Background: The COVID-19 pandemic relies on real-time polymerase chain reaction (qRT-PCR) for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to facilitate roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers' recommendations to sustain the testing capability in a resource-limited setting. Methods: We used a SARS-CoV-2 positive control RNA sample to generate several 10-fold dilution series that were used for optimization and comparison of the performance of the four qRT-PCR assays: i) Charité Berlin primer-probe set, ii) European Virus Archive - GLOBAL (EVAg) primer-probe set, iii) DAAN premixed commercial kit and iv) Beijing Genomics Institute (BGI) premixed commercial kit. We adjusted the manufacturer- and protocol-recommended reaction component volumes for these assays and assessed the impact on cycle threshold (Ct) values. Results: The Berlin and EVAg E gene and RdRp assays reported mean Ct values within range of each other across the different titrations and with less than 5% difference. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit improved in performance following a reduction of the reaction components. Conclusion: We achieved a 2.6-fold and 4-fold increase in the number of tests per kit for the commercial kits and the primer-probe sets, respectively. All the assays had optimal performance when the primers and probes were used at 0.375X, except for the Berlin N gene assay. The DAAN kit was a reliable assay for primary screening of SARS-CoV-2 whereas the BGI kit's performance was dependent on the volumes and concentrations of both the reaction buffer and enzyme mix. Our recommendation for SARS-CoV-2 diagnostic testing in resource-limited settings is to optimize the assays available to establish the lowest volume and suitable concentration of reagents required to produce valid results. Copyright:
Background: The COVID-19 pandemic relies on real-time polymerase chain reaction (qRT-PCR) for the detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), to facilitate roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers' recommendations to sustain the testing capability in a resource-limited setting. Methods: We used a SARS-CoV-2 positive control RNA sample to generate several 10-fold dilution series that were used for optimization and comparison of the performance of the four qRT-PCR assays: i) Charité Berlin primer-probe set, ii) European Virus Archive - GLOBAL (EVAg) primer-probe set, iii) DAAN premixed commercial kit and iv) Beijing Genomics Institute (BGI) premixed commercial kit. We adjusted the manufacturer- and protocol-recommended reaction component volumes for these assays and assessed the impact on cycle threshold (Ct) values. Results: The Berlin and EVAg E gene and RdRp assays reported mean Ct values within range of each other across the different titrations and with less than 5% difference. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit improved in performance following a reduction of the reaction components. Conclusion: We achieved a 2.6-fold and 4-fold increase in the number of tests per kit for the commercial kits and the primer-probe sets, respectively. All the assays had optimal performance when the primers and probes were used at 0.375X, except for the Berlin N gene assay. The DAAN kit was a reliable assay for primary screening of SARS-CoV-2 whereas the BGI kit's performance was dependent on the volumes and concentrations of both the reaction buffer and enzyme mix. Our recommendation for SARS-CoV-2 diagnostic testing in resource-limited settings is to optimize the assays available to establish the lowest volume and suitable concentration of reagents required to produce valid results. Copyright:
Authors: Charles N Agoti; Lynette Isabella Ochola-Oyier; Simon Dellicour; Khadija Said Mohammed; Arnold W Lambisia; Zaydah R de Laurent; John M Morobe; Maureen W Mburu; Donwilliams O Omuoyo; Edidah M Ongera; Leonard Ndwiga; Eric Maitha; Benson Kitole; Thani Suleiman; Mohamed Mwakinangu; John K Nyambu; John Otieno; Barke Salim; Jennifer Musyoki; Nickson Murunga; Edward Otieno; John N Kiiru; Kadondi Kasera; Patrick Amoth; Mercy Mwangangi; Rashid Aman; Samson Kinyanjui; George Warimwe; My Phan; Ambrose Agweyu; Matthew Cotten; Edwine Barasa; Benjamin Tsofa; D James Nokes; Philip Bejon; George Githinji Journal: Elife Date: 2022-06-14 Impact factor: 8.713
Authors: James Nyagwange; Leonard Ndwiga; Kelvin Muteru; Kevin Wamae; James Tuju; Covid Testing Team; Bernadette Kutima; John Gitonga; Henry Karanja; Daisy Mugo; Kadondi Kasera; Patrick Amoth; Nickson Murunga; Lawrence Babu; Edward Otieno; George Githinji; D J Nokes; Benjamin Tsofa; Benedict Orindi; Edwine Barasa; George Warimwe; Charles N Agoti; Philip Bejon; Lynette Isabella Ochola-Oyier Journal: Wellcome Open Res Date: 2022-02-23