A methodology for deriving the sensitivity of pooled testing, based on viral load progression and pooling dilution

Abstract

Background Pooled testing, in which biological specimens from multiple subjects are combined into a testing pool and tested via a single test, is a common testing method for both surveillance and screening activities. The sensitivity of pooled testing for various pool sizes is an essential input for surveillance and screening optimization, including testing pool design. However, clinical data on test sensitivity values for different pool sizes are limited, and do not provide a functional relationship between test sensitivity and pool size. We develop a novel methodology to accurately compute the sensitivity of pooled testing, while accounting for viral load progression and pooling dilution. We demonstrate our methodology on the nucleic acid amplification testing (NAT) technology for the human immunodeficiency virus (HIV). Methods Our methodology integrates mathematical models of viral load progression and pooling dilution to derive test sensitivity values for various pool sizes. This methodology derives the conditional test sensitivity, conditioned on the number of infected specimens in a pool, and uses the law of total probability, along with higher dimensional integrals, to derive pooled test sensitivity values. We also develop a highly accurate and easy-to-compute approximation function for pooled test sensitivity of the HIV ULTRIO Plus NAT Assay. We calibrate model parameters using published efficacy data for the HIV ULTRIO Plus NAT Assay, and clinical data on viral RNA load progression in HIV-infected patients, and use this methodology to derive and validate the sensitivity of the HIV ULTRIO Plus Assay for various pool sizes. Results We demonstrate the value of this methodology through optimal testing pool design for HIV prevalence estimation in Sub-Saharan Africa. This case study indicates that the optimal testing pool design is highly efficient, and outperforms a benchmark pool design. Conclusions The proposed methodology accounts for both viral load progression and pooling dilution, and is computationally tractable. We calibrate this model for the HIV ULTRIO Plus NAT Assay, show that it provides highly accurate sensitivity estimates for various pool sizes, and, thus, yields efficient testing pool design for HIV prevalence estimation. Our model is generic, and can be calibrated for other infections.

Group testing for SARS-CoV-2 allows for up to 10-fold efficiency increase across realistic scenarios and testing strategies

“…43 Nevertheless, an appropriate quantitative understanding of pooling effects and viral load progression on the sensitivity is still an active discussion. 68 For a PCR sensitivity of 99%, we observe that the reduction caused by the use of a pooling method is very small (97% for D3, A2, and A1 3 ; 98% for D2 and A1 2 ). Only a single PCR procedure showed a low sensitivity of 90% when choosing a specific gene target (compared to 100% when choosing another target).…”

Evaluation of efficiency and sensitivity of 1D and 2D sample pooling strategies for SARS-CoV-2 RT-qPCR screening purposes

“…However, appraising the performance of a pooling method exclusively by its efficiency would ignore one of the major drawbacks of pooling: loss of sensitivity due to dilution of the target. This issue becomes most pertinent when the viral load is low [9][10][11]13,14 . Our results confirm that all tested pooling methods suffer from false negatives, to a variable degree (Figure 2).…”

Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools

“…In this method, first suggested by Dorfman in 1943 10 and perfected over the years [11][12][13] , samples are mixed and tested at a single pool, and subsequent individual tests are made only if the pool tests positive. In addition to being used in the clinic for infectious disease diagnostics in previous epidemics 14,15 , pooling has been proven to work for RT-qPCR 16,17 , a time-consuming step for which the reagents are expected to be in short supply 18 .…”