Faecal shedding of SARS-CoV-2 from patients with asymptomatic and mild COVID-19 without gastrointestinal symptoms in Ghana.

OBJECTIVE: In this study, we sought to determine whether faecal shedding occurs among SARS-COV-2 positive Ghanaians, as reported elsewhere. Hence we assayed for SARS-COV-2 in the stools of 48 SARS-COV-2 confirmed patients at the Ho Municipal Hospital in Ghana. RESULTS: Of the 48 COVID-19 patients, 45 (93.8%) had positive tests for SARS-CoV-2 faecal shedding. About 60% reported no respiratory symptoms, while only 2% (1 patient) reported gastrointestinal (GI) symptoms in the form of nausea. Other symptoms reported included headache (57.9%), weakness (57.9%), cough (52.6%), blocked/runny nose (47.4%), fever (31.6%), sore throat (31.6%), and shortness of breath (21.1%). One person complained of nausea (5.3%) Semi-quantitative comparison of the SARS COV-2 viral loads in matched respiratory and faecal samples using the cycle threshold (CT) values revealed no statistical differences. Furthermore, the duration between collection of respiratory and faecal samples did not have any direct influence on the differences in the CT values. This suggests that treatment and use of sewage for environmental surveillance of SARS COV-2 could be a potential public health countermeasure.

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.

Pre-vaccination seroprevalence of SARS-CoV-2 antibodies in the Volta Region, Ghana.

Objectives: Before administration of the first dose of the AstraZeneca 2019 SARS-CoV-2 vaccine to selected prioritized groups in the Volta regional capital of Ghana, we determined the pre-vaccination status of prospective recipients and established the baseline exposure status 1 year after the first case was reported. Methods: After informed consent, blood samples were collected for the detection of SARS-CoV-2 immunoglobulin (Ig) M/IgG antibodies using rapid diagnostic test kits. A total of 409 individuals (mean age 27 years) consented and participated in the study, comprising 70% students and others were health staff and educators who presented themselves for vaccination. Results: The overall exposure rate of SARS-CoV-2 was 12.7% (95% confidence interval [CI] 9.6-16.3). The prevalence of SARS-CoV-2 IgM and IgG were 4.2% (95% CI 2.4-6.6) and 5.6% (95% CI 3.6-8.3), respectively. IgM and IgG were detected in 2.9% (95% CI 1.5-5.1) of the respondents. The exposure rates were higher in participants over 40 years old (15.5%). Participants without a history of COVID-19-like symptoms had an exposure rate of 13.0% and those without any chronic diseases was 13.2%. Conclusion: Pre-vaccination exposure was relatively low and underscored the need for vaccination i to increase protection in communities and disease outcomes.

Genetic diversity of SARS-CoV-2 infections in Ghana from 2020-2021.

The COVID-19 pandemic is one of the fastest evolving pandemics in recent history. As such, the SARS-CoV-2 viral evolution needs to be continuously tracked. This study sequenced 1123 SARS-CoV-2 genomes from patient isolates (121 from arriving travellers and 1002 from communities) to track the molecular evolution and spatio-temporal dynamics of the SARS-CoV-2 variants in Ghana. The data show that initial local transmission was dominated by B.1.1 lineage, but the second wave was overwhelmingly driven by the Alpha variant. Subsequently, an unheralded variant under monitoring, B.1.1.318, dominated transmission from April to June 2021 before being displaced by Delta variants, which were introduced into community transmission in May 2021. Mutational analysis indicated that variants that took hold in Ghana harboured transmission enhancing and immune escape spike substitutions. The observed rapid viral evolution demonstrates the potential for emergence of novel variants with greater mutational fitness as observed in other parts of the world.

Molecular detection of Mycobacterium tuberculosis in blood stained sputum samples using GeneXpert PCR assay.

This study investigated the possibility of detecting Mycobacterium tuberculosis (MTB) in blood-stained sputa with GeneXpert assay (Xpert MTB/Rif G4 v.5) using appropriate blood lysing (distilled water and distilled water/carbon tetrachloride) and protein denaturing (guanidinium chloride and heat) agents. Blood free sputa were collected from individuals infected with MTB. Various levels of blood-spiked sputa (0%-50%) were prepared and subsequently assayed with GeneXpert analyzer. GeneXpert was found to be reproducible at less than 2% blood contamination. However, beyond 5% blood contamination, GeneXpert was unreliable with absolute PCR inhibition at 20% of blood contamination. Boiling at 95°C for 5 minutes was able to recover MTB DNA in previously undetectable levels as well as in 57% of patients' sputa with blood stains with previous negative GeneXpert results.

Molecular Characterization and Immuno-Reactivity Patterns of a Novel Plasmodium falciparum Armadillo-Type Repeat Protein, PfATRP.

Nearly half of the genes in the Plasmodium falciparum genome have not yet been functionally investigated. We used homology-based structural modeling to identify multiple copies of Armadillo repeats within one uncharacterized gene expressed during the intraerythrocytic stages, PF3D7_0410600, subsequently referred to as P. falciparum Armadillo-Type Repeat Protein (PfATRP). Soluble recombinant PfATRP was expressed in a bacterial expression system, purified to apparent homogeneity and the identity of the recombinant PfATRP was confirmed by mass spectrometry. Affinity-purified α-PfATRP rabbit antibodies specifically recognized the recombinant protein. Immunofluorescence assays revealed that α-PfATRP rabbit antibodies reacted with P. falciparum schizonts. Anti-PfATRP antibody exhibited peripheral staining patterns around the merozoites. Given the localization of PfATRP in merozoites, we tested for an egress phenotype during schizont arrest assays and demonstrated that native PfATRP is inaccessible on the surface of merozoites in intact schizonts. Dual immunofluorescence assays with markers for the inner membrane complex (IMC) and microtubules suggest partial colocalization in both asexual and sexual stage parasites. Using the soluble recombinant PfATRP in a screen of plasma samples revealed that malaria-infected children have naturally acquired PfATRP-specific antibodies.

Plasmodium falciparum strains spontaneously switch invasion phenotype in suspension culture.

The extensive redundancy in the use of invasion ligands by Plasmodium falciparum, and its unique ability to switch between invasion pathways have hampered vaccine development. P. falciparum strains Dd2 and W2mef have been shown to change from sialic acid (SA)-dependent to SA-independent phenotypes when selected on neuraminidase-treated erythrocytes. Following an observation of increasing ability of Dd2 to invade neuraminidase-treated cells when cultured for several weeks, we systematically investigated this phenomenon by comparing invasion phenotypes of Dd2, W2mef and 3D7 strains of P. falciparum that were cultured with gentle shaking (Suspended) or under static (Static) conditions. While Static Dd2 and W2mef remained SA-dependent for the entire duration of the investigation, Suspended parasites spontaneously and progressively switched to SA-independent phenotype from week 2 onwards. Furthermore, returning Suspended cultures to Static conditions led to a gradual reversal to SA-dependent phenotype. The switch to SA-independent phenotype was accompanied by upregulation of the key invasion ligand, reticulocyte-binding homologue 4 (RH4), and the increased invasion was inhibited by antibodies to the RH4 receptor, CR1. Our data demonstrates a novel mechanism for inducing the switching of invasion pathways in P. falciparum parasites and may provide clues for understanding the mechanisms involved.

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.