Pooled sample testing for COVID-19 diagnosis: Evaluation of bi-directional matrix pooling strategies

Abstract

In the on-going COVID-19 pandemic, pooled testing of samples by RT-PCR has been recommended at certain scenarios to increase labs' testing capacity and reduce cost of testing. This paper describes the evaluation of bi-directional matrix pooling strategies with clinical samples in a 5 × 5 and 10 × 10 matrix. Nasopharyngeal swab samples in viral transport medium (VTM) previously tested (positive or negative) by real time RT-PCR for SARS-CoV-2 were used for these experiments. Ten sets of 5 × 5 (250 samples) and ten sets of 10 × 10 (1000 samples) pooling of samples in both directions was done with known positive samples introduced at random positions. Extracted nucleic acid was tested for SARS-CoV-2 E-gene by RT-PCR. Sensitivity or concordance and feasibility of matrix pooling were assessed in comparison to direct RT-PCR testing. In comparison to direct testing, the overall concordance was 86.6% for 5 × 5 pooling, 73.3% for 10 × 10 with 200 µL extraction volume and 86.6% for 10 × 10 with 400 µL extraction volume. Bi-directional matrix pooling can be adopted with advantage over conventional direct or pool testing for COVID-19 by RT-PCR under the following conditions: i) sample positivity rate of ≤ 5%, ii) matrix pool size of 8-10 samples, iii) use of min. 40 µL VTM from each sample and iv) utilization of automated liquid handling equipment, if available, for sample addition to avoid human errors.

Keywords: COVID-19; Diagnosis; Pooling; RT-PCR; SARS-CoV-2; Screening.

Fig. 1

(a) 5×5 sample pool: A total of 25 samples were included in 5 × 5 matrix pool. Forty µL from each sample was added in two pools i.e row and column. e.g. 40 µL VTM from sample no. 1 was added to pool no. C1 and R1; Sample no.6 was added in pool no. C1 and R2, and so on. One to three positives were introduced at random positions in an operator- blinded manner; (b) 10 × 10 sample pool: A total of 100 samples were included in 10 × 10 pool. Twenty or Forty µL from each VTM was added in two pools. e.g. 40 µL VTM from Sample no. 1 was added in pool no. C1 and R1; Sample no.11 was added in pool no. C1 and R2, and so on. 1–3 positives were introduced at random positions in an operator-blinded manner [C- column, R-Row, numerical- sample nos.].

Fig. 2

Identification of intersects of positive pools in 5-sample matrix. (a) 1 × 1 = 1 intersect (red) directly identified as positive; (b) 2 × 2 = 4 intersects (yellow) to be tested to identify true positives; (c) 3 × 3 = 9 intersects (yellow) to be tested to identify true positives.

Fig. 3

Identification of intersects of positive pools in 10-sample matrix. 2 × 2 = 4 intersects (yellow) to be tested to identify true positives.