Development of an Affordable ELISA Targeting the SARS-CoV-2 Nucleocapsid and Its Application to Samples from the Ongoing COVID-19 Epidemic in Ghana.

INTRODUCTION: The true nature of the population spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations is often not fully known as most cases, particularly in Africa, are asymptomatic. Finding the true magnitude of SARS-CoV-2 spread is crucial to provide actionable data about the epidemiological progress of the disease for researchers and policymakers. This study developed and optimized an antibody enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid antigen expressed in-house using a simple bacterial expression system. METHODS: Nucleocapsid protein from SARS-CoV-2 was expressed and purified from Escherichia coli. Plasma samples used for the assay development were obtained from Ghanaian SARS-CoV-2 seropositive individuals during the pandemic, while seronegative controls were plasma samples collected from blood donors before the coronavirus disease 2019 (COVID-19) pandemic. Another set of seronegative controls was collected during the COVID-19 pandemic. Antibody detection and levels within the samples were validated using commercial kits and Luminex. Analyses were performed using GraphPad Prism, and the sensitivity, specificity and background cut-off were calculated. RESULTS AND DISCUSSION: This low-cost ELISA (£0.96/test) assay has a high prediction of 98.9%, and sensitivity and specificity of 97% and 99%, respectively. The assay was subsequently used to screen plasma from SARS-CoV-2 RT-PCR-positive Ghanaians. The assay showed no significant difference in nucleocapsid antibody levels between symptomatic and asymptomatic, with an increase of the levels over time. This is in line with our previous publication. CONCLUSION: This study developed a low-cost and transferable assay that enables highly sensitive and specific detection of human anti-SARS-CoV-2 IgG antibodies. This assay can be modified to include additional antigens and used for continuous monitoring of sero-exposure to SARS-CoV-2 in West Africa.

Structure-Based Identification of Natural-Product-Derived Compounds with Potential to Inhibit HIV-1 Entry.

Broadly neutralizing antibodies (bNAbs) are potent in neutralizing a wide range of HIV strains. VRC01 is a CD4-binding-site (CD4-bs) class of bNAbs that binds to the conserved CD4-binding region of HIV-1 envelope (env) protein. Natural products that mimic VRC01 bNAbs by interacting with the conserved CD4-binding regions may serve as a new generation of HIV-1 entry inhibitors by being broadly reactive and potently neutralizing. This study aimed to identify compounds that mimic VRC01 by interacting with the CD4-bs of HIV-1 gp120 and thereby inhibiting viral entry into target cells. Libraries of purchasable natural products were virtually screened against clade A/E recombinant 93TH057 (PDB: 3NGB) and clade B (PDB ID: 3J70) HIV-1 env protein. Protein-ligand interaction profiling from molecular docking and dynamics simulations showed that the compounds had intermolecular hydrogen and hydrophobic interactions with conserved amino acid residues on the CD4-binding site of recombinant clade A/E and clade B HIV-1 gp120. Four potential lead compounds, NP-005114, NP-008297, NP-007422, and NP-007382, were used for cell-based antiviral infectivity inhibition assay using clade B (HXB2) env pseudotype virus (PV). The four compounds inhibited the entry of HIV HXB2 pseudotype viruses into target cells at 50% inhibitory concentrations (IC50) of 15.2 µM (9.7 µg/mL), 10.1 µM (7.5 µg/mL), 16.2 µM (12.7 µg/mL), and 21.6 µM (12.9 µg/mL), respectively. The interaction of these compounds with critical residues of the CD4-binding site of more than one clade of HIV gp120 and inhibition of HIV-1 entry into the target cell demonstrate the possibility of a new class of HIV entry inhibitors.

Impacts of the Filoviridae family.

Over 40 filovirus disease outbreaks have been reported since the discovery of the first member of the Filoviridae family, and most of the outbreaks have occurred in Africa. In addition to deaths (primary impacts), there have also been health, social, economic, and political effects (secondary impacts) due to the outbreaks. Two large filovirus disease outbreaks have occurred in West and Central Africa in recent times, and direct and indirect repercussions resulting from the outbreaks underscores the need to strengthen the capacity of health services in disease hotspots.

Longitudinal Secretion of Paramyxovirus RNA in the Urine of Straw-Coloured Fruit Bats (Eidolon helvum).

The straw-coloured fruit bat (Eidolon helvum) is widespread in sub-Saharan Africa and is widely hunted for bushmeat. It is known to harbour a range of paramyxoviruses, including rubuloviruses and henipaviruses, but the zoonotic potential of these is unknown. We previously found a diversity of paramyxoviruses within a small, captive colony of E. helvum after it had been closed to contact with other bats for 5 years. In this study, we used under-roost urine collection to further investigate the paramyxovirus diversity and ecology in this colony, which had been closed to the outside for 10 years at the time of sampling. By sampling urine weekly throughout an entire year, we investigated possible seasonal patterns of shedding of virus or viral RNA. Using a generic paramyxovirus L-gene PCR, we detected eight distinct paramyxovirus RNA sequences. Six distinct sequences were detected using a Henipavirus-specific PCR that targeted a different region of the L-gene. Sequence detection had a bi-annual pattern, with the greatest peak in July, although different RNA sequences appeared to have different shedding patterns. No significant associations were detected between sequence detection and birthing season, environmental temperature or humidity, and no signs of illness were detected in any of the bats in the colony during the period of sample collection.

Genotypic Characterization of Epstein Barr Virus in Blood of Patients with Suspected Nasopharyngeal Carcinoma in Ghana.

Nasopharyngeal cancer (NPC) is associated with Epstein Barr virus (EBV) infection. However different viral strains have been implicated in NPC worldwide. This study aimed to detect and characterize EBV in patients diagnosed with NPC in Ghana. A total of 55 patients diagnosed with NPC by CT scan and endoscopy were age-matched with 53 controls without a known oncological disease. Venous blood was collected from the study participants and DNA extracted from the blood samples. Detection of EBV and genotyping were done by amplifying Epstein Barr nuclear antigen 1 (EBNA-1) and Epstein Barr nuclear antigen 2 (EBNA-2), respectively, using specific primers. Viral load in patients and controls was determined using real-time polymerase chain reaction. EBV positivity in controls (92%) was significantly greater than that of NPC patients (67%) (χ2 = 19.17, p < 0.0001), and viral infection was independent of gender (χ2 = 1.770, p = 0.1834). The predominant EBV genotypes in patients and controls were genotype 2 (52%) and genotype 1 (62%), respectively. Median EBV load was significantly higher in NPC patients than the control group (p < 0.01). In summary, prevalence of EBV genotype 2 infection was higher in NPC patients than the control group. Assessment of EBV load may be used as a biomarker for the diagnosis of NPC.

Incidence and mortality of cancer in the Volta Region of Ghana.

IMPACT STATEMENT: Region-specific cancer reports are essential in knowing the common cancers in specific populations. This study reports all cancer types recorded in the major hospitals in the Volta Region of Ghana, a population that is sparsely captured in the national cancer registries. The study identified the common cancers as well as the death rates in the Volta Region, hence contributing to the national effort to report cancer data. Although not comprising all the elements of a cancer registry, our data will augment the effort of the two national cancer registries in Ghana to provide the national cancer reports.

Patterns of Frequently Diagnosed Pediatric Morbidities in Hospitalized Children in the Volta Region of Ghana.

Background. The aim of this study was to identify the frequently diagnosed pediatric diseases in the Volta Region of Ghana, as well as to examine the burden of these diseases. The top pediatric diseases that were frequently diagnosed were malaria, gastroenteritis, systemic infection, anemia, pneumonia, and respiratory tract infection. Methods. Clearance was obtained from the Volta Regional Directorate of the Ghana Health Service to collect primary data on pediatric hospitalizations in the major hospitals in the Region. Diseases with more than 1000 recorded cases of hospitalizations were considered among the top frequently diagnosed childhood morbidities. Results. The data suggest that the Northern sector had different seasonal patterns of recorded diagnosed pediatric cases compared with the Central and Southern sectors, which had similar patterns of the reported diseases. Most of the pediatric diseases in the Volta Region were more prevalent during the dry seasons compared with the rainy seasons and resulting in seasonal patterns of hospitalizations. Conclusion. Although the frequently diagnosed pediatric diseases can be prevented and/or treated, many children are hospitalized, with a proportion of them dying. It is, therefore, important that efforts are made to reduce the burden of pediatric hospitalization.

Filovirus Disease Outbreaks: A Chronological Overview.

Filoviruses cause outbreaks which lead to high fatality in humans and non-human primates, thus tagging them as major threats to public health and species conservation. In this review, we give account of index cases responsible for filovirus disease outbreaks that have occurred over the past 52 years in a chronological fashion, by describing the circumstances that led to the outbreaks, and how each of the outbreaks broke out. Since the discovery of Marburg virus and Ebola virus in 1967 and 1976, respectively, more than 40 filovirus disease outbreaks have been reported; majority of which have occurred in Africa. The chronological presentation of this review is to provide a concise overview of filovirus disease outbreaks since the discovery of the viruses, and highlight the patterns in the occurrence of the outbreaks. This review will help researchers to better appreciate the need for surveillance, especially in areas where there have been no filovirus disease outbreaks. We conclude by summarizing some recommendations that have been proposed by health and policy decision makers over the years.

In Vitro Evaluation of Cytotoxic Activities of Marketed Herbal Products in Ghana.

There are numerous herbal products on the Ghanaian market that are purported to cure various ailments, including cancer. However, scientific investigations on efficacy and toxicity of most of these products are not done. The aim of the study was to assess the anticancer potentials of herbal products on the Ghanaian market. Antiproliferative effects of Kantinka BA (K-BA), Kantinka Herbaltics (K-HER), Centre of Awareness (COA), a stomach (STO) and multicancer (MUT) product were evaluated in vitro using liver (Hep G2), breast (MCF-7), prostate (PC-3 and LNCaP), and blood (Jurkat) cancer cell lines. Cytotoxicity of the medicinal products was assessed using tetrazolium-based colorimetric assay, and total phenolic content and antioxidant activity of the products were determined using Folin-Ciocalteau and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays, respectively. Phytochemical screening resulted in the detection of terpenoids and flavonoids in most of the products, and alkaloids were detected in only MUT. Tannins were absent from all the products. The highest and lowest concentrations of phenolics were recorded for MUT and K-BA, respectively. The highest and lowest antioxidant activities were measured for MUT and K-HER, respectively. Only 2 products (STO and MUT) were cytotoxic to Hep G2 cells; with MUT being the only product that was cytotoxic to MCF-7 cells. All but K-BA were cytotoxic to PC-3 cells, while all products except K-HER were cytotoxic to LNCaP and Jurkat cells. The study thus confirms that the herbal products have selective cytotoxic activities against the tested cancer cell lines. However, comprehensive toxicity studies must be conducted to establish their safety.