Pooled RT-qPCR testing for SARS-CoV-2 surveillance in schools - a cluster randomised trial

Abstract

Background: The 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.

Methods: In 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).

Findings: 5,537 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.

Interpretation: In 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.

Funding: Federal Ministry of education and research (BMBF; Project B-FAST in "NaFoUniMedCovid19"; registration number: 01KX2021).

Keywords: Covid-19; Pooled testing; RT-qPCR; SARS-CoV-2; School; Surveillance.

Fig. 1

CONSORT chart for students by randomised groups. Students were randomised into the three factors sampling technique (saliva swabs using the ‘lolli method’ vs. oropharyngeal or buccal swabs), testing frequency (two vs. three times per week), and number of pooled tests per class (one pooled test containing all swabs of a class vs. two pooled tests each containing half of the swabs taken in the respective class) using a 2 × 2 × 2 incomplete block design. See Fig. 4b for staff participation and testing.

Fig. 2

SARS-CoV-2 incidence at study sites and study design. a. Number of SARS-CoV-2 infections/100,000 inhabitants in the participating cities/counties from calendar week 14–53 in 2020. The B-FAST study period is highlighted in grey. b. B-FAST study design in which swabs were taken from an entire class several times per week (Monday, Wednesday and Friday (shown) or Monday and Thursday). Swabs were pooled and tested by RT-qPCR. Detection of a positive pooled test (red dot) triggered individual testing of the entire class by RT-qPCR and subsequent identification of a single or several positive individuals who were quarantined. The remaining students continued with school lessons and scheduled pooled testing (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 3

Overall study results for students. A total of 21,978 swabs were taken and combined in 2218 pooled RT-qPCR tests. We detected 41 positive pooled tests leading to 36 SARS-CoV-2 cases which could be identified by individual re-testing.

Fig. 4

Selected study results for sampling method in students and staff. a. Flow diagram showing selected study results for one of three randomised factors which tested for three age-adjusted swab sampling methods (oropharyngeal swabs, buccal swabs and saliva swabs (lolli method). Number of eligible and enroled students and study results for pooled and individual RT-qPCR testing. (yo: years old). For CONSORT flow diagram of the entire study see Fig. 1. b. Number of eligible and enroled staff members and study results for pooled and individual RT-qPCR testing.

Fig. 5

Positivity rate dependent on sampling method and incidence rates. a. Rate of positive students identified with different sampling methods used for pooled testing in total numbers and percentage. Lolli method: red pie; buccal swab: yellow pie; oropharyngeal swab: blue pie. b. Number of SARS-CoV-2 positive students (right Y-axis) and incidence rate (left Y-axis, per 100,000 individuals) per week in secondary and primary schools. Note that week 1 contains asymptomatic prevalent individuals many of whom were identified on the first day of testing. Secondary schools: blue bars; primary schools white bars. Incidence rate: hatched bars; cases: open bars. c. Cumulative incidence (per 100,000; three-week period) identified in a high burden school situated in a city district with lower socioeconomic status (secondary school 4, blue bar), a low burden school (secondary school 5, white bar), in the entire study (all 14 schools, light grey bar), the general population of the respective Cologne district of secondary school 4 (medium grey bar), the general population of the city of Cologne (dark grey bar) and the entire country (Germany, black bar) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).