ABSTRACT
Early in the pandemic, a simple, open-source, RNA extraction-free RT-qPCR protocol for SARS-CoV-2 detection in saliva was developed and made widely available. This simplified approach (SalivaDirect) requires only sample treatment with proteinase K prior to PCR testing. However, feedback from clinical laboratories highlighted a need for a flexible workflow that can be seamlessly integrated into their current health and safety requirements for the receiving and handling of potentially infectious samples. To address these varying needs, we explored additional pre-PCR workflows. We built upon the original SalivaDirect workflow to include an initial incubation step (95{degrees}C for 30 minutes, 95{degrees}C for 5 minutes or 65{degrees}C for 15 minutes) with or without addition of proteinase K. The limit of detection for the workflows tested did not significantly differ from that of the original SalivaDirect workflow. When tested on de-identified saliva samples from confirmed COVID-19 individuals, these workflows also produced comparable virus detection and assay sensitivities, as determined by RT-qPCR analysis. Exclusion of proteinase K did not negatively affect the sensitivity of the assay. The addition of multiple heat pretreatment options to the SalivaDirect protocol increases the accessibility of this cost-effective SARS-CoV-2 test as it gives diagnostic laboratories the flexibility to implement the workflow which best suits their safety protocols.
ABSTRACT
Genomic sequencing provides critical information to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments and vaccines, and guide public health responses. To investigate the spatiotemporal heterogeneity in the global SARS-CoV-2 genomic surveillance, we estimated the impact of sequencing intensity and turnaround times (TAT) on variant detection in 167 countries. Most countries submit genomes >21 days after sample collection, and 77% of low and middle income countries sequenced <0.5% of their cases. We found that sequencing at least 0.5% of the cases, with a TAT <21 days, could be a benchmark for SARS-CoV-2 genomic surveillance efforts. Socioeconomic inequalities substantially impact our ability to quickly detect SARS-CoV-2 variants, and undermine the global pandemic preparedness. One-Sentence SummarySocioeconomic inequalities impacted the SARS-CoV-2 genomic surveillance, and undermined the global pandemic preparedness.
ABSTRACT
BackgroundSARS-CoV-2 infections are characterized by viral proliferation and clearance phases and can be followed by low-level persistent viral RNA shedding. The dynamics of viral RNA concentration, particularly in the early stages of infection, can inform clinical measures and interventions such as test-based screening. MethodsWe used prospective longitudinal RT-qPCR testing to measure the viral RNA trajectories for 68 individuals during the resumption of the 2019-20 National Basketball Association season. For 46 individuals with acute infections, we inferred the peak viral concentration and the duration of the viral proliferation and clearance phases. FindingsAccording to our mathematical model, we found that viral RNA concentrations peaked an average of 3.3 days (95% credible interval [2.5, 4.2]) after first possible detectability at a cycle threshold value of 22.3 [20.5, 23.9]. The viral clearance phase lasted longer for symptomatic individuals (10.9 days [7.9, 14.4]) than for asymptomatic individuals (7.8 days [6.1, 9.7]). A second test within 2 days after an initial positive PCR substantially improves certainty about a patients infection phase. The effective sensitivity of a test intended to identify infectious individuals declines substantially with test turnaround time. ConclusionsSARS-CoV-2 viral concentrations peak rapidly regardless of symptoms. Sequential tests can help reveal a patients progress through infection stages. Frequent rapid-turnaround testing is needed to effectively screen individuals before they become infectious.
