A Sanger approach based on overlapping fragments to screen SARS-CoV-2 variants.

Since the beginning of the COVID-19 pandemic, the SARS-CoV-2 virus has undergone various genetic mutations which have led to the emergence of variants. The World Health Organization (WHO) defines Variants of Concern (VOCs) and Variants of Interest (VOIs) according to several criteria. These include significant changes in the transmissibility and pathogenicity of the virus characterized by mutations in the spike gene coding the spike glycoprotein. In this study, we designed ten Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) assays in order to identify mutations of SARS-CoV-2 in overlapping fragments. Each assay contained mutations on the fragments sequenced by a Sanger method. The genomic analysis of the fragments allowed to identify the variant according to the position of the mutations. The assembly of the 10 fragments refined the analysis, highlighting all the mutations present in the S gene. Finally, a comparison of methods using a Next-Generation Sequencing (NGS) approches for samples enabled the method to be validated. By this method we have highlighted a characteristic mutation of the lineage B of SARS-CoV-2. We showed the circulation of SARS-CoV-2 belonging to lineage A and B in the beginning of the pandemic in Gabon. We have identified the Alpha, Delta and Omicron variants. This method would allow laboratories with limited financial means or without NGS instrument to obtain sequences of the S gene. This method wase very effective to highlight the circulation of variants, in particular VOCs and VOIs, in this developing country, Gabon, during the COVID-19 pandemic.

Epidemiology of first cases of SARS-CoV-2 infection, from March to April 2020, in Gabon.

Background After the first cases of coronaviruses disease 2019 (COVID-19) in China in January 2020, we conducted an epidemiological surveillance of COVID-19 in Gabon. Methods We led molecular investigations on nasopharyngeal and oropharyngeal samples from the 1161 first suspected cases of COVID-19. We diagnosed the first case of COVID-19 on March, 12 2020. Results Among those suspected cases, 83 were confirmed cases. There was no significant difference in prevalence of SARS-CoV-2 between age groups (p=0.14). 73% were asymptomatic. The viral loads were significantly higher in the nasopharyngeal samples than in the oropharyngeal samples (p=0.03). There was no significant difference in viral loads between age groups (p=0.9895) and no correlation between clinical symptoms and viral loads (p=0.06042). A phylogenetic analysis performed with five sequences of the spike S gene showed that two sequences had the D614G mutation. Conclusion In conclusion, this study provides the first molecular data from Gabon concerning the COVID-19 pandemic. The data showed that most of the infected people were asymptomatic. The viral load was higher in the nasopharyngeal samples. The S gene analyzed suggested both introduction of the D614 and G614 variant in Gabon.

Screening and Whole Genome Sequencing of SARS-CoV-2 Circulating During the First Three Waves of the COVID-19 Pandemic in Libreville and the Haut-Ogooué Province in Gabon.

Since the onset of the COVID-19 pandemic, the SARS-CoV-2 viral dynamics in Africa have been less documented than on other continents. In Gabon, a Central African country, a total number of 37,511 cases of COVID-19 and 281 deaths have been reported as of December 8, 2021. After the first COVID-19 case was reported on March 12, 2020, in the capital Libreville, the country experienced two successive waves. The first one, occurred in March 2020 to August 2020, and the second one in January 2021 to May 2021. The third wave began in September 2021 and ended in November 2021. In order to reduce the data gap regarding the dynamics of SARS-CoV-2 in Central Africa, we performed a retrospective genotyping study using 1,006 samples collected from COVID-19 patients in Gabon from 2020 to 2021. Using SARS-CoV-2 variant screening by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and whole genome sequencing (WGS), we genotyped 809 SARS-CoV-2 samples through qRT-PCR and identified to generated 291 new genomes. It allowed us to describe specific mutations and changes in the SARS-CoV-2 variants in Gabon. The qRT-PCR screening of 809 positive samples from March 2020 to September 2021 showed that 119 SARS-CoV-2 samples (14.7%) were classified as VOC Alpha (Pangolin lineage B.1.1.7), one (0.1%) was a VOC Beta (B.1.351), and 198 (24.5 %) were VOC Delta (B.1.617.2), while 491 samples (60.7%) remained negative for the variants sought. The B1.1 variant was predominant during the first wave while the VOC Alpha dominated the second wave. The B1.617.2 Delta variant is currently the dominant variant of the third wave. Similarly, the analysis of the 291 genome sequences indicated that the dominant variant during the first wave was lineage B.1.1, while the dominant variants of the second wave were lineages B.1.1.7 (50.6%) and B.1.1.318 (36.4%). The third wave started with the circulation of the Delta variant (B.1.617). Finally, we compared these results to the SARS-CoV-2 sequences reported in other African, European, American and Asian countries. Sequences of Gabonese SARS-CoV-2 strains presented the highest similarities with those of France, Belgium and neighboring countries of Central Africa, as well as West Africa.