A Low-Cost and High-Efficiency 10-in-1 Test for the PCR-Based Screening of SARS-CoV-2 Infection in Low-Risk Areas
The global impact of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been profound, with millions of infections and deaths reported worldwide. As of August 9, 2020, the virus had infected over 19 million people and caused more than 720,000 deaths globally. To curb the spread of the virus, timely identification of infected individuals through nucleic acid detection screenings has been critical, especially in high-risk areas such as Wuhan, Beijing, and Xinjiang. However, large-scale screening presents significant challenges, including high costs, resource consumption, and testing capacity limitations. To address these issues, a novel 10-in-1 test was developed, offering a low-cost and high-efficiency solution for SARS-CoV-2 screening in low-risk areas.
Challenges in Large-Scale Screening
Large-scale nucleic acid screening for SARS-CoV-2 is essential for identifying infected individuals and implementing quarantine measures to prevent transmission. However, this approach has several limitations. The consumption of equipment and reagents for specimen collection and detection is substantial, leading to elevated screening costs. Additionally, as the number of samples increases, the testing capabilities of medical institutions often reach saturation, hindering the speed and scope of screenings. To mitigate these challenges, pooling strategies have been proposed, such as mixing swab transfer buffers before nucleic acid extraction or pooling RNA after extraction. However, these methods inevitably dilute samples, reducing detection sensitivity. There is a lack of research on how to effectively improve screening efficiency while controlling the sensitivity loss caused by dilution.
Development of the 10-in-1 Test
To overcome these challenges, a novel 10-in-1 test was designed. This test involves pooling ten pharyngeal swab samples from ten individuals into a single custom-made virus collection tube (CMT) for nucleic acid extraction and testing. The CMT was optimized to accommodate ten swabs without causing inconvenience to the collector. The tube had an outer diameter of 14.8 ± 0.2 mm and a height of 100.5 ± 0.4 mm, containing 6 mL of preservation solution with guanidinium salt. This volume was double that of the individual acquisition tube (IAT), which contained 3 mL of the same solution. The pharyngeal swab used in the test was a polypropylene flocking swab with a head length of 2 cm, a diameter of 3 to 5 mm, and a breaking point 3 cm from the head.
Evaluation of the 10-in-1 Test
The study protocol was approved by the institutional review boards of Shengjing Hospital of China Medical University and Dalian Sixth People’s Hospital. Data from a nucleic acid screening in Dalian, which enrolled 7 million people, were used for evaluation. A total of 2.15 million subjects were included in the study, with 82,000 people used to evaluate the sampling tube usage experience and 640 people used to assess the consistency of the methods. Among the 640 individuals, 64 were confirmed COVID-19 cases, and 576 were volunteers who tested negative for the virus.
For the 10-in-1 test, pharyngeal swabs from 19 SARS-CoV-2-positive patients and asymptomatic volunteers were collected using IAT on the first day after hospital admission. Swabs from 171 SARS-CoV-2-negative volunteers were stored in a CMT (nine swabs per tube). Two hundred microliters of preservation solution from each IAT and CMT was collected, mixed, and subjected to RNA extraction. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the viral open reading frame 1ab (ORF1ab) and nucleocapsid protein (NP) gene fragments. The RT-PCR kits used were from Guangzhou Da’an Gene Biotechnology Co., Ltd. and Wuhan Mingde Biotechnology Co., Ltd., both with a detection limit of 500 copies/mL.
The cycle threshold (Ct) values for ORF1ab and NP gene amplicons from the IAT samples were 28.89 ± 5.21 and 29.02 ± 5.74, respectively. For the 10-in-1 simulation, where positive samples were mixed with nine negative samples, the Ct values were 30.00 ± 5.07 and 30.03 ± 5.56, respectively. The IAT samples were positive in all 19 cases, while the 10-in-1 simulation yielded 18 positive results, with one case showing an uncertain result (positive only for ORF1ab). The positive coincidence rate between IAT and the 10-in-1 simulation was 94.7%.
Real 10-in-1 Test Performance
To validate the 10-in-1 test in real-world conditions, samples were collected from 405 volunteers and 45 COVID-19-positive patients. Each volunteer provided two pharyngeal swabs: one for IAT and one for CMT (containing nine different swabs). The Ct values for ORF1ab and NP genes from IAT and CMT samples were consistent, with Kappa values of 0.863 (95% confidence interval [CI]: 0.7552, 0.9708) and 0.973 (95% CI: 1.02592, 0.92008) for the Da’an and Mingde kits, respectively. The Kappa value for 450 IAT samples tested using both kits was 0.856.
Large-Scale Screening in Low-Risk Areas
The 10-in-1 test was deployed in large-scale screenings in Dalian, Qingdao, and Shenyang, covering 2.15 million, 11 million, and 7.7 million people, respectively. No positive cases were identified in Dalian and Qingdao, and no new COVID-19 patients were diagnosed in the screened populations after the screenings were completed. In Shenyang, a single SARS-CoV-2-positive case was identified through the 10-in-1 test, which was later confirmed as an asymptomatic patient. These results demonstrated the accuracy and applicability of the 10-in-1 test for COVID-19 screening in low-risk areas.
Design Considerations and Future Directions
The development of the 10-in-1 test addressed three main challenges. First, the CMT was designed to accommodate ten pharyngeal swabs without causing inconvenience to collectors. The tube’s dimensions, swab material, and breaking point were optimized for safe and convenient sampling. Second, the volume of preservation fluid was increased to ensure adequate elution of up to ten swabs while maintaining assay accuracy. The two-fold dilution caused by the increased volume increased the Ct value by only one, preserving the test’s reliability. Third, the study initially lacked enough new cases for a real 10-in-1 test, so samples from treated patients were used. However, the high viral load in newly infected or asymptomatic patients suggests that the 10-in-1 test would perform even better in primary case screening.
Conclusion
The 10-in-1 test offers a low-cost and high-efficiency solution for large-scale SARS-CoV-2 screening in low-risk areas. By optimizing the pooling technique and addressing the challenges of sample dilution and resource consumption, this method enables the rapid and accurate identification of infected individuals. Future research will focus on further optimizing the pooling technique and exploring the suitability of pooling more samples for even larger-scale population screenings.
doi.org/10.1097/CM9.0000000000001627
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