S-variant SARS-CoV-2 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-QPCR

Abstract

Birmingham University Turnkey laboratory is part of the Lighthouse network responsible for testing clinical samples under the UK government ‘Test & Trace’ scheme. Samples are analysed for the presence of SARS-CoV-2 in respiratory samples using the Thermofisher TaqPath RT-QPCR test, which is designed to co-amplify sections of three SARS-CoV-2 viral genes.Since more recent information became available regarding the presence of SARS-CoV-2 variants of concern (S-VoC), which can show a suboptimal profile in RT-QPCR tests such as the ThermoFisher TaqPath used at the majority of Lighthouse laboratories, we analysed recently published data for trends and significance of the S-gene ‘dropout’ variant.Results showed that:the population of S-gene dropout samples had significantly lower median Ct values of ORF and N-gene targets compared to samples where S-gene was detectedon a population basis, S-gene dropout samples clustered around very low Ct values for ORF and N targetslinked Ct values for individual samples showed that a low Ct for ORF and N were clearly associated with an S-dropout characteristicwhen conservatively inferring relative viral load from Ct values, approximately 35% of S-dropout samples had high viral loads between 10 and 10,000-fold greater than 1 × 106, compared to 10% of S-positive samples.This analysis suggests that patients whose samples exhibit the S-dropout profile in the TaqPath test are more likely to have high viral loads at the time of sampling. The relevance of this to epidemiological reports of fast spread of the SARS-CoV-2 in regions of the UK is discussed.

Clinical, Serological, Whole Genome Sequence Analyses to Confirm SARS-CoV-2 Reinfection in Patients From Mumbai, India

“…Recent detection of SARS-CoV-2 variants has raised important questions about the impact of S gene mutations and deletions on increased transmissibility, ACE-2 receptor affinity, viral loads, immune escape, and severity. S variants of SARS-CoV-2 have been associated with significantly lower median Ct values suggesting that changes in the S protein RBD may result in increased viral loads (30). While our sample size and absence of viral culture studies does not allow us to make determinations about the impact of S gene mutations and deletions on clinical severity and viral load, it is possible that mutations at the Spike gene may explain lower Ct values and increased severity in the second episodes.…”

Modelling pooling strategies for SARS-CoV-2 testing in a university setting

“…It is also of note that the new Variant of Concern 202012/01 (VOC), which is thought to have originated in the UK in late Summer or early Autumn 2020, and which has spread rapidly in the UK since then (even during the November lockdown), is estimated to be between 1.4 and 1.8 times more transmissible than the original (wild-type) SARS-CoV-2 36 . Early results suggest that this new variant is associated with significantly higher viral loads in the upper respiratory tract, than was the case with wild-type SARS-CoV-2 37 . If a higher viral load is maintained across the infection course, then all tests will gain in sensitivity, and adopting pooling approaches (or the LFD) will have a reduced adverse impact on sensitivity when compared against the single RT-qPCR test.…”

Rapid diagnostics of novel coronavirus infection by loop-mediated isothermal amplification

“…В связи с тем что суммарная область покрытия праймерами существенно больше, чем при классической ПЦР, заметно выше становится и вероятность появления мутаций в местах посадки, особенно у РНК-вирусов, к которым относится и SARS-CoV-2. Это явление может привести к меньшей эффективности отжига праймеров с получением ложнонегативных результатов [51]. Поэтому необходимо постоянно отслеживать появление новых мутационных изменений в геномах исследуемых патогенов, чтобы своевременно корректировать ситуацию посредством замены олигонуклеотидов либо введением так называемых вырождений.…”

“…Далее, создана тест-система на основе RT-LAMP для определения одновременно 2 генов SARS-CoV-2 (S и RdRP), имеющая параметры аналитической чувствительности и специфичности, предел обнаружения и точность, сравнимые с таковыми ОТ-ПЦР [62]. Такой протокол, использующий сразу 2 мишени, может быть особенно востребован с учётом постоянно появляющихся новых мутаций возбудителя, потенциально способных привести к снижению эффективности некоторых тестов (что уже произошло с рядом диагностикумов вследствие «британской мутации») [51]. Наконец, описан ещё один подход с мультиплексированием RT-LAMP и проверена его чувствительность [63].…”