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.

Role of CTX-M-15 gene in spread of extended-spectrum beta-lactamases among immunocompetent patients in Ghana.

Background: Patients with faecal carriage of extended-spectrum beta-lactamases (ESBL)-producing Enterobacterales serve as reservoirs and sources of dissemination and infection. Objective: This report examined immunocompetent patients for faecal carriage of ESBL-producing Enterobacterales in a district care hospital setting in Ghana. Methods: Between March 2019 and May 2020, cross-sectional sampling was performed to enrol patients and conduct questionnaire-structured interviews for factors that predispose patients to ESBL faecal carriage. Faecal samples from study patients were quantified for ESBL-producing Enterobacterales. The ESBL genes were characterised by polymerase chain reaction and sequencing. Results: The overall proportion of ESBL faecal carriage was 35.5% (n = 38/107). The blaCTX-M gene, mostly CTX-M-15, was detected in 89.5% (n = 34/38) of the ESBL-producing isolates. The other ESBL types included blaSHV (n = 3) and blaOXA (n = 1). The CTX-M-15-positive isolates, when present in a faecal sample compared to the non-ESBL-CTX-M-15 isolates, constituted the predominant faecal Enterobacterales, with significantly higher colony counts than all other enterobacteria in that sample. In multivariate regression, independent risk factors for faecal carriage of ESBL-producing Enterobacterales were hospitalisation in the past year, infections since admission, use of antibiotics in the past 6 weeks, and admission from another hospital. Conclusion: The study found that CTX-M-15-producing isolates were the predominant faecal Enterobacterales, and that further investigations are needed to determine the reasons behind this dominance. What this study adds: The CTX-M-15-producing isolates dominance in this study shows the misuse and abuse of antibiotics in an African medical facility and indicates the potential role of immunity in controlling ESBL spread, which is to be investigated further.

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.

Cocoa-associated filamentous fungi for the biocontrol of aflatoxigenic Aspergillus flavus.

Aflatoxin and other mycotoxin contamination are major threats to global food security and present an urgent need to secure the global food crop against spoilage by mycotoxigenic fungi. Cocoa material is noted for naturally low aflatoxin contamination. This study was designed to assess the potential for harnessing cocoa-associated filamentous fungi for the biocontrol of aflatoxigenic Aspergillus flavus. The candidate fungi were isolated from fermented cocoa beans collected from four cocoa-growing areas in Ghana. Molecular characterization included Internal Transcribed Spacer (ITS)-sequencing for identification and polymer chain reaction (PCR) to determine mating type. Effects of the candidate isolates on growth and aflatoxin-production by an aflatoxigenic A. flavus isolate (BANGA1) were assessed. Aflatoxin production was monitored by UV fluorescence and quantified by enzyme-linked immunosorbent assay (ELISA). Thirty-six filamentous fungi were cultured and identified as Aspergillus, Cladosporium, Lichtheimia, or Trichoderma spp. isolates. The isolates generally interacted negatively with BANGA1 growth and aflatoxin production. The Aspergillus niger and Aspergillus aculeatus biocontrol candidates showed the strongest colony antagonism (54%-94%) and reduction in aflatoxin production (12%-50%) on agar. In broth, the A. niger isolates reduced aflatoxin production by up to 97%. Metabolites from the A. niger isolates showed the strongest inhibition of growth by BANGA1 and inhibited aflatoxin production. Four of the candidate isolates belonged to the MAT1-1 mating type and 12 identified as MAT1-2. This may be indicative of the potential for genetic recombination events between fungi in the field, a finding which is particularly relevant to the risk posed by A. flavus biocontrol measures that rely on atoxigenic A. flavus strains.

Prophylactic or therapeutic administration of Holarrhena floribunda hydro ethanol extract suppresses complete Freund's adjuvant-induced arthritis in Sprague-Dawley rats.

BACKGROUND: A hydro ethanol extract of the stem bark of Holarrhena floribunda (HFE) has been shown to be effective in the management of acute inflammation. This study was to evaluate usefulness of the extract for the management of chronic inflammation in a murine model. METHODS: Arthritis was induced in Sprague-Dawley rats using Complete Freund's Adjuvant. Anti-arthritic effect of the extract was evaluated in prophylactic and therapeutic treatment models at doses of 50, 200 and 500 mg/kg. Parameters assessed included oedema, serology of inflammatory response, bone tissue histology and haematology. Data were analysed by ANOVA and Tukey's multiple comparisons post hoc test. RESULTS: HFE at 50-500 mg/kg dose-dependently [P ≥ 0.0354 (prophylactic) and P ≥ 0.0001 (therapeutic) inhibited swelling of the injected paw upon prophylactic [≤ 81.26% (P < 0.0001) or therapeutic [≤ 67.92% (P < 0.01) administration - and prevented spread of arthritis to the contralateral paw. The inflammation alleviation activity was further demonstrated by decrease in arthritis score, radiologic score and erythrocyte sedimentation rate. HFE at all doses significantly reduced serum interleukin (IL)-1α (P < 0.0197), and 500 mg/kg HFE reduced IL-6 (P = 0.0032). In contrast, serum concentrations of IL-10, protein kinase A and cyclic adenosine monophosphate were enhanced (P ≤ 0.0436). HFE consistently showed better prophylactic than therapeutic activity. CONCLUSION: HFE strongly suppressed Complete Freund's Adjuvant-induced arthritis and modulated regulators of inflammation, including IL-1α, - 6 and - 10. Taken together, the data suggest that HFE has potential for use as an agent for modulation of the inflammatory response.

Hydro-ethanol extract of Holarrhena floribunda stem bark exhibits anti-anaphylactic and anti-oedematogenic effects in murine models of acute inflammation.

BACKGROUND: Holarrhena floribunda (G.Don) T.Durand & Schinz stem bark has anecdotal use in Ghanaian folk medicine for the management of inflammatory conditions. This study was conducted to investigate the in vivo anti-inflammatory activity of the bark extract using models of acute inflammation in male Sprague Dawley rats, C57BL/6 mice and ICR mice. METHODS: A 70% hydro-ethanol extract of the stem bark (HFE) was evaluated at doses of 5-500 mg/kg bw. Local anaphylaxis was modelled by the pinnal cutaneous anaphylactic test. Systemic anaphylaxis or sepsis were modeled by compound 48/80 or lipopolysaccharide, respectively. Clonidine-induced catalepsy was used to investigate the effect on histamine signaling. Anti-oedematogenic effect was assessed by induction with carrageenan. Effects on mediators of biphasic acute inflammation were studied using histamine and serotonin (early phase) or prostaglandin E2 (late phase). RESULTS: HFE demonstrated anti-inflammatory and/or anti-oedematogenic activity comparable to standard doses of aspirin and diclofenac (inhibitors of cyclooxygenases-1 and -2), chlorpheniramine (histamine H1-receptor antagonist), dexamethasone (glucocorticoid receptor agonist), granisetron (serotonin receptor antagonist) and sodium cromoglycate (inhibitor of mast cell degranulation). All observed HFE bioactivities increased with dose. CONCLUSIONS: The data provide evidence that the extract of H. floribunda stem bark has anti-anaphylactic and anti-oedematogenic effects; by interfering with signalling or metabolism of histamine, serotonin and prostaglandin E2 which mediate the progression of inflammation. The anti-inflammatory and antihistaminic activities of HFE may be relevant in the context of the management of COVID-19.

Explaining the unexpected COVID-19 trends and potential impact across Africa.

Official COVID-19 case counts and mortality rates across Africa are lower than had been anticipated. Research reports, however, indicate far higher exposure rates than the official counts in some countries. Particularly in Western and Central Africa, where mortality rates are disproportionately lower than the rest of the continent, this occurrence may be due to immune response adaptations resulting from (1) frequent exposure to certain pro-inflammatory pathogens, and (2) a prevalence of low-grade inflammation coupled with peculiar modifications to the immune response based on one's immunobiography. We suggest that the two factors lead to a situation where post infection, there is a rapid ramp-up of innate immune responses, enough to induce effective defense and protection against plethora pathogens. Alongside current efforts at procuring and distributing vaccines, we draw attention to the need for work towards appreciating the impact of the apparently widespread, asymptomatic SARS-CoV-2 infections on Africa's populations vis a vis systemic inflammation status and long-term consequences for public health.

Nutritional value and safety of animal feed supplemented with Talaromyces verruculosus-treated cocoa pod husks.

Theobromine exerts deleterious effects on animal physiology. Removal of theobromine from the millions of metric tons of cocoa pod husks (CPH) discarded annually could allow for the production of cheap, CPH-based animal feed. The aim of this study was to evaluate safety and nutritional value of bio-detheobrominated CPH in Sprague-Dawley rats. Theobromine was removed from CPH by treatment with an isolate of Talaromyces verruculosus (TvTD). Substituted feeds containing CPH were formulated by replacing 30% or 50% of the maize content of regular rat feed with TvTD-treated or inactivated TvTD-treated CPH. Feeding groups included control groups without or with theobromine administration. Effects of the feed formulations on water and feed intake, weight gain, blood biochemistry and organ-specific toxicity were assessed. Rats ingesting theobromine in inactivated TvTD-treated CPH-based diet or by oral gavage variably exhibited marked deleterious effects, mainly evident in body weight, thymus wet weight and tissue histology. In contrast, substitution with TvTD-treated CPH caused significant increase in body weight. Substitution at 30% did not cause mortality or organ-specific toxicity with reference to the testes, kidneys, spleen or liver, unlike substitution at 50%. The data demonstrate that detheobrominated CPH may safely replace up to 30% of maize in animal feed formulations.

Household cockroaches carry CTX-M-15-, OXA-48- and NDM-1-producing enterobacteria, and share beta-lactam resistance determinants with humans.

BACKGROUND: This study was designed to investigate whether household cockroaches harbor cephalosporin-resistant enterobacteria that share resistance determinants with human inhabitants. From February through July 2016, whole cockroach homogenates and human fecal samples from 100 households were cultured for cephalosporin-resistant enterobacteria (CRe). The CRe were examined for plasmid-mediated AmpC, ESBL, and carbapenemase genes; antibiotic susceptibility patterns; and conjugative transfer of antibiotic resistance mechanisms. Clonal associations between CRe were determined by multi-locus sequence typing (MLST). RESULTS: Twenty CRe were recovered from whole cockroach homogenates from 15 households. The prevalence of households with cockroaches that harbored CRe, AmpC- (based on phenotype, with no identifiable blaAmpC genes), ESBL-, and carbapenemase-producers were 15, 4, 5%(2 blaCTX-M-15/TEM-1; 1 blaCTX-M-15/TEM-4; 1 blaTEM-24; 1 blaSHV-4) and 3%(2 blaNDM-1 genes and 1 blaOXA-48 gene), respectively. Overall, 20 CRe were recovered from 61 fecal samples of inhabitants from all 15 households that had cockroach samples positive for CRe. Of these, 5CRe (1 per household) were positive for ESBLs (blaTEM-24, blaTEM-14, blaCTX-M-15/TEM-4, blaSHV-3, blaCTX-M-15/TEM-1) and none carried AmpCs or carbapenemases. From 4% of households, the pair of cockroach and human CRe shared the same sequence type (ST), clonal complex (CC), antibiogram, and conjugable bla gene sequence (house 34, E. coli ST9/CC20-blaTEM-4; house 37, E. coli ST44/CC10-blaCTX-15/TEM-4; house 41, E. coli ST443/CC205-blaCTX-15/TEM-1; house 49, K. pneumoniae ST231/CC131-blaSHV-13). CONCLUSION: The findings provide evidence that household cockroaches may carry CTX-M-15-, OXA-48- and NDM-1-producers, and share clonal relationship and beta-lactam resistance determinants with humans.

Bio-detheobromination of cocoa pod husks: reduction of ochratoxin A content without change in nutrient profile.

BACKGROUND: Utilization of cocoa pod husks (CPH) in animal feed is hindered by the presence of theobromine, which is variably toxic to animals. Treatment of this agro-waste to remove theobromine, while preserving its nutrient content, would allow beneficial use of the millions of metric tonnes discarded annually. The aim of this study was to assess the suitability of selected theobromine-degrading filamentous fungi for use as bio-tools in degradation of theobromine in CPH. RESULTS: The candidate fungi assessed in this study were an Aspergillus niger (AnTD) and three Talaromyces spp. (TmTD-1, TmTD-2, TvTD) isolates. All the fungi eliminated CPH theobromine, 0.15% w/w starting concentration, within 7 days of start of treatment, and were capable of degrading caffeine and theophylline. The fungi decreased CPH ochratoxin A content by 31-74%. Pectin was not detectable in fungus-treated CPH whereas parameters assessed for proximate composition were not affected. CONCLUSIONS: The data provide ample evidence that the four isolates can be applied to CPH for the purpose of eliminating theobromine and decreasing ochratoxin A content without affecting nutrient profile. Comparatively, Talaromyces verruculosus TvTD was considered as most suitable for use as a bio-tool in detheobromination of CPH for animal feed.

Isolation and characterisation of theobromine-degrading filamentous fungi.

Strategies for achieving global food security include identification of alternative feedstock for use as animal feed, to contribute towards efforts at increasing livestock farming. The presence of theobromine in cocoa pod husks, a major agro-waste in cocoa-producing countries, hinders its utilisation for this purpose. Cheap treatment of cocoa pod husks to remove theobromine would allow largescale beneficial use of the millions of metric tonnes generated annually. The aim of this study was to isolate theobromine-degrading filamentous fungi that could serve as bioremediation agents for detheobromination of cocoa pod husks. Filamentous fungi were screened for ability to degrade theobromine. The most promising isolates were characterized with respect to optimal environmental conditions for theobromine degradation. Secretion of theobromine-degrading enzymes by the isolates was investigated. Theobromine degradation was monitored by HPLC. Of fourteen theobromine-degrading isolates collected and identified by rDNA 5.8S and ITS sequences, seven belonged to Aspergillus spp. and six were Talaromyces spp. Based on the extent of theobromine utilization, four isolates; Aspergillus niger, Talaromyces verruculosus and two Talaromyces marneffei, showed the best potential for use as bioagents for detheobromination. First-time evidence was found of the use of xanthine oxidase and theobromine oxidase in degradation of a methylxanthine by fungal isolates. Metabolism of theobromine involved initial demethylation at position 7 to form 3-methylxanthine, or initial oxidation at position 8 to form 3,7-dimethyuric acid. All four isolates degraded theobromine beyond uric acid. The data suggest that the four isolates can be applied to substrates, such as cocoa pod husks, for elimination of theobromine.

Genetic characterization of TEM-type ESBL-associated antibacterial resistance in Enterobacteriaceae in a tertiary hospital in Ghana.

BACKGROUND: Antibiotic resistance due to the presence of extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae is a worldwide problem. Data from Ghana regarding this resistance mechanism is limited. This study was designed to investigate the presence of TEM-type ESBL genes, their locations and their conjugabilities in clinical isolates of enterobacteria collected from the Korle-Bu Teaching Hospital in Ghana. METHODS: Study isolates were characterized with respect to ESBL phenotype, TEM-type ESBL gene detection, location of the ESBL gene(s) and conjugability of the ESBL phenotype using nalidixic acid-resistant Escherichia coli K-12 as recipient. Phenotyping was by Kirby Bauer disk diffusion using cefpodoxime, ceftazidime, cefotaxime and their combinations with clavulanate. Gene detections were by PCR using blaTEM primers. RESULTS: Overall, 37.96 % of 137 clinical isolates showed ESBL phenotype. The ESBLs occurred mostly in Klebsiella spp. (42.3 %) and then Escherichia coli (34.6 %). The TEM gene was detected in 48.1 % of ESBL-positive isolates and was determined to be plasmid-borne in 24 of 25 blaTEM detections. Overall, 62.7 % of TEM-producing isolates transferred the ESBL phenotype by conjugation. CONCLUSIONS: The results highlight the presence of TEM-type ESBLs in the Korle-Bu Teaching Hospital and show considerable risk of environmental contamination through the urine of infected persons. An inhibition zone chart was generated which indicates the possible presence of complex beta-lactamase types. The data points to the fact that the ESBL-producing bacteria may disseminate this resistance mechanism via conjugation.