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Pooling RT-qPCR testing for SARS-CoV-2 in 1000 individuals of healthy and infection-suspected patients.

ABSTRACT: Severe acute respiratory coronavirus 2 (SARS-CoV-2) testing reagents are expected to become scarce worldwide. However, little is known regarding whether pooling of samples accurately detects SARS-CoV-2. To validate the feasibility of pooling samples, serial dilution analysis and spike-in experiments were conducted using synthetic DNA and nucleic acids extracted from SARS-CoV-2-positive and -negative patients. Furthermore, we studied 1000 individuals, 667 of whom were "healthy" individuals (195 healthcare workers and 472 hospitalized patients with disorders other than COVID-19 infection), and 333 infection-suspected patients with cough and fever. Serial dilution analysis showed a limit of detection of around 10-100 viral genome copies according to the protocol of the National Institute of Infectious Diseases, Japan. Spike-in experiments demonstrated that RT-qPCR detected positive signals in pooled samples with SARS-CoV-2-negative and -positive patients at 5-, 10-, 20-fold dilutions. By screening with this pooling strategy, by the end of April 2020 there were 12 SARS-CoV-2-positive patients in 333 infection-suspected patients (3.6%) and zero in 667 "healthy" controls. We obtained these results with a total of 538 runs using the pooling strategy, compared with 1000 standard runs. In a prospective study, we successfully detected SARS-CoV-2 using 10- to 20-fold diluted samples of nasopharyngeal swabs from eighteen COVID-19 patients with wide ranges of viral load. Pooling sample is feasible for conserving test reagents and detecting SARS-CoV-2 in clinical settings. This strategy will help us to research the prevalence infected individuals and provide infected-status information to prevent the spread of the virus and nosocomial transmission.

SUBMITTER: Hirotsu Y

PROVIDER: S-EPMC7641135 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Pooling RT-qPCR testing for SARS-CoV-2 in 1000 individuals of healthy and infection-suspected patients.

Hirotsu Yosuke Y Maejima Makoto M Shibusawa Masahiro M Nagakubo Yuki Y Hosaka Kazuhiro K Amemiya Kenji K Sueki Hitomi H Hayakawa Miyoko M Mochizuki Hitoshi H Tsutsui Toshiharu T Kakizaki Yumiko Y Miyashita Yoshihiro Y Omata Masao M

Scientific reports 20201103 1

Severe acute respiratory coronavirus 2 (SARS-CoV-2) testing reagents are expected to become scarce worldwide. However, little is known regarding whether pooling of samples accurately detects SARS-CoV-2. To validate the feasibility of pooling samples, serial dilution analysis and spike-in experiments were conducted using synthetic DNA and nucleic acids extracted from SARS-CoV-2-positive and -negative patients. Furthermore, we studied 1000 individuals, 667 of whom were "healthy" individuals (195 h ...[more]

PMID: 33144632

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Project description:BackgroundThe extent to which children and adolescents contribute to SARS-CoV-2 transmission remains not fully understood. Novel high-capacity testing methods may provide real-time epidemiological data in educational settings helping to establish a rational approach to prevent and minimize SARS-CoV-2 transmission. We investigated whether pooling of samples for SARS-CoV-2 detection by RT-qPCR is a sensitive and feasible high-capacity diagnostic strategy for surveillance of SARS-CoV-2 infections in schools.MethodsIn this study, students and school staff of 14 educational facilities in Germany were tested sequentially between November 9 and December 23, 2020, two or three times per week for at least three consecutive weeks. Participants were randomized for evaluation of two different age adjusted swab sampling methods (oropharyngeal swabs or buccal swabs compared to saliva swabs using a 'lolli method'). Swabs were collected and pooled for SARS-CoV-2 RT-qPCR. Individuals of positive pooled tests were retested by RT-qPCR the same or the following day. Positive individuals were quarantined while the SARS-CoV-2 negative individuals remained in class with continued pooled RT-qPCR surveillance. The study is registered with the German Clinical Trials register (registration number: DRKS00023911).Findings5,537 individuals were eligible and 3970 participants were enroled and included in the analysis. In students, a total of 21,978 swabs were taken and combined in 2218 pooled RT-qPCR tests. We detected 41 positive pooled tests (1·8%) leading to 36 SARS-CoV-2 cases among students which could be identified by individual re-testing. The cumulative 3-week incidence for primary schools was 564/100,000 (6/1064, additionally 1 infection detected in week 4) and 1249/100,000 (29/2322) for secondary schools. In secondary schools, there was no difference in the number of SARS-CoV-2 positive students identified from pooled oropharyngeal swabs compared to those identified from pooled saliva samples (lolli method) (14 vs. 15 cases; 1·3% vs. 1·3%; OR 1.1; 95%-CI 0·5-2·5). A single secondary school accounted for 17 of 36 cases (47%) indicating a high burden of asymptomatic prevalent SARS-CoV-2 cases in the respective school and community.InterpretationIn educational settings, SARS-CoV-2 screening by RT-qPCR-based pooled testing with easily obtainable saliva samples is a feasible method to detect incident cases and observe transmission dynamics.FundingFederal Ministry of education and research (BMBF; Project B-FAST in "NaFoUniMedCovid19"; registration number: 01KX2021).

Dataset Information

Pooling RT-qPCR testing for SARS-CoV-2 in 1000 individuals of healthy and infection-suspected patients.

Publications

Pooling RT-qPCR testing for SARS-CoV-2 in 1000 individuals of healthy and infection-suspected patients.

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