Evaluation of Sars-cov-2 molecular assays and circulating variants in Cameroon

The COVID-19 pandemic occurred in March 2020 and has since spread across countries and territories, causing over 5 million deaths and raising serious public health concerns. SARS-CoV-2 testing is a weapon for ensuring appropriate clinical care for patients and for contact tracing to break the chains of transmission. However, Sub-Saharan Africa still faces low capacity to test and reporting results. The management and control of the pandemic depends, essentially, on the performance and quality of diagnostic tools and on genomic surveillance of the insurgence and circulation of SARS-CoV-2 variants. Thus, in order to optimize the response to this outbreak in the field, we aimed to evaluate the diagnostic performance of molecular assays widely used in Cameroon and to determine the molecular epidemiology of the circulating virus in our context. Methods This was a laboratory-based study conducted at the Chantal BIYA International Reference Centre (CIRCB) in Yaounde-Cameroon. Firstly, a comparative study was conducted on nasopharyngeal samples tested in parallel with Abbott and DaAn gene rRT-PCR. The performance characteristics were evaluated by Cohen's coefficient and Spearman’s correlation. Secondly, a study comparing a rapid reverse transcription loop-mediated isothermal amplification PCR (RT-LAMP) assay and the Abbott rRT-PCR was conducted on 366 nasopharyngeal samples. The sensitivity and specificity of RT-LAMP were determined at 30 minutes (T30) and 45 minutes (T45) relative to the Abbott RT-PCR as a reference test. The Cohen’s Kappa coefficient (k) was used to measure inter-rater reliability. Thirdly, a study was conducted on 125 nasopharyngeal specimens from COVID-19 positive individuals diagnosed by PCR between April 2020 and October 2021. Partial sequences of the SARS-CoV-2 spike region were obtained using the Sanger sequencing method. Descriptive statistics were performed for socio-demographic and biological data. The generated sequences were analysed for mutations using Stanford University database. Results Diagnostic Concordance between a manual and automated assay for the molecular diagnosis of SARS-CoV-2 in Cameroon: A total of 273 participants (median age [IQR] 36 [26-46] years and 14 EQA specimens were included in the study. The overall agreement was 82.6% (237/287), with k=0.82 (95% CI: 0.777-0.863), indicating an excellent diagnostic agreement. Regarding viral load (VL), positive agreement was 100% for samples with high VLs (CT<20). The agreement (>80%) between the Abbott and DaAn gene rRT-PCR platforms supports interoperability between the two assays. Diagnostic performance of a rapid reverse transcription loop-mediated isothermal amplification PCR (RT-LAMP) assay compared to the Abbott rRT-PCR: The overall sensitivity and specificity of RT-LAMP at T30 were 83.4% (95% CI: 81.5-83.5) and 100% (95% CI: 97.7-100.0), respectively. The overall sensitivity and specificity of RT-LAMP at T45 were 86.1% (95% CI: 83.1-88.0) and 93.7% (95% CI: 89.1-96.7), respectively. Good sensitivity 97.67% (95% CI: 93-100) was observed in samples with Abbott RT-PCR cycle number CN value <25, but significantly decreased 63% (95% CI: 53-73) in samples with CN value >25; indicating that the sensitivity of the assay decreases with low quantity of viral RNA. RT-LAMP appears to be a reliable test, comparable to RT-PCR, especially at T30 and in patients with high viral loads (Ct < 25). Evaluation of circulating variants in Cameroon: A total of 99 (79.2%) samples were successfully sequenced using Sanger method. According to the Stanford database analyses, variants of concern were found in 48 (48.97%) of 98 samples, the Delta variant (B.1.617.2) was found in 33 (33.67%) of 98 samples, 11 (11.22%) of 98 samples had Alpha variant (B.1.1.7), 3 (3.06%) had the Beta variant (B.1.351) and 1 (1.02%) of 98 samples had Gamma variant (P.1). No mutations were identified by the database in one sequence. According to disease wave, during the first wave of the pandemic, the predominant circulating virus was from the lineage of origin (wild type) 96.87% (31/32). During the second wave, the wild type 50% (16/32) and Alpha variant 31.25% (10/32) were the most present, while in the third wave, the Delta variant was highly dominant 97.14% (34/35). The D614G mutation was the most prevalent, indicating the increased risk of disease infectivity during all three waves of the pandemic. The study population was mainly asymptomatic, suggesting a reduced severity of the disease despite the emergence of new variants. Our findings highlight a significant presence of SARS-CoV-2 variants of concern (VOC) (Delta, Alpha, Beta and Gamma variants) in the Cameroonian context, which greatly vary across disease waves. Conclusion The overall outcomes of this thesis provide excellent agreement between the Abbott and DaAn gene rRT-PCR platforms, supporting interoperability between the two assays. Moreover, the reliability of RT-LAMP compared to rRT-PCR, this test can therefore be deployed in a real-life setting for rapid management and prevention of COVID-19 without compromising the quality of the results and finally, the third study reveals a significant presence of SARS-CoV-2 variants of concern (Delta, Alpha, Beta Gamma) in the Cameroonian population which greatly vary across disease waves. The D614G mutation was the most prevalent, indicating infectivity of the disease during these three phases of the pandemic. Interestingly, in a context of limited resources as in Cameroon, the possibility of using, when available, Abbott or DaAn gene PCR tests with accurate results, RT-LAMP in remote areas, and of having a means of genomic surveillance is a great advance in the fight against the spread of the pandemic on a national scale.