Detection of SARS-CoV-2 in Terrestrial Animals in Southern Nigeria: Potential Cases of Reverse Zoonosis.

Since SARS-CoV-2 caused the COVID-19 pandemic, records have suggested the occurrence of reverse zoonosis of pets and farm animals in contact with SARS-CoV-2-positive humans in the Occident. However, there is little information on the spread of the virus among animals in contact with humans in Africa. Therefore, this study aimed to investigate the occurrence of SARS-CoV-2 in various animals in Nigeria. Overall, 791 animals from Ebonyi, Ogun, Ondo, and Oyo States, Nigeria were screened for SARS-CoV-2 using RT-qPCR (n = 364) and IgG ELISA (n = 654). SARS-CoV-2 positivity rates were 45.9% (RT-qPCR) and 1.4% (ELISA). SARS-CoV-2 RNA was detected in almost all animal taxa and sampling locations except Oyo State. SARS-CoV-2 IgGs were detected only in goats from Ebonyi and pigs from Ogun States. Overall, SARS-CoV-2 infectivity rates were higher in 2021 than in 2022. Our study highlights the ability of the virus to infect various animals. It presents the first report of natural SARS-CoV-2 infection in poultry, pigs, domestic ruminants, and lizards. The close human-animal interactions in these settings suggest ongoing reverse zoonosis, highlighting the role of behavioral factors of transmission and the potential for SARS-CoV-2 to spread among animals. These underscore the importance of continuous monitoring to detect and intervene in any eventual upsurge.

Adherence to COVID-19 preventive measures in Sub-Saharan Africa during the 1st year of the pandemic: Pooled analysis of the International Citizen Project on COVID-19 (ICPCovid) surveys.

Introduction: While most governments instituted several interventions to stall the spread of COVID-19, little is known regarding the continued observance of the non-pharmaceutical COVID-19 preventive measures particularly in Sub-Saharan Africa (SSA). We investigated adherence to these preventive measures during the initial 6 months of the COVID-19 outbreak in some SSA countries. Methods: Between March and August 2020, the International Citizen Project on COVID-19 consortium (www.icpcovid.com) conducted online surveys in six SSA countries: Benin, Cameroon, Democratic Republic of Congo, Mozambique, Somalia, and Uganda. A five-point individual adherence score was constituted by scoring respondents' observance of the following measures: mask use, physical distancing, hand hygiene, coughing hygiene, and avoiding to touch one's face. Community behaviors (going to public places, traveling during the pandemic) were also assessed. Data were analyzed in two time periods: Period 1 (March-May) and Period 2 (June-August). Results: Responses from 26,678 respondents were analyzed (mean age: 31.0 ± 11.1 years; 54.1% males). Mean individual adherence score decreased from 3.80 ± 1.37 during Period 1, to 3.57 ± 1.43 during Period 2; p < 0.001. At the community level, public events/places were significantly more attended with increased travels during Period 2 compared to Period 1 (p < 0.001). Using linear mixed models, predictors of increased individual adherence included: higher age (Coef = 0.005; 95% CI: 0.003-0.007), female gender (Coef = 0.071; 95% CI: 0.039-0.104), higher educational level (Coef = 0.999; 95% CI: 0.885-1.113), and working in the healthcare sector (Coef = 0.418; 95% CI: 0.380-0.456). Conclusion: Decreasing adherence to non-pharmaceutical measures over time constitutes a risk for the persistence of COVID-19 in SSA. Younger persons and those with lower education levels constitute target groups for improving adherence to such measures.

Emergence and Genomic Characterization of Multidrug Resistant Candida auris in Nigeria, West Africa.

Candida auris is an emerging multidrug-resistant fungal pathogen that has become a worldwide public health threat due to the limitations of treatment options, difficulty in diagnosis, and its potential for clonal transmission. Four ICU patients from three different healthcare facilities in Southern Nigeria presented features suggestive of severe sepsis and the blood cultures yielded the growth of Candida spp., which was identified using VITEK 2 as C. auris. Further confirmation was performed using whole genome sequencing (WGS). From the genomic analysis, two had mutations that conferred resistance to the antifungal azole group and other non-synonymous mutations in hotspot genes, such as ERG2, ERG11, and FKS1. From the phylogenetic analysis, cases 2 and 4 had a confirmed mutation (ERG11:Y132F) that conferred drug resistance to azoles clustered with clade 1, whilst cases 1 and 3 clustered with clade 4. Three of the patients died, and the fourth was most likely a case of colonization since he received no antifungals and was discharged home. These first cases of C. auris reported from Nigeria were most likely introduced from different sources. It is of public health importance as it highlights diagnostic gaps in our setting and the need for active disease surveillance in the region.

Simplified Cas13-based assays for the fast identification of SARS-CoV-2 and its variants.

The widespread transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for rapid nucleic acid diagnostics that are easy to use outside of centralized clinical laboratories. Here we report the development and performance benchmarking of Cas13-based nucleic acid assays leveraging lyophilised reagents and fast sample inactivation at ambient temperature. The assays, which we named SHINEv.2 (for 'streamlined highlighting of infections to navigate epidemics, version 2'), simplify the previously reported RNA-extraction-free SHINEv.1 technology by eliminating heating steps and the need for cold storage of the reagents. SHINEv.2 detected SARS-CoV-2 in nasopharyngeal samples with 90.5% sensitivity and 100% specificity (benchmarked against the reverse transcription quantitative polymerase chain reaction) in less than 90 min, using lateral-flow technology and incubation in a heat block at 37 °C. SHINEv.2 also allows for the visual discrimination of the Alpha, Beta, Gamma, Delta and Omicron SARS-CoV-2 variants, and can be run without performance losses by using body heat. Accurate, easy-to-use and equipment-free nucleic acid assays could facilitate wider testing for SARS-CoV-2 and other pathogens in point-of-care and at-home settings.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

VGEA: an RNA viral assembly toolkit.

Next generation sequencing (NGS)-based studies have vastly increased our understanding of viral diversity. Viral sequence data obtained from NGS experiments are a rich source of information, these data can be used to study their epidemiology, evolution, transmission patterns, and can also inform drug and vaccine design. Viral genomes, however, represent a great challenge to bioinformatics due to their high mutation rate and forming quasispecies in the same infected host, bringing about the need to implement advanced bioinformatics tools to assemble consensus genomes well-representative of the viral population circulating in individual patients. Many tools have been developed to preprocess sequencing reads, carry-out de novo or reference-assisted assembly of viral genomes and assess the quality of the genomes obtained. Most of these tools however exist as standalone workflows and usually require huge computational resources. Here we present (Viral Genomes Easily Analyzed), a Snakemake workflow for analyzing RNA viral genomes. VGEA enables users to map sequencing reads to the human genome to remove human contaminants, split bam files into forward and reverse reads, carry out de novo assembly of forward and reverse reads to generate contigs, pre-process reads for quality and contamination, map reads to a reference tailored to the sample using corrected contigs supplemented by the user's choice of reference sequences and evaluate/compare genome assemblies. We designed a project with the aim of creating a flexible, easy-to-use and all-in-one pipeline from existing/stand-alone bioinformatics tools for viral genome analysis that can be deployed on a personal computer. VGEA was built on the Snakemake workflow management system and utilizes existing tools for each step: fastp (Chen et al., 2018) for read trimming and read-level quality control, BWA (Li & Durbin, 2009) for mapping sequencing reads to the human reference genome, SAMtools (Li et al., 2009) for extracting unmapped reads and also for splitting bam files into fastq files, IVA (Hunt et al., 2015) for de novo assembly to generate contigs, shiver (Wymant et al., 2018) to pre-process reads for quality and contamination, then map to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences, SeqKit (Shen et al., 2016) for cleaning shiver assembly for QUAST, QUAST (Gurevich et al., 2013) to evaluate/assess the quality of genome assemblies and MultiQC (Ewels et al., 2016) for aggregation of the results from fastp, BWA and QUAST. Our pipeline was successfully tested and validated with SARS-CoV-2 (n = 20), HIV-1 (n = 20) and Lassa Virus (n = 20) datasets all of which have been made publicly available. VGEA is freely available on GitHub at: https://github.com/pauloluniyi/VGEA under the GNU General Public License.

Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2.

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE's results can be visualized with an in-tube fluorescent readout - reducing contamination risk as amplification reaction tubes remain sealed - and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.