The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Genomic RFLP analysis using different restriction enzymes and 3 RAPD markers unassigned by <i>in silico</i> analysis.

<p>The hybridization patterns were revealed by the different probes, A: LEM536/320/OPAY8, B: LEM496/300/OPAD17 and C: M106/200/OPAY9. Tested isolates correspond to <i>L. major</i> (FMH; <i>L. maj</i>), <i>L. archibaldi</i> (GEBRE; <i>L. arch</i>), <i>L. donovani</i> (HU3; <i>L. don</i>), <i>L. tropica</i> (DBKM; <i>L. tro</i>) and <i>L. infantum</i> (LEM1163; <i>L. inf</i>). <i>EcoR</i>I, <i>Pst</i>I, <i>Hind</i>III, <i>Xho</i>I on top of the panel indicate the restriction enzyme used to digest the total <i>Leishmania</i> DNAs.</p

Homology analysis of the sequences of the cloned RAPD markers in 3 <i>Leishmania</i> genomes.

<p>NF: Not found. BPK282A1, JPCM5 and Friedlin are names of <i>L. donovani</i>, <i>L. infantum</i> and <i>L. major</i> strains, respectively used for genome sequencing and available in GeneDB.</p><p>Homology analysis of the sequences of the cloned RAPD markers in 3 <i>Leishmania</i> genomes.</p

Screening and Characterization of RAPD Markers in Viscerotropic <i>Leishmania</i> Parasites

<div><p>Visceral leishmaniasis (VL) is mainly due to the <i>Leishmania donovani</i> complex. VL is endemic in many countries worldwide including East Africa and the Mediterranean region where the epidemiology is complex. Taxonomy of these pathogens is under controversy but there is a correlation between their genetic diversity and geographical origin. With steady increase in genome knowledge, RAPD is still a useful approach to identify and characterize novel DNA markers. Our aim was to identify and characterize polymorphic DNA markers in VL <i>Leishmania</i> parasites in diverse geographic regions using RAPD in order to constitute a pool of PCR targets having the potential to differentiate among the VL parasites. 100 different oligonucleotide decamers having arbitrary DNA sequences were screened for reproducible amplification and a selection of 28 was used to amplify DNA from 12 <i>L. donovani</i>, <i>L. archibaldi</i> and <i>L. infantum</i> strains having diverse origins. A total of 155 bands were amplified of which 60.65% appeared polymorphic. 7 out of 28 primers provided monomorphic patterns. Phenetic analysis allowed clustering the parasites according to their geographical origin. Differentially amplified bands were selected, among them 22 RAPD products were successfully cloned and sequenced. Bioinformatic analysis allowed mapping of the markers and sequences and priming sites analysis. This study was complemented with Southern-blot to confirm assignment of markers to the kDNA. The bioinformatic analysis identified 16 nuclear and 3 minicircle markers. Analysis of these markers highlighted polymorphisms at RAPD priming sites with mainly 5′ end transversions, and presence of inter– and intra– taxonomic complex sequence and microsatellites variations; a bias in transitions over transversions and indels between the different sequences compared is observed, which is however less marked between <i>L. infantum</i> and <i>L. donovani</i>. The study delivers a pool of well-documented polymorphic DNA markers, to develop molecular diagnostics assays to characterize and differentiate VL causing agents.</p></div

Lesionia: a digital data management system for epidemiological and clinical data collected from patients suspected for cutaneous leishmaniasis

Abstract Background. Digital systems for data management (DSDM) are considered nowadays of high importance in the field of biomedical sciences. Such systems ensure that data meet the standards of FAIR (Findability, Accessibility, Interoperability and Reusability). Our group is interested in implementing a DSDM for data collected from patients suspected of having cutaneous leishmaniasis (CL) in the frame of diagnostics evaluation. The data is collected in multiple sites and countries by different partners in the frame of a project supported by the USAID-NAS PEER program. We capitalized on the thorough clinical and field expertise of some partners to assess needs. Then, we further refined these needs consortium-wide to define the data to be collected by the clinicians and biologists during the data life cycle. This led to the development of a questionnaire form for data collection and the implementation of a web-based application, called Lesionia.Results. Based on the questionnaire, we developed Lesionia, a digital system for the management and the analysis of clinical and epidemiological data. It consists of a relational database and a web-based user interface (WUI). The database was conceived to be expandable to new collaborators and projects. It allows for data handling from the consented patient interview and sample collection to the samples storage and investigation. The WUI permits data entry, fetching, visualization and analysis. Rigorous controls on data entry were implemented to reduce discrepancies. It also offers a set of analysis tools that range from descriptive statistics to variable correlation analysis. Lesionia is accessible in a secure manner to all users of the consortium through a web browser connected to the Internet. Conclusion. Lesionia is a valuable tool for clinical and epidemiological data management. It is an open source software that can broadly serve the scientific community interested in studying, controlling, reporting and diagnosing CL and similar cutaneous diseases.</jats:p

Panel of <i>Leishmania</i> strains used for screening of RAPD markers.

<p>WHO that summarizes Host, geographical origin, year of isolation and laboratory code is presented together with pathology and zymodeme code whenever available. MON– corresponds to zymodeme code attributed by the reference center in Montpellier. The table also gathers study codes assigned to some of the isolates in other studies: D21, D28, D29, D31 and D32: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Mauricio1" target="_blank">[20]</a>; DON-39 and ARC-43: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Kuhls1" target="_blank">[21]</a>; Devi, H9, LRC-L57, ADDIS 164: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Jamjoom1" target="_blank">[18]</a>; Devi, GEBRE1 and KA-Jeddah: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Thiel1" target="_blank">[53]</a>; DON-81and ARC-43 (LG11): strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Kuhls2" target="_blank">[28]</a>; DON-09, DON-31, DON-39 and ARC-11: strains used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109773#pone.0109773-Chocholov1" target="_blank">[25]</a>. Country abbreviations are shown as specified by WHO recommendations (SD: Sudan; TN: Tunisia; ET: Ethiopia; SA: Saudi Arabia; KE: Kenya; IN: India). ND: Not Determined; CL: cutaneous leishmaniasis; VL: visceral leishmaniasis; PKDL: Post Kala azar Dermal Leishmaniasis.</p><p>Panel of <i>Leishmania</i> strains used for screening of RAPD markers.</p