A method for campus-wide SARS-CoV-2 surveillance at a large public university

The systematic screening of asymptomatic and pre-symptomatic individuals is a powerful tool for controlling community transmission of infectious disease on college campuses. Faced with a paucity of testing in the beginning of the COVID-19 pandemic, many universities developed molecular diagnostic laboratories focused on SARS-CoV-2 diagnostic testing on campus and in their broader communities. We established the UC Santa Cruz Molecular Diagnostic Lab in early April 2020 and began testing clinical samples just five weeks later. Using a clinically-validated laboratory developed test (LDT) that avoided supply chain constraints, an automated sample pooling and processing workflow, and a custom laboratory information management system (LIMS), we expanded testing from a handful of clinical samples per day to thousands per day with the testing capacity to screen our entire campus population twice per week. In this report we describe the technical, logistical, and regulatory processes that enabled our pop-up lab to scale testing and reporting capacity to thousands of tests per day.

Diagnostic Performance of RT-PCR-Based Sample Pooling Strategy for the Detection of SARS-CoV-2

Background: The rapid spread of SARS-CoV-2 has created a shortage of supplies of reagents for its detection throughout the world, especially in Latin America. The pooling of samples consists of combining individual patient samples in a block and analyzing the group as a particular sample. This strategy has been shown to reduce the burden of laboratory material and logistical resources by up to 80%. Therefore, we aimed to evaluate the diagnostic performance of the pool of samples analyzed by RT-PCR to detect SARS-CoV-2.Methods: A cross-sectional study of diagnostic tests was carried out. We individually evaluated 420 samples, and 42 clusters were formed, each one with ten samples. These clusters could contain 0, 1 or 2 positive samples to simulate a positivity of 0, 10 and 20%, respectively. RT-PCR analyzed the groups for the detection of SARS-CoV-2. The area under the ROC curve (AUC), the Youden index, the global and subgroup Sensitivity and specificity were calculated according to their Ct values that were classified as high (H: ≤25), moderate (M: 26-30) and low (L: 31-35) concentration of viral RNA.Results: From a total of 42 pools, 41 (97.6%) obtained the same result as the samples they contained (positive or negative). The AUC for pooling, Youden index, sensitivity, and specificity were 0.98 (95% CI, 0.95-1); 0.97 (95% CI, 0.90-1.03); 96.67% (95% CI; 88.58-100%) and 100% (95% CI; 95.83-100%) respectively. In the stratified analysis of the pools containing samples with CT <31, the Sensitivity was 100% (95% CI; 90-100%), while with the pools containing samples with Ct ≥31, the Sensitivity was 80% (95% CI, 34.94% - 100%). Finally, a median greater than 2.32 (IQR: 1.12 - 3.03) in the Ct was observed in the pools concerning the Ct of the individual samples (p <0.001).Conclusions: The strategy of pooling nasopharyngeal swab samples for analysis by SARS-CoV-2 RT-PCR showed high diagnostic performance.

Highly-specific early detection of colorectal cancer by novel non-invasive serum methylation biomarkers

Early detection has proven to be the most effective strategy to reduce the incidence and mortality of colorectal cancer (CRC). Nevertheless, most current screening programs suffer from low participation rates. A blood test may improve both the adherence to screening and the selection to colonoscopy. In this study, we conducted a serum-based discovery and validation of cfDNA methylation biomarkers for CRC screening in a multicentre cohort of 453 serum samples including healthy controls, benign pathologies, advanced adenomas (AA), and CRC. First, we performed an epigenome-wide methylation analysis with the MethylationEPIC array using a sample pooling approach, followed by a robust prioritization of candidate biomarkers for the detection of advanced neoplasia (AN: AA and CRC). Then, candidate biomarkers were validated by pyrosequencing in independent individual cfDNA samples. We report GALNT9, UPF3A, WARS, and LDB2 as new non-invasive biomarkers for the early detection of AN. The combination of GALNT9/UPF3A by logistic regression discriminated AN with 78.8% sensitivity and 100% specificity, outperforming the commonly used fecal immunochemical test and the methylated SEPT9 blood test. Overall, our results suggest that the combination methylated GALNT9/UPF3A has the potential to serve as a highly specific and sensitive blood-based test for screening and early detection of CRC.

Evaluation of Sample Pooling for SARS-CoV-2 Detection in Nasopharyngeal Swab and Saliva Samples with the Idylla SARS-CoV-2 Test

To control outbreaks of coronavirus disease 2019 (COVID-19) and to avoid reagent shortages, testing strategies must be adapted and maintained for the foreseeable future. We analyzed the feasibility of pooling NPS and saliva samples for SARS-CoV-2 testing with the Idylla SARS-CoV-2 test, and we found that sensitivity was dependent on the pool size.

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

Immediate pools of malaria infections at diagnosis combined with targeted deep sequencing accurately quantifies frequency of drug resistance mutations

Antimalarial resistance surveillance in sub-Saharan Africa is often constrained by logistical and financial challenges limiting its breadth and frequency. At two sites in Ghana, we have piloted a streamlined sample pooling process created immediately by sequential addition of positive malaria cases at the time of diagnostic testing. This streamlined process involving a single tube minimized clinical and laboratory work and provided accurate frequencies of all known drug resistance mutations after high-throughput targeted sequencing using molecular inversion probes. Our study validates this method as a cost-efficient, accurate and highly-scalable approach for drug resistance mutation monitoring that can potentially be applied to other infectious diseases such as tuberculosis.

Pooling Nasopharyngeal Swab Specimens to Improve Testing Capacity for SARS-CoV-2 by Real-Time RT-PCR

Background The detection of SARS-CoV-2 using qRT-PCR with the pooling of samples can reduce workload and costs especially when the prevalence rate of COVID-19 in a population is low. To analyse the effect of pooling samples on the sensitivity of RT-qPCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we compared the cycle threshold (Ct) values of pools of 5 and 10 that tested positive with Ct values of individual samples that tested positive in that pool. Twenty positive nasopharyngeal (NP) specimens with low to high viral load were selected and pooled individually with four and nine negative NP. Results In NP specimens, the sensitivity of pools of 5 and 10 were 90 and 85%, compared to individual sample testing, respectively. The RT-qPCR sensitivity of pools of 5 and 10 against individual testing were not significantly different (p > 0.05). Detection of positive samples with low Ct values (< 36) was consistently achieved in pools of 5 and 10. However, there were higher false negatives when samples with high ct values (> 36) were pooled and tested. The mean Ct values obtained with the 5-sample pooled testing exceeded individual sample testing by 1.85 ± 1.09 cycles, while Ct values obtained with the 10-sample pooling exceeded individual sample testing by 3.4 ± 1.65 cycles. Conclusions In a low prevalence setting, testing capacity can be increased by pooling 5 or 10 samples, but the risk of additional false negatives needs to be considered

Optimised multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

Objective: Rapid, cost-effective identification of genetic variants in small candiate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. Application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We have developed a multiplexing assay which pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals which could be applied to gene-targeted diagnostics. Methods: A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons, spanning 30 loci in DNA isolated from 82 neurodevelopmental cases and family members. The target regions were chosen for further interegation because a potentially disease-causative variants had been identified in affected individuals by Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the guppy aligner software. Results: Our multiplexing approach produced interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interstingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (139x - 21,499x; mean = 9,050, std err = 538.21) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. Conclusion: We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 140x depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory.

Implementation of an efficient SARS-CoV-2 specimen pooling strategy for high throughput diagnostic testing

The rapid identification and isolation of infected individuals remains a key strategy for controlling the spread of SARS-CoV-2. Frequent testing of populations to detect infection early in asymptomatic or presymptomatic individuals can be a powerful tool for intercepting transmission, especially when the viral prevalence is low. However, RT-PCR testing—the gold standard of SARS-CoV-2 diagnosis—is expensive, making regular testing of every individual unfeasible. Sample pooling is one approach to lowering costs. By combining samples and testing them in groups the number of tests required is reduced, substantially lowering costs. Here we report on the implementation of pooling strategies using 3-d and 4-d hypercubes to test a professional sports team in South Africa. We have shown that infected samples can be reliably detected in groups of 27 and 81, with minimal loss of assay sensitivity for samples with individual Ct values of up to 32. We report on the automation of sample pooling, using a liquid-handling robot and an automated web interface to identify positive samples. We conclude that hypercube pooling allows for the reliable RT-PCR detection of SARS-CoV-2 infection, at significantly lower costs than lateral flow antigen (LFA) tests.

Implementation of Sample Pooling Procedure Using a Rapid SARS-CoV-2 Diagnostic Real-Time PCR Test Performed Prior to Hospital Admission of People with Intellectual Disabilities

Reliability, accuracy, and timeliness of diagnostic testing for SARS-CoV-2 infection have allowed adequate public health management of the disease, thus notably helping the timely mapping of viral spread within the community. Furthermore, the most vulnerable populations, such as people with intellectual disability and dementia, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions, lower health maintenance, and a propensity for rapid community spread. This led to an urgent need for reliable in-house rapid testing to be performed prior to hospital admission. In the present study, we describe a pooling procedure in which oropharyngeal and nasopharyngeal swabs for SARS-CoV-2 detection (performed prior to hospital admission using rapid RT-PCR assay) are pooled together at the time of sample collection. Sample pooling (groups of 2–4 samples per tube) allowed us to significantly reduce response times, consumables, and personnel costs while maintaining the same test sensitivity.