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Figure.  Operating Characteristics of Pooled Testing Stratified by Prevalence
Operating Characteristics of Pooled Testing Stratified by Prevalence

k indicates the number of patients in a pool; and k0 (circles), the optimal pool size at which maximal cost-savings was achieved for various prevalence levels. These examples assumed a test sensitivity of 70%.

Table.  Optimal Pool Size and Corresponding Efficiency, Probability of False Negative, Savings, and Expected Number of False Negatives With Respect to Prevalence and Test Errors for a Pool of 94 Patients
Optimal Pool Size and Corresponding Efficiency, Probability of False Negative, Savings, and Expected Number of False Negatives With Respect to Prevalence and Test Errors for a Pool of 94 Patients
1.
Yelin  I, Aharony  N, Shaer Tamar  E,  et al.  Evaluation of COVID-19 RT-qPCR test in multi-sample pools.   Clin Infect Dis. Published online May 2, 2020;ciaa531. doi:10.1093/cid/ciaa531PubMedGoogle Scholar
2.
Dorfman  R.  The detection of defective numbers of large populations.   Ann Math Stat. 1943;14:436-440. doi:10.1214/aoms/1177731363Google ScholarCrossref
3.
Johnson  NL, Kotz  S, Wu  X.  Inspection Errors for Attributes in Quality Control. Chapman and Hall Ltd; 1991. doi:10.1007/978-1-4899-3196-2
4.
Wang  W, Xu  Y, Gao  R,  et al.  Detection of SARS-CoV-2 in different types of clinical specimens.   JAMA. 2020;323(18):1843-1844. doi:10.1001/jama.2020.3786PubMedGoogle Scholar
5.
Zou  L, Ruan  F, Huang  M,  et al.  SARS-CoV-2 viral load in upper respiratory specimens of infected patients.   N Engl J Med. 2020;382(12):1177-1179. doi:10.1056/NEJMc2001737PubMedGoogle ScholarCrossref
6.
Zenios  SA, Wein  LM.  Pooled testing for HIV prevalence estimation: exploiting the dilution effect.   Stat Med. 1998;17(13):1447-1467. doi:10.1002/(SICI)1097-0258(19980715)17:13<1447::AID-SIM862>3.0.CO;2-KPubMedGoogle ScholarCrossref
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    1 Comment for this article
    EXPAND ALL
    Pool Testing of COVID-19 with Limited Test Availability
    Michael McAleer, PhD(Econometrics),Queen's | Asia University, Taiwan
    In the research letter on global health, the experts evaluate real-time reverse transcriptase–polymerase chain reaction (RT-PCR) testing for COVID-19 with simulations of pool testing under conditions of limited test availability, that is, in the presence of local testing shortages.

    Pool testing uses a pooled sample of several patients, with pooled results leading to all patients declared as negative or as positive, with the latter leading to individual testing.

    All patients declared as negative leads to the likelihood of false negative diagnoses.

    The experts developed a useful probabilistic model to estimate the risk of false negatives using simulations
    based on three determining factors, namely COVID-19 prevalence, test sensitivity, and patient pool size.

    Among others, it was assumed that all sequential tests are identically distributed based on independent Bernoulli trials, which did not seem to be tested.

    Under certain stated conditions, the simulations showed that a pool testing strategy was an improvement over individual testing.

    The test outcomes are random variables, so it would have been useful to consider the confidence intervals of the estimated probabilities of false negative outcomes under a variety of distributional assumptions that might be observed in practice, such as through alternative bootstrapping techniques of the numerical simulations.

    This would lead to even greater confidence in the invaluable numerical experiments based on simulated pooling of the RT-PCR tests.
    CONFLICT OF INTEREST: None Reported
    READ MORE
    Research Letter
    Global Health
    June 23, 2020

    Simulation of Pool Testing to Identify Patients With Coronavirus Disease 2019 Under Conditions of Limited Test Availability

    Author Affiliations
    • 1Research Division, Renal Research Institute, New York, New York
    • 2Icahn School of Medicine at Mount Sinai, New York, New York
    JAMA Netw Open. 2020;3(6):e2013075. doi:10.1001/jamanetworkopen.2020.13075
    Introduction

    Specimens from patients with suspected coronavirus disease 2019 (COVID-19) undergo real-time reverse transcriptase–polymerase chain reaction (RT-PCR) testing for qualitative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA detection. The high demand for SARS-CoV-2 RT-PCR tests during the COVID-19 pandemic has resulted in local shortages, prompting researchers to consider pool testing strategies.1

    Pool testing strategies build on testing a pooled sample from several patients: if the results from the pool test are negative, all patients in the pooled sample are declared not to have COVID-19; if the results of the pool are positive, each patient sample is tested individually. The pooled testing strategy is appealing, particularly when test availability is limited. However, any test sensitivity less than 100% bears the risk of a false-negative result for the entire pool. To support informed decision-making regarding the implementation of pool testing for COVID-19, we have developed a probabilistic model to estimate the risk of false negatives considering 3 determining factors: COVID-19 prevalence, test sensitivity, and patient pool size.

    Methods

    This decision analytical model study did not require institutional review board review because it used simulation-based research, per Common Rule 45 CFR §102 (e). This study followed the Strengthening the Reporting of Empirical Simulation Studies (STRESS) reporting guideline.

    We considered a 2-stage pool testing2,3 in the presence of imperfect testing, in which p is prevalence, Se is test sensitivity, and Sp is specificity. We assumed that the probability of a true-positive result pool test equals Se, the probability of a false-positive result equals 1 − Sp, test Se and Sp would be unaffected by the number of patients in a pool (k), and all tests are identically distributed. Let Z be a random variable denoting the number of tests needed to complete the pooling strategy. Let N be the total number of patients, then there are N/k subgroups, each with k members. Assuming independent Bernoulli trials, let Pk be the probability of having at least 1 positive test in k patients. If 1 patient of the subgroup has RT-PCR results positive for SARS-CoV-2, then there will be (k + 1) tests necessary; otherwise, only 1 test will be needed. The expected number of tests for each subgroup with k patients is (k + 1)Pk + (1 − Pk). Then, for N/k subgroups, the expected number of tests needed is E(Z) = (N/k)[(k + 1)Pk + (1 − Pk)] = (N/k)[kPk + 1], in which Pk = (1 − Sp)(1 − p)k + Se(1 − (1 − p)k), which incorporates the effects of test sensitivity and specificity. The optimal pool size is achieved for k that minimizes E(Z). To characterize the pooled test strategy, we define efficiency of the pool as Ef = E(Z)/N = [kPk + 1]/k and expected number of false negatives (ENFN) as kp(1 − S2e) This method does not include a dilution effect on the pooling strategy sensitivity, PS = S2e, as a function of pool size, where S2e provides the upper bound of the pooling sensitivity.

    Results

    For demonstration purposes, we reported the results for a typical number of RT-PCR tests for 94 patients, a specificity of 100%, a prevalence from 0.001% to 40%, and sensitivities from 60% to 100% (Table). Mathematical simulations showed that a pool testing strategy was an improvement over individual testing for a prevalence less than 30% and that the optimal pool size, k0, was approximately 1 + 1/√(pSe),p ∈ (0.1%, 30%). For a realistic scenario, such as a sensitivity of 70% and prevalence of 1%, the optimal strategy required 13 patients per subgroup. With this optimal pool size, only 16% as many tests would be required by subgroup tests than by individual tests. The Figure shows test efficiency, cost savings, probability of false negatives, and the expected number of false negatives for k and p.

    Discussion

    This decision analytical model study found that pool testing efficiency varied with prevalence, test sensitivity, and patient pool size. Therefore, pool testing may be considered as an alternative, especially in circumstances of limited SARS-CoV-2 test availability and a COVID-19 prevalence less 30%. One potential limitation of pool testing is that the false-negative rate may increase owing to dilution of positive samples. The mean viral load of more than 1.5 × 104 RNA copies per mL in nasal swabs4 spikes within the first week after clinical onset and reaches 1.5×107 RNA copies per mL.5 In our example with an optimal pool size of 13 patients, high-sensitivity RT-PCR assays with a lower detection limit of more than 1100 RNA copies per mL will detect SARS-CoV-2 in pooled samples. If sensitivity of the test at hand is a concern, test swabs can be collected and eluted into 1 virus transportation medium container, thereby increasing reliability. However, this requires re-collecting individual samples if a pool tests positive. The mathematical underpinning of the proposed method is generic and can be applied to other infectious diseases.6

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    Article Information

    Accepted for Publication: May 26, 2020.

    Published: June 23, 2020. doi:10.1001/jamanetworkopen.2020.13075

    Open Access: This is an open access article distributed under the terms of the CC-BY-NC-ND License. © 2020 Cherif A et al. JAMA Network Open.

    Corresponding Author: Alhaji Cherif, PhD, Research Division, Renal Research Institute, 315 E 62nd St, 4th Floor, New York, NY 10065 (alhaji.cherif@rriny.com).

    Author Contributions: Dr Cherif had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

    Concept and design: Cherif, Grobe, Kotanko.

    Acquisition, analysis, or interpretation of data: All authors.

    Drafting of the manuscript: Cherif, Grobe, Kotanko.

    Critical revision of the manuscript for important intellectual content: All authors.

    Statistical analysis: Cherif.

    Administrative, technical, or material support: Grobe, Kotanko.

    Supervision: Kotanko.

    Conflict of Interest Disclosures: All authors are employees of the Renal Research Institute, a wholly owned subsidiary of Fresenius Medical Care North America. Dr Kotanko reported owning stock in Fresenius Medical Care.

    References
    1.
    Yelin  I, Aharony  N, Shaer Tamar  E,  et al.  Evaluation of COVID-19 RT-qPCR test in multi-sample pools.   Clin Infect Dis. Published online May 2, 2020;ciaa531. doi:10.1093/cid/ciaa531PubMedGoogle Scholar
    2.
    Dorfman  R.  The detection of defective numbers of large populations.   Ann Math Stat. 1943;14:436-440. doi:10.1214/aoms/1177731363Google ScholarCrossref
    3.
    Johnson  NL, Kotz  S, Wu  X.  Inspection Errors for Attributes in Quality Control. Chapman and Hall Ltd; 1991. doi:10.1007/978-1-4899-3196-2
    4.
    Wang  W, Xu  Y, Gao  R,  et al.  Detection of SARS-CoV-2 in different types of clinical specimens.   JAMA. 2020;323(18):1843-1844. doi:10.1001/jama.2020.3786PubMedGoogle Scholar
    5.
    Zou  L, Ruan  F, Huang  M,  et al.  SARS-CoV-2 viral load in upper respiratory specimens of infected patients.   N Engl J Med. 2020;382(12):1177-1179. doi:10.1056/NEJMc2001737PubMedGoogle ScholarCrossref
    6.
    Zenios  SA, Wein  LM.  Pooled testing for HIV prevalence estimation: exploiting the dilution effect.   Stat Med. 1998;17(13):1447-1467. doi:10.1002/(SICI)1097-0258(19980715)17:13<1447::AID-SIM862>3.0.CO;2-KPubMedGoogle ScholarCrossref
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