Assembly and publication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome in January 2020 enabled the immediate development of tests to detect the new virus. This began the largest global testing programme in history, in which hundreds of millions of individuals have been tested to date. The unprecedented scale of testing has driven innovation in the strategies, technologies and concepts that govern testing in public health. This Review describes the changing role of testing during the COVID-19 pandemic, including the use of genomic surveillance to track SARS-CoV-2 transmission around the world, the use of contact tracing to contain disease outbreaks and testing for the presence of the virus circulating in the environment. Despite these efforts, widespread community transmission has become entrenched in many countries and has required the testing of populations to identify and isolate infected individuals, many of whom are asymptomatic. The diagnostic and epidemiological principles that underpin such population-scale testing are also considered, as are the high-throughput and point-of-care technologies that make testing feasible on a massive scale.

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Testing at scale during the COVID-19 pandemic.

Large-scale implementation of pooled RNA extraction and RT-PCR for SARS-CoV-2 detection.

Suppressing infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will probably require the rapid identification and isolation of individuals infected with the virus on an ongoing basis. Reverse-transcription polymerase chain reaction (RT-PCR) tests are accurate but costly, which makes the regular testing of every individual expensive. These costs are a challenge for all countries around the world, but particularly for low-to-middle-income countries. Cost reductions can be achieved by pooling (or combining) subsamples and testing them in groups1-7. A balance must be struck between increasing the group size and retaining test sensitivity, as sample dilution increases the likelihood of false-negative test results for individuals with a low viral load in the sampled region at the time of the test8. Similarly, minimizing the number of tests to reduce costs must be balanced against minimizing the time that testing takes, to reduce the spread of the infection. Here we propose an algorithm for pooling subsamples based on the geometry of a hypercube that, at low prevalence, accurately identifies individuals infected with SARS-CoV-2 in a small number of tests and few rounds of testing. We discuss the optimal group size and explain why, given the highly infectious nature of the disease, largely parallel searches are preferred. We report proof-of-concept experiments in which a positive subsample was detected even when diluted 100-fold with negative subsamples (compared with 30-48-fold dilutions described in previous studies9-11). We quantify the loss of sensitivity due to dilution and discuss how it may be mitigated by the frequent re-testing of groups, for example. With the use of these methods, the cost of mass testing could be reduced by a large factor. At low prevalence, the costs decrease in rough proportion to the prevalence. Field trials of our approach are under way in Rwanda and South Africa. The use of group testing on a massive scale to monitor infection rates closely and continually in a population, along with the rapid and effective isolation of people with SARS-CoV-2 infections, provides a promising pathway towards the long-term control of coronavirus disease 2019 (COVID-19).

/pdf/a-pooled-testing-strategy-for-identifying-sars-cov-2-at-low-houuoxh1b4.pdf

A pooled testing strategy for identifying SARS-CoV-2 at low prevalence.

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, from antigen production to data analyses, that helps define thresholds for IgG and IgM seropositivity with high specificities. Validation of this protocol is performed using traditionally collected serum as well as dried blood on mail-in blood sampling kits. Archival (pre-2019) samples are used as negative controls, and convalescent, PCR-diagnosed COVID-19 patient samples serve as positive controls. Using this protocol, minimal cross-reactivity is observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses, and no cross reactivity is observed with anti-influenza A H1N1 HAI. Our protocol may thus help provide standardized, population-based data on the extent of SARS-CoV-2 seropositivity, immunity and infection.

/pdf/standardization-of-elisa-protocols-for-serosurveys-of-the-1l6q5xc7e8.pdf

Standardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling.

Systematic testing of large population groups by RT-PCR is mandatory to Covid-19 case identification and contact tracing in order to minimize the likelyhood of resurgence in contagion. Sample pooling for RT-PCR has been effectively used to detect community transmission of SARS CoV-2. Nevertheless, this procedure may decrease the sensitivity of RT-PCR assays due to specimen dilution. We evaluated the efficacy of this strategy for diagnosis of Covid-19 using a sensitive commercially-available RT-PCR targeting SARS CoV-2 E and RdRp genes in a single reaction. A total of 20 mini-pools containing either 5 (n=10) or 10 (n=10) nasopharyngeal exudates collected in universal transport medium were made, each of which including a unique positive NP specimen. Positive specimens yielding CT 35.8 for the E gene or 35.7 for the RdRP gene remained undetected in mini-pools of 5 specimens (3/4 and 2/3, respectively) or in mini-pools of 10 samples (4/4 and 3/3, respectively.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/jmv.25971

Pooling of nasopharyngeal swab specimens for SARS-CoV-2 detection by RT-PCR.

Recent reports suggest that 10 to 30% of severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) infected patients are asymptomatic and that viral shedding may occur before symptom onset. Therefore, there is an urgent need to increase diagnostic testing capabilities to prevent disease spread. We developed P-BEST, a method for Pooling-Based Efficient SARS-CoV-2 Testing, which identifies all positive subjects within a set of samples using a single round of testing. Each sample is assigned into multiple pools using a combinatorial pooling strategy based on compressed sensing. We pooled sets of 384 samples into 48 pools, providing both an eightfold increase in testing efficiency and an eightfold reduction in test costs, while identifying up to five positive carriers. We then used P-BEST to screen 1115 health care workers using 144 tests. P- BEST provides an efficient and easy-to-implement solution for increasing testing capacity that can be easily integrated into diagnostic laboratories.

Michal Goldhirsh

Papers

Efficient high-throughput SARS-CoV-2 testing to detect asymptomatic carriers.