Detection of naturally acquired, strain-transcending antibodies against rosetting Plasmodium falciparum strains in humans.

Strain-transcending antibodies against virulence-associated subsets of P. falciparum-infected erythrocyte surface antigens could protect children from severe malaria. However, the evidence supporting the existence of such antibodies is incomplete and inconsistent. One subset of surface antigens associated with severe malaria, rosette-mediating Plasmodium falciparum Erythrocyte Membrane Protein one (PfEMP1) variants, cause infected erythrocytes to bind to uninfected erythrocytes to form clusters of cells (rosettes) that contribute to microvascular obstruction and pathology. Here, we tested plasma from 80 individuals living in malaria-endemic regions for IgG recognition of the surface of four P. falciparum rosetting strains using flow cytometry. Broadly reactive plasma samples were then used in antibody elution experiments in which intact IgG was eluted from the surface of infected erythrocytes and transferred to heterologous rosetting strains to look for strain-transcending antibodies. We found that seroprevalence (percentage of positive plasma samples) against allopatric rosetting strains was high in adults (63%-93%) but lower in children (13%-48%). Strain-transcending antibodies were present in nine out of eleven eluted antibody experiments, with six of these recognizing multiple heterologous rosetting parasite strains. One eluate had rosette-disrupting activity against heterologous strains, suggesting PfEMP1 as the likely target of the strain-transcending antibodies. Naturally acquired strain-transcending antibodies to rosetting P. falciparum strains in humans have not been directly demonstrated previously. Their existence suggests that such antibodies could play a role in clinical protection and raises the possibility that conserved epitopes recognized by strain-transcending antibodies could be targeted therapeutically by monoclonal antibodies or vaccines.

Upper Airway Epithelial Tissue Transcriptome Analysis Reveals Immune Signatures Associated with COVID-19 Severity in Ghanaians.

The immunological signatures driving the severity of coronavirus disease 19 (COVID-19) in Ghanaians remain poorly understood. We performed bulk transcriptome sequencing of nasopharyngeal samples from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected Ghanaians with mild and severe COVID-19, as well as healthy controls to characterize immune signatures at the primary SARS-CoV-2 infection site and identify drivers of disease severity. Generally, a heightened antiviral response was observed in SARS-CoV-2-infected Ghanaians compared with uninfected controls. COVID-19 severity was associated with immune suppression, overexpression of proinflammatory cytokines, including CRNN, IL1A, S100A7, and IL23A, and activation of pathways involved in keratinocyte proliferation. SAMD9L was among the differentially regulated interferon-stimulated genes in our mild and severe disease cohorts, suggesting that it may play a critical role in SARS-CoV-2 pathogenesis. By comparing our data with a publicly available dataset from a non-African (Indians) (GSE166530), an elevated expression of antiviral response-related genes was noted in COVID-19-infected Ghanaians. Overall, the study describes immune signatures driving COVID-19 severity in Ghanaians and identifies immune drivers that could serve as potential prognostic markers for future outbreaks or pandemics. It further provides important preliminary evidence suggesting differences in antiviral response at the upper respiratory interface in sub-Saharan Africans (Ghanaians) and non-Africans, which could be contributing to the differences in disease outcomes. Further studies using larger datasets from different populations will expand on these findings.

Association of symptomatic upper respiratory tract infections with the alteration of the oropharyngeal microbiome in a cohort of school children in Côte d'Ivoire.

Introduction: The oropharyngeal microbiome plays an important role in protection against infectious agents when in balance. Despite use of vaccines and antibiotic therapy to prevent respiratory tract infections, they remain one of the major causes of mortality and morbidity in Low- and middle-income countries. Hence the need to explore other approaches to prevention by identifying microbial biomarkers that could be leveraged to modify the microbiota in order to enhance protection against pathogenic bacteria. The aim of this study was to analyze the oropharyngeal microbiome (OPM) of schoolchildren in Côte d'Ivoire presenting symptoms of upper respiratory tract infections (URTI) for better prevention strategy. Methods: Primary schools' children in Korhogo (n = 37) and Abidjan (n = 39) were followed for six months with monthly oropharyngeal sampling. Clinical diagnostic of URT infection was performed and nucleic acid extracted from oropharyngeal swabs were used for 16S rRNA metagenomic analysis and RT-PCR. Results: The clinical examination of children's throat in Abidjan and Korhogo identified respectively 17 (43.59%) and 15 (40.54%) participants with visible symptoms of URTIs, with 26 episodes of infection in Abidjan and 24 in Korhogo. Carriage of Haemophilus influenzae (12%), Streptococcus pneumoniae (6%) and SARS-CoV-2 (6%) was confirmed by PCR. A significant difference in alpha diversity was found between children colonized by S. pneumoniae and those that were not (p = 0.022). There was also a significant difference in alpha diversity between children colonised with H. influenzae and those who were not (p = 0.017). No significant difference was found for SARS-CoV-2. Sphingomonas, Ralstonia and Rothia were significantly enriched in non-carriers of S. pneumoniae; Actinobacillus was significantly enriched in non-carriers of H. influenzae; Actinobacillus and Porphyromonas were significantly enriched in non-carriers of SARS-CoV-2 (p < 0.001). Discussion: Nearly 40% of children showed clinical symptoms of infection not related to geographical location. The OPM showed an imbalance during H. influenzae and S. pneumoniae carriage. This study provides a baseline understanding of microbiome markers in URTIs in children for future research, to develop targeted interventions aimed at restoring the microbial balance and reducing the symptoms associated with RTIs.

Putative molecular markers of Plasmodium falciparum resistance to antimalarial drugs in malaria parasites from Ghana.

Introduction: Antimalarial drugs including artemisinin-based combination therapy (ACT) regimens and sulphadoxine-pyrimethamine (SP) are used in Ghana for malaria therapeutics and prophylaxis respectively. The genetic basis of Plasmodium falciparum development of drug resistance involves single nucleotide polymorphisms in genes encoding proteins for multiple cellular and metabolic processes. The prevalence of single nucleotide polymorphisms in nine P. falciparum genes linked to ACT and SP resistance in the malaria parasite population was determined. Methods: Archived filter paper blood blot samples from patients aged 9 years and below with uncomplicated malaria reporting at 10 sentinel sites located in three ecological zones for the Malaria Therapeutic Efficacy Studies were used. The samples used were collected from 2007-2018 malaria transmission seasons and mutations in the genes were detected using PCR and Sanger sequencing. Results: In all 1,142 samples were used for the study. For falcipain-2 gene (pffp2), Sanger sequencing was successful for 872 samples and were further analysed. The prevalence of the mutants was 45% (392/872) with pffp2 markers V51I and S59F occurring in 15.0% (128/872) and 3.0% (26/872) of the samples respectively. Prevalence of other P. falciparum gene mutations: coronin (pfcoronin) was 44.8% (37/90); cysteine desulfurase (pfnfs) was 73.9% (68/92); apicoplast ribosomal protein S10 (pfarps10) was 36.8% (35/95); ferredoxin (pffd) was 8.8% (8/91); multidrug resistance protein-1 (pfmrp1) was 95.2.0% (80/84); multidrug resistance protein-2 (pfmrp2) was 91.4% (32/35); dihydrofolate reductase (pfdhfr) was 99.0% (84/85); dihydropteroate synthase (pfdhps) was 72% (68/95). Discussion: The observation of numerous mutations in these genes of interest in the Ghanaian isolates, some of which have been implicated in delayed parasite clearance is of great interest. The presence of these genotypes may account for the decline in the efficacies of ACT regimens being used to treat uncomplicated malaria in the country. The need for continuous monitoring of these genetic markers to give first-hand information on parasite susceptibility to antimalarial drugs to inform policy makers and stakeholders in malaria elimination in the country is further discussed.

Development and utility of a SARS-CoV-2 pseudovirus assay for compound screening and antibody neutralization assays.

Background: The highly infectious nature of SARS-CoV-2 necessitates using bio-containment facilities to study viral pathogenesis and identify potent antivirals. However, the lack of high-level bio-containment laboratories across the world has limited research efforts into SARS-CoV-2 pathogenesis and the discovery of drug candidates. Previous research has reported that non-replicating SARS-CoV-2 Spike-pseudotyped viral particles are effective tools to screen for and identify entry inhibitors and neutralizing antibodies. Methods: To generate SARS-CoV-2 pseudovirus, a lentiviral packaging plasmid p8.91, a luciferase expression plasmid pCSFLW, and SARS-CoV-2 Spike expression plasmids (Wild-type (D614G) or Delta strain) were co-transfected into HEK293 cells to produce a luciferase-expressing non-replicating pseudovirus which expresses SARS-CoV-2 spike protein on the surface. For relative quantitation, HEK293 cells expressing ACE2 (ACE2-HEK293) were infected with the pseudovirus, after which luciferase activity in the cells was measured as a relative luminescence unit. The ACE2-HEK293/Pseudovirus infection system was used to assess the antiviral effects of some compounds and plasma from COVID-19 patients to demonstrate the utility of this assay for drug discovery and neutralizing antibody screening. Results: We successfully produced lentiviral-based SARS-CoV2 pseudoviruses and ACE2-expressing HEK293 cells. The system was used to screen compounds for SARS-CoV-2 entry inhibitors and identified two compounds with potent activity against SARS-CoV-2 pseudovirus entry into cells. The assay was also employed to screen patient plasma for neutralizing antibodies against SARS-CoV-2, as a precursor to live virus screening, using successful hits. Significance: This assay is scalable and can perform medium-to high-throughput screening of antiviral compounds with neither severe biohazard risks nor the need for higher-level containment facilities. Now fully deployed in our resource-limited laboratory, this system can be applied to other highly infectious viruses by swapping out the envelope proteins in the plasmids used in pseudovirus production.

Knowledge, attitude and perception towards COVID-19 among representative educated sub-Saharan Africans: A cross-sectional study during the exponential phase of the pandemic.

Coronavirus disease 2019 (COVID-19) pandemic, caused by the Severe Acute Coronavirus 2 (SARS-CoV-2), is a global health threat with extensive misinformation and conspiracy theories. Therefore, this study investigated the knowledge, attitude and perception of sub-Saharan Africans (SSA) on COVID-19 during the exponential phase of the pandemic. In this cross-sectional survey, self-administered web-based questionnaires were distributed through several online platforms. A total of 1046 respondents from 35 SSA countries completed the survey. The median age was 33 years (18-76 years) and about half (50.5%) of them were males. More than 40% across all socio-demographic categories except the Central African region (21.2%), vocational/secondary education (28.6%), student/unemployed (35.5%), had high COVID-19 knowledge score. Socio-demographic factors and access to information were associated with COVID-19 knowledge. Bivariate analysis revealed that independent variables, including the region of origin, age, gender, education and occupation, were significantly (p < 0.05) associated with COVID-19 knowledge. Multivariate analysis showed that residing in East (odds ratio [OR]: 7.9, 95% confidence interval (CI): 4.7-14, p<0.001), Southern (OR: 3.7, 95% CI: 2.1-6.5, p<0.001) and West (OR: 3.9, 95% CI: 2.9-5.2, p<0.001) Africa was associated with high COVID-19 knowledge level. Apart from East Africa (54.7%), willingness for vaccine acceptance across the other SSA regions was <40%. About 52%, across all socio-demographic categories, were undecided. Knowledge level, region of origin, age, gender, marital status and religion were significantly (p < 0.05) associated with COVID-19 vaccine acceptance. About 67.4% were worried about contracting SARS-CoV-2, while 65.9% indicated they would consult a health professional if exposed. More than one-third of the respondents reported that their governments had taken prompt measures to tackle the pandemic. Despite high COVID-19 knowledge in our study population, most participants were still undecided regarding vaccination, which is critical in eliminating the pandemic. Therefore, extensive, accurate, dynamic and timely education in this aspect is of ultimate priority.

Cellular immune response to SARS-CoV-2 and clinical presentation in individuals exposed to endemic malaria.

Ghana and other parts of West Africa have experienced lower COVID-19 mortality rates than other regions. This phenomenon has been hypothesized to be associated with previous exposure to infections such as malaria. This study investigated the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the influence of previous malaria exposure. Blood samples were collected from individuals with asymptomatic or symptomatic COVID-19 (n = 217). A variety of assays were used to characterize the SARS-CoV-2-specific immune response, and malaria exposure was quantified using Plasmodium falciparum ELISA. The study found evidence of attenuated immune responses to COVID-19 among asymptomatic individuals, with elevated proportions of non-classical monocytes and greater memory B cell activation. Symptomatic patients displayed higher P. falciparum-specific T cell recall immune responses, whereas asymptomatic individuals demonstrated elevated P. falciparum antibody levels. Summarily, this study suggests that P. falciparum exposure-associated immune modulation may contribute to reduced severity of SARS-CoV-2 infection among people living in malaria-endemic regions.

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 3,960,704 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of March 2023, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. Phase 2 of our project will address the fact that the data in the public archives is heavily geographically biased towards the Global North. We therefore have contributed new raw data to ENA/SRA from many countries including Ghana, Thailand, Laos, Sri Lanka, India, Argentina and Singapore. We will incorporate these, along with all public raw data submitted between March 2023 and the current day, into an updated set of assemblies, and phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

Comparison of malaria diagnostic methods in four hospitals in the Volta region of Ghana.

Background: Rapid diagnostic tests (RDTs) and microscopy are routinely used for the diagnosis of malaria in Ghana. DNA-based polymerase chain reaction (PCR) is not yet used routinely. We compared diagnostic methods and tested the sensitivities of different malaria diagnostic methods against PCR. Materials and methods: Study participants from four hospitals with a suspicion of malaria donated finger -prick blood for RDT and blood film examination. In addition, a blood spot was collected for PCR analysis, prior to treatment. Retrospective species-specific PCR was performed on all samples collected. Results: Using PCR we found an overall malaria prevalence of 39% among the 211 evaluable blood spots (83/211) and this ranged between 6-61% across the four hospitals. Of the 164 participants with RDT data, malaria prevalence was 57% (94/164), ranging from 3-100% from the four hospitals. Microscopy was the least sensitive with a parasite prevalence of 21% (25/119) of the evaluable 119 participants, varying from 9 to 35% across three health facilities. By comparison, we found the sensitivities and specificities of RDT results when compared to PCR to be slightly higher than microscopy compared to PCR. These were 56.4% versus 41.7% and 90% versus 81.9%, respectively, but generally lower than expected. Ninety-five percent of the PCR-detected infections were P. falciparum, while 4% were mixed species infections of P. falciparum and P. malariae, with the remaining being a mono-infection of P. malariae. Conclusions: While using PCR as a gold standard, we found RDT to be more reliable in diagnosing malaria than microscopy. In addition, a majority of malaria-treated cases were not supported by PCR diagnosis, leading to possible overtreatment. Pragmatic strategies are needed to ensure suspected malaria cases are accurately diagnosed before treatment.