An Observational Laboratory-Based Assessment of SARS-CoV-2 Molecular Diagnostics in Benin, Western Africa

Abstract

Information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread in Africa is limited by insufficient diagnostic capacity. Here, we assessed the coronavirus disease (COVID-19)-related diagnostic workload during the onset of the pandemic in the central laboratory of Benin, Western Africa; characterized 12 SARS-CoV-2 genomes from returning travelers; and validated the Da An RT-PCR-based diagnostic kit that is widely used across Africa. We found a 15-fold increase in the monthly laboratory workload due to COVID-19, dealt with at the cost of routine activities. Genomic surveillance showed near-simultaneous introduction of distinct SARS-CoV-2 lineages termed A.4 and B.1, including the D614G spike protein variant potentially associated with higher transmissibility from travelers from six different European and African countries during March-April 2020. We decoded the target regions within the ORF1ab and N genes of the Da An dual-target kit by MinION-based amplicon sequencing. Despite relatively high similarity between SARS-CoV-2 and endemic human coronaviruses (HCoVs) within the ORF1ab target domain, no cross-detection of high-titered cell culture supernatants of HCoVs was observed, suggesting high analytical specificity. The Da An kit was highly sensitive, detecting 3.2 to 9.0 copies of target-specific in vitro transcripts/reaction. Although discrepant test results were observed in low-titered clinical samples, clinical sensitivity of the Da An kit was at least comparable to that of commercial kits from affluent settings. In sum, virologic diagnostics are achievable in a resource-limited setting, but unprecedented pressure resulting from COVID-19-related diagnostics requires rapid and sustainable support of national and supranational stakeholders addressing limited laboratory capacity.IMPORTANCE Months after the start of the COVID-19 pandemic, case numbers from Africa are surprisingly low, potentially because the number of SARS-CoV-2 tests performed in Africa is lower than in other regions. Here, we show an overload of COVID-19-related diagnostics in the central laboratory of Benin, Western Africa, with a stagnating average number of positive samples irrespective of daily sample counts. SARS-CoV-2 genomic surveillance confirmed a high genomic diversity in Benin introduced by travelers returning from Europe and other African countries, including early circulation of the D614G spike mutation associated with potentially higher transmissibility. We validated a widely used RT-PCR kit donated by the Chinese Jack Ma Foundation and confirmed high analytical specificity and clinical sensitivity equivalent to tests used in affluent settings. Our assessment shows that although achievable in an African setting, the burden from COVID-19-related diagnostics on national reference laboratories is very high.

Keywords: Benin; COVID-19; RT-PCR; SARS-CoV-2; West Africa; coronavirus.

FIG 1

Africa’s development status and COVID-19 workload at the reference laboratory of Benin. (A) Total number of SARS-CoV-2 tests performed relative to the size of population, received as freely available data from https://ourworldindata.org/coronavirus-testing#the-scale-of-testing-compared-to-the-scale-of-the-outbreak (accessed 14 September 2020). The newest entry for each country was selected, and countries were allocated to defined regions. Bars represent mean tests performed for the region. (B) Regional human development indices. Longevity (orange), income (red), and education (gray) indices and the human development index (HDI) (blue) as the geometric mean of the three aforementioned indices are shown. Data were obtained as freely available data from http://hdr.undp.org/en/data, accessed 15 June 2020. (C) Population density map constructed using freely available data from https://www.worldpop.org/doi/10.5258/SOTON/WP00004 (accessed 9 June 2020). Benin (BJ) is shown below. BF, Burkina Faso; TG, Togo; NG, Nigeria. The red arrow denotes the location of the Laboratoire des Fièvres Hémorragiques Virales du Benin (LFHB) in Cotonou. (D) COVID-19 workload at the LFHB. Overall SARS-CoV-2 daily diagnostic requests received at the LFHB until 28 April 2020 (black) and positive cases confirmed per day at the LFHB (red). Dotted lines denote the range of maximal daily diagnostic capacity of LFHB. Marked is 14 March 2020, the day of the first confirmed SARS-CoV-2 case in Benin.

FIG 2

Phylogenetic analyses of SARS-CoV-2 in Africa and molecular diagnostic test validation. (A) SARS-CoV-2 sequence entries from Africa and affluent settings in GISAID on 10 November 2020. (B) Phylogenetic tree inferred using BEAST2 showing 23 complete SARS-CoV-2 genomes globally sampled from humans. Posterior support is shown for nodes >0.90 as filled circles. Benin-derived sequences are shown in bold. Only sequences without missing information were used for this analysis. Sequences are designated with GISAID accession IDs/country of origin/lineage according to reference . (C) Alignment showing all variable sites across Benin derived SARS-CoV-2 genomes from this study. Empty spaces indicate lack of sequence information. Red boxes denote variable nucleotide positions in at least three or more sequences. Gray boxes denote groups belonging to lineage A.4 or B.1 according to the work of Rambaut et al. (7). Asterisks indicate nonsynonymous substitutions. Nucleotide positions correspond to the SARS-CoV-2 reference genome (GenBank accession number MN908947). BEL, Belgium; BFA, Burkina Faso; COD, Democratic Republic of the Congo; FRA, France; ITA, Italy; TOG, Togo; NSP, nonstructural protein; ORF, open reading frame. (D) Nucleotide (nt) identity plot within the ORF1ab (left) and N (right) genes between SARS-CoV-2 and endemic HCoVs. Top, schematic representation of the ORF1ab and N gene. Highlighted are the locations of the ORF1ab and N gene target regions of the Da An kit. (E) Alignments showing the ORF1ab (top) and N (bottom) gene target regions of SARS-CoV-2 and endemic HCoVs. Shown are the 100% consensus sequences of all sequences in the final data sets of each HCoV. nt, nucleotide. (F) Analytical sensitivity of the Da An real-time RT-PCR kit for both assays using target region-specific IVTs. The solid line shows predicted proportion of positive results at a given input; colored lines show the 95% CI. The genome illustration highlights the target genes of the assays. CI, confidence interval; LOD, lower limit of detection. (G) SARS-CoV-2 assay comparison using clinical samples.