Highly Sensitive Spatial Glycomics at Near-Cellular Resolution by On-Slide Derivatization and Mass Spectrometry Imaging.

Glycans on proteins and lipids play important roles in maturation and cellular interactions, contributing to a variety of biological processes. Aberrant glycosylation has been associated with various human diseases including cancer; however, elucidating the distribution and heterogeneity of glycans in complex tissue samples remains a major challenge. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is routinely used to analyze the spatial distribution of a variety of molecules including N-glycans directly from tissue surfaces. Sialic acids are nine carbon acidic sugars that often exist as the terminal sugars of glycans and are inherently difficult to analyze using MALDI-MSI due to their instability prone to in- and postsource decay. Here, we report on a rapid and robust method for stabilizing sialic acid on N-glycans in FFPE tissue sections. The established method derivatizes and identifies the spatial distribution of α2,3- and α2,6-linked sialic acids through complete methylamidation using methylamine and PyAOP ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate). Our in situ approach increases the glycans detected and enhances the coverage of sialylated species. Using this streamlined, sensitive, and robust workflow, we rapidly characterize and spatially localize N-glycans in human tumor tissue sections. Additionally, we demonstrate this method's applicability in imaging mammalian cell suspensions directly on slides, achieving cellular resolution with minimal sample processing and cell numbers. This workflow reveals the cellular locations of distinct N-glycan species, shedding light on the biological and clinical significance of these biomolecules in human diseases.

Association between early viral lower respiratory tract infections and subsequent asthma development.

BACKGROUND: The association between hospitalization for human respiratory syncytial virus (HRSV) bronchiolitis in early childhood and subsequent asthma is well established. The long-term prognosis for non-bronchiolitis lower respiratory tract infections (LRTI) caused by viruses different from HRSV and rhinovirus, on the other hand, has received less interest. AIM: To investigate the relationship between infant LRTI and later asthma and examine the influence of confounding factors. METHODS: The PubMed and Global Index Medicus bibliographic databases were used to search for articles published up to October 2021 for this systematic review. We included cohort studies comparing the incidence of asthma between patients with and without LRTI at ≤ 2 years regardless of the virus responsible. The meta-analysis was performed using the random effects model. Sources of heterogeneity were assessed by stratified analyses. RESULTS: This review included 15 articles (18 unique studies) that met the inclusion criteria. LRTIs at ≤ 2 years were associated with an increased risk of subsequent asthma up to 20 years [odds ratio (OR) = 5.0, 95%CI: 3.3-7.5], with doctor-diagnosed asthma (OR = 5.3, 95%CI: 3.3-8.6), current asthma (OR = 5.4, 95%CI: 2.7-10.6), and current medication for asthma (OR = 1.2, 95%CI: 0.7-3.9). Our overall estimates were not affected by publication bias (P = 0.671), but there was significant heterogeneity [I 2 = 58.8% (30.6-75.5)]. Compared to studies with hospitalized controls without LRTI, those with ambulatory controls had a significantly higher strength of association between LRTIs and subsequent asthma. The strength of the association between LRTIs and later asthma varied significantly by country and age at the time of the interview. The sensitivity analyses including only studies with similar proportions of confounding factors (gender, age at LRTI development, age at interview, gestational age, birth weight, weight, height, smoking exposure, crowding, family history of atopy, and family history of asthma) between cases and controls did not alter the overall estimates. CONCLUSION: Regardless of the causative virus and confounding factors, viral LRTIs in children < 2 years are associated with an increased risk of developing a subsequent asthma. Parents and pediatricians should be informed of this risk.

Global epidemiology of occult hepatitis B virus infections in blood donors, a systematic review and meta-analysis.

This study aimed to assess the global prevalence of occult hepatitis B in blood donors. We searched PubMed, Web of Science, Global Index Medicus, and Excerpta Medica Database. Study selection and data extraction were performed by at least two independent investigators. Heterogeneity (I2) was assessed using the χ2 test on the Cochran Q statistic and H parameters. Sources of heterogeneity were explored by subgroup analyses. This study is registered with PROSPERO, number CRD42021252787. We included 82 studies in this meta-analysis. The overall prevalence of OBI was 6.2% (95% CI: 5.4-7.1) in HBsAg negative and anti-HBc positive blood donors. Only sporadic cases of OBI were reported in HBsAg negative and anti-HBc negative blood donors. The overall prevalence of OBI was 0.2% (95% CI: 0.1-0.4) in HBsAg negative blood donors. The prevalence of OBI was generally higher in countries with low-income economic status. The results of this study show that despite routine screening of blood donors for hepatitis B, the transmission of HBV by blood remains possible via OBI and/or a seronegative window period; hence there is a need for active surveillance and foremost easier access to molecular tests for the screening of blood donors before transfusion.

Epidemiology of yellow fever virus in humans, arthropods, and non-human primates in sub-Saharan Africa: A systematic review and meta-analysis.

Yellow fever (YF) has re-emerged in the last two decades causing several outbreaks in endemic countries and spreading to new receptive regions. This changing epidemiology of YF creates new challenges for global public health efforts. Yellow fever is caused by the yellow fever virus (YFV) that circulates between humans, the mosquito vector, and non-human primates (NHP). In this systematic review and meta-analysis, we review and analyse data on the case fatality rate (CFR) and prevalence of YFV in humans, and on the prevalence of YFV in arthropods, and NHP in sub-Saharan Africa (SSA). We performed a comprehensive literature search in PubMed, Web of Science, African Journal Online, and African Index Medicus databases. We included studies reporting data on the CFR and/or prevalence of YFV. Extracted data was verified and analysed using the random effect meta-analysis. We conducted subgroup, sensitivity analysis, and publication bias analyses using the random effect meta-analysis while I2 statistic was employed to determine heterogeneity. This review was registered with PROSPERO under the identification CRD42021242444. The final meta-analysis included 55 studies. The overall case fatality rate due to YFV was 31.1% (18.3-45.4) in humans and pooled prevalence of YFV infection was 9.4% (6.9-12.2) in humans. Only five studies in West and East Africa detected the YFV in mosquito species of the genus Aedes and in Anopheles funestus. In NHP, YFV antibodies were found only in members of the Cercopithecidae family. Our analysis provides evidence on the ongoing circulation of the YFV in humans, Aedes mosquitoes and NHP in SSA. These observations highlight the ongoing transmission of the YFV and its potential to cause large outbreaks in SSA. As such, strategies such as those proposed by the WHO's Eliminate Yellow Fever Epidemics (EYE) initiative are urgently needed to control and prevent yellow fever outbreaks in SSA.

Global prevalence of occult hepatitis C virus: A systematic review and meta-analysis.

BACKGROUND: Occult hepatitis C infection (OCI) is characterized by the presence of hepatitis C virus (HCV) RNA in the liver, peripheral blood mononuclear cells (PBMC) and/or ultracentrifuged serum in the absence of detectable HCV-RNA in serum. OCI has been described in several categories of populations including hemodialysis patients, patients with a sustained virological response, immunocompromised individuals, patients with abnormal hepatic function, and apparently healthy subjects. AIM: To highlight the global prevalence of OCI. METHODS: We performed a systematic and comprehensive literature search in the following 4 electronic databases PubMed, EMBASE, Global Index Medicus, and Web of Science up to 6th May 2021 to retrieve relevant studies published in the field. Included studies were unrestricted population categories with known RNA status in serum, PBMC, liver tissue and/or ultracentrifuged serum. Data were extracted independently by each author and the Hoy et al tool was used to assess the quality of the included studies. We used the random-effect meta-analysis model to estimate the proportions of OCI and their 95% confidence intervals (95%CI). The Cochran's Q-test and the I 2 test statistics were used to assess heterogeneity between studies. Funnel plot and Egger test were used to examine publication bias. R software version 4.1.0 was used for all analyses. RESULTS: The electronic search resulted in 3950 articles. We obtained 102 prevalence data from 85 included studies. The pooled prevalence of seronegative OCI was estimated to be 9.61% (95%CI: 6.84-12.73) with substantial heterogeneity [I² = 94.7% (95%CI: 93.8%-95.4%), P < 0.0001]. Seropositive OCI prevalence was estimated to be 13.39% (95%CI: 7.85-19.99) with substantial heterogeneity [I 2 = 93.0% (90.8%-94.7%)]. Higher seronegative OCI prevalence was found in Southern Europe and Northern Africa, and in patients with abnormal liver function, hematological disorders, and kidney diseases. Higher seropositive OCI prevalence was found in Southern Europe, Northern America, and Northern Africa. CONCLUSION: In conclusion, in the present study, it appears that the burden of OCI is high and variable across the different regions and population categories. Further studies on OCI are needed to assess the transmissibility, clinical significance, long-term outcome, and need for treatment.

Epidemiology of hepatitis B virus and/or hepatitis C virus infections among people living with human immunodeficiency virus in Africa: A systematic review and meta-analysis.

INTRODUCTION: Due to their common routes of transmission, human immunodeficiency virus (HIV) coinfection with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) has become a major public health problem worldwide, particularly in Africa, where these viruses are endemic. Few systematic reviews report the epidemiological data of HBV and/or HCV coinfection with HIV in Africa, and none provided data on the case fatality rate (CFR) associated with this coinfection. This study was conducted to investigate the prevalence and case fatality rate of HBV and/or HCV infections among people living with human immunodeficiency virus (PLHIV) in Africa. METHODS: We conducted a systematic review of published articles in PubMed, Web of Science, African Journal Online, and African Index Medicus up to January 2022. Manual searches of references from retrieved articles and grey literature were also performed. The meta-analysis was performed using a random-effects model. Sources of heterogeneity were investigated using subgroup analysis, while funnel plots and Egger tests were performed to assess publication bias. RESULTS: Of the 4388 articles retrieved from the databases, 314 studies met all the inclusion criteria. The overall HBV case fatality rate estimate was 4.4% (95% CI; 0.7-10.3). The overall seroprevalences of HBV infection, HCV infection, and HBV/HCV coinfection in PLHIV were 10.5% [95% CI = 9.6-11.3], 5.4% [95% CI = 4.6-6.2], and 0.7% [95% CI = 0.3-1.0], respectively. The pooled seroprevalences of current HBsAg, current HBeAg, and acute HBV infection among PLHIV were 10.7% [95% CI = 9.8-11.6], 7.0% [95% CI = 4.7-9.7], and 3.6% [95% CI = 0.0-11.0], respectively. Based on HBV-DNA and HCV-RNA detection, the seroprevalences of HBV and HCV infection in PLHIV were 17.1% [95% CI = 11.5-23.7] and 2.5% [95% CI = 0.9-4.6], respectively. Subgroup analysis showed substantial heterogeneity. CONCLUSIONS: In Africa, the prevalence of hepatotropic viruses, particularly HBV and HCV, is high in PLHIV, which increases the case fatality rate. African public health programs should emphasize the need to apply and comply with WHO guidelines on viral hepatitis screening and treatment in HIV-coinfected patients. REVIEW REGISTRATION: PROSPERO, CRD42021237795.

Pneumococcal Phasevarions Control Multiple Virulence Traits, Including Vaccine Candidate Expression.

Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule are only effective against a limited number of the >100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. All S. pneumoniae strains encode the SpnIII Restriction-Modification system. This system contains a phase-variable methyltransferase that switches specificity, and controls expression of multiple genes-a phasevarion. We examined the role of this phasevarion during pneumococcal pathobiology, and determined if phase variation resulted in differences in expression of currently investigated conserved protein antigens. Using locked strains that express a single methyltransferase specificity, we found differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. We also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine. IMPORTANCE S. pneumoniae is the world's foremost bacterial pathogen. S. pneumoniae encodes a phasevarion (phase-variable regulon), that results in differential expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population. Here, we show that this phasevarion generates multiple phenotypic differences relevant to pathobiology. Importantly, expression of conserved protein antigens varies with phasevarion switching. As capsule expression, a major pneumococcal virulence factor, is also controlled by the phasevarion, our work will inform the selection of the best candidates to include in a rationally designed, universal pneumococcal vaccine.

Characterization of the Phase-Variable Autotransporter Lav Reveals a Role in Host Cell Adherence and Biofilm Formation in Nontypeable Haemophilus influenzae.

Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in nontypeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA(n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to human lung cells and denser biofilm formation. A survey of Haemophilus species genome sequences showed lav is present in ∼60% of NTHi strains, but lav is not present in most typeable H. influenzae strains. Sequence analysis revealed a total of five distinct variants of the Lav passenger domain present in Haemophilus spp., with these five variants showing a distinct lineage distribution. Determining the role of Lav in NTHi will help understand the role of this protein during distinct pathologies.

Global prevalence and case fatality rate of Enterovirus D68 infections, a systematic review and meta-analysis.

A substantial amount of epidemiological data has been reported on Enterovirus D68 (EV-D68) infections after the 2014 outbreak. Our goal was to map the case fatality rate (CFR) and prevalence of current and past EV-D68 infections. We conducted a systematic review (PROSPERO, CRD42021229255) with published articles on EV-68 infections in PubMed, Embase, Web of Science and Global Index Medicus up to January 2021. We determined prevalences using a model random effect. Of the 4,329 articles retrieved from the databases, 89 studies that met the inclusion criteria were from 39 different countries with apparently healthy individuals and patients with acute respiratory infections, acute flaccid myelitis and asthma-related diseases. The CFR estimate revealed occasional deaths (7/1353) related to EV-D68 infections in patients with severe acute respiratory infections. Analyses showed that the combined prevalence of current and past EV-D68 infections was 4% (95% CI = 3.1-5.0) and 66.3% (95% CI = 40.0-88.2), respectively. The highest prevalences were in hospital outbreaks, developed countries, children under 5, after 2014, and in patients with acute flaccid myelitis and asthma-related diseases. The present study shows sporadic deaths linked to severe respiratory EV-D68 infections. The study also highlights a low prevalence of current EV-D68 infections as opposed to the existence of EV-D68 antibodies in almost all participants of the included studies. These findings therefore highlight the need to implement and/or strengthen continuous surveillance of EV-D68 infections in hospitals and in the community for the anticipation of the response to future epidemics.

Combinatorial liposomal peptide vaccine induces IgA and confers protection against influenza virus and bacterial super-infection.

OBJECTIVES: The upper respiratory tract is the major entry site for Streptococcus pyogenes and influenza virus. Vaccine strategies that activate mucosal immunity could significantly reduce morbidity and mortality because of these pathogens. The severity of influenza is significantly greater if a streptococcal infection occurs during the viraemic period and generally viral infections complicated by a subsequent bacterial infection are known as super-infections. We describe an innovative vaccine strategy against influenza virus:S. pyogenes super-infection. Moreover, we provide the first description of a liposomal multi-pathogen-based platform that enables the incorporation of both viral and bacterial antigens into a vaccine and constitutes a transformative development. METHODS: Specifically, we have explored a vaccination strategy with biocompatible liposomes that express conserved streptococcal and influenza A virus B-cell epitopes on their surface and contain encapsulated diphtheria toxoid as a source of T-cell help. The vaccine is adjuvanted by inclusion of the synthetic analogue of monophosphoryl lipid A, 3D-PHAD. RESULTS: We observe that this vaccine construct induces an Immunoglobulin A (IgA) response in both mice and ferrets. Vaccination reduces viral load in ferrets from influenza challenge and protects mice from both pathogens. Notably, vaccination significantly reduces both mortality and morbidity associated with a super-infection. CONCLUSION: The vaccine design is modular and could be adapted to include B-cell epitopes from other mucosal pathogens where an IgA response is required for protection.

A Sulfonozanamivir Analogue Has Potent Anti-influenza Virus Activity.

Influenza virus infection continues to cause significant, often severe, respiratory illness worldwide. A validated target for the development of anti-influenza agents is the virus surface protein sialidase. In the current study, we have discovered a highly potent inhibitor of influenza virus sialidase, based on a novel sialosyl sulfonate template. The synthesised 3-guanidino sialosyl α-sulfonate, a sulfonozanamivir analogue, inhibits viral replication in vitro at the nanomolar level, comparable to that of the anti-influenza drug zanamivir. Using protein X-ray crystallography we show that the sialosyl α-sulfonate template binds within the sialidase active site in a 1 C4 chair conformation. The C1-sulfonate moiety forms crucial and strong-binding interactions with the active site's triarginyl cluster, while the 3-guanidino moiety interacts significantly with conserved active site residues. This sulfonozanamivir analogue provides a new direction in anti-influenza virus drug development.

Cyclotriveratrylene-Containing Porphyrins.

The C3-symmetric cyclotriveratrylene (CTV) was covalently bonded via click chemistry to 1, 2, 3, and 6 zinc(II) porphyrin units to various host for C60. The binding constants, Ka, were measured from the quenching of the porphyrin fluorescence by C60. These constants vary between 400 and 4000 M(-1) and are considered weak. Computer modeling demonstrated that the zinc(II) porphyrin units, [Zn], exhibit a strong tendency to occupy the CTV cavity, hence blocking the access for C60 to land on this site. Instead, the pincer of the type [Zn]----[Zn] and in one case [Zn]----CTV, were found to be the most probable geometry to promote host-guest associations in these systems.