an important role in protection against (re)infection, the dynamics of mucosal antibodies during primary infection and their potential impact on viral load and the resolution of disease symptoms remain unclear. During the first pandemic wave, we assessed the longitudinal nasal antibody response in index cases with mild COVID-19 and their household contacts. Nasal and serum antibody responses were analysed for up to nine months. Higher nasal receptor binding domain and spike protein-specific antibody levels at study inclusion were associated with lower viral load. Older age was correlated with more frequent COVID-19 related symptoms. Receptor binding domain and spike protein-specific mucosal antibodies were associated with the resolution of systemic, but not respiratory symptoms. Finally, receptor binding domain and spike protein-specific mucosal antibodies remained elevated up to nine months after symptom onset.

Infection Manager System (IMS) as a new hemocytometry-based bacteremia detection tool: A diagnostic accuracy study in a malaria-endemic area of Burkina Faso.

BACKGROUND: New hemocytometric parameters can be used to differentiate causes of acute febrile illness (AFI). We evaluated a software algorithm-Infection Manager System (IMS)-which uses hemocytometric data generated by Sysmex hematology analyzers, for its accuracy to detect bacteremia in AFI patients with and without malaria in Burkina Faso. Secondary aims included comparing the accuracy of IMS with C-reactive protein (CRP) and procalcitonin (PCT). METHODS: In a prospective observational study, patients of ≥ three-month-old (range 3 months- 90 years) presenting with AFI were enrolled. IMS, blood culture and malaria diagnostics were done upon inclusion and additional diagnostics on clinical indication. CRP, PCT, viral multiplex PCR on nasopharyngeal swabs and bacterial- and malaria PCR were batch-tested retrospectively. Diagnostic classification was done retrospectively using all available data except IMS, CRP and PCT results. FINDINGS: A diagnosis was affirmed in 549/914 (60.1%) patients and included malaria (n = 191) bacteremia (n = 69), viral infections (n = 145), and malaria-bacteremia co-infections (n = 47). The overall sensitivity, specificity, and negative predictive value (NPV) of IMS for detection of bacteremia in patients of ≥ 5 years were 97.0% (95% CI: 89.8-99.6), 68.2% (95% CI: 55.6-79.1) and 95.7% (95% CI: 85.5-99.5) respectively, compared to 93.9% (95% CI: 85.2-98.3), 39.4% (95% CI: 27.6-52.2), and 86.7% (95% CI: 69.3-96.2) for CRP at ≥20mg/L. The sensitivity, specificity and NPV of PCT at 0.5 ng/ml were lower at respectively 72.7% (95% CI: 60.4-83.0), 50.0% (95% CI: 37.4-62.6) and 64.7% (95% CI: 50.1-77.6) The diagnostic accuracy of IMS was lower among malaria cases and patients <5 years but remained equal to- or higher than the accuracy of CRP. INTERPRETATION: IMS is a new diagnostic tool to differentiate causes of AFI. Its high NPV for bacteremia has the potential to improve antibiotic dispensing practices in healthcare facilities with hematology analyzers. Future studies are needed to evaluate whether IMS, combined with malaria diagnostics, may be used to rationalize antimicrobial prescription in malaria endemic areas. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02669823) https://clinicaltrials.gov/ct2/show/NCT02669823.

Viral-bacterial (co-)occurrence in the upper airways and the risk of childhood pneumonia in resource-limited settings.

OBJECTIVE: To examine the association between bacterial-viral co-occurrence in the nasopharynx and the risk of community acquired pneumonia (CAP) in young children living in resource-limited settings. METHODS: A case-control study was conducted between January and December 2017 in Moshi, Tanzania. Children 2-59 months with CAP and healthy controls were enrolled. RSV and Influenza A/B were detected with a standardized polymerase chain reaction (PCR) method, and a simplified real-time quantitative PCR method, without sample pre-processing, was developed to detect bacterial pathogens in nasopharyngeal samples. RESULTS: A total of 109 CAP patients and 324 healthy controls were enrolled. Co-detection of H. influenzae and S. pneumoniae in nasopharyngeal swabs was linked with higher odds of CAP (aOR=3.2, 95% CI=1.1-9.5). The majority of the H. influenzae isolated in cases and controls (95.8%) were non-typeable. Of the viruses examined, respiratory syncytial virus (RSV) was most common (n = 31, 7.2%) in cases and controls. Children with RSV had 8.4 times higher odds to develop pneumonia than healthy children (aOR=8.4, 95%CI= 3.2 - 22.1). CONCLUSIONS: Co-occurence of H. influenzae and S. pneumoniae in the nasopharynx was strongly associated with CAP. The high prevalence of non-typeable H. influenzae might be a sign of replacement as a consequence of Haemophilus influenzae type b vaccination.

Exploring metal availability in the natural niche of Streptococcus pneumoniae to discover potential vaccine antigens.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of S. pneumoniae. Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.