Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020

Pandemic Containment through Universal High-Frequency Low-Latency Saliva-Based Screening via Low-Cost Adaptive Strategies, With Application to SARS-CoV-2/COVID-191

Eli M. Rabani NanoCybernetics Corp., and NanoTechnology International Corp. [email protected]

Executive Summary:

Pandemics caused by emergent zoonotic pathogens present significant challenges to public health, societies and economies, as underlined by the presently ongoing COVID-19 pandemic. Although public health measures, where vigorously implemented, have yielded successful control, due to resource limitations and/or suboptimal management, the pandemic rages in many places. Due to the high degree of global interconnectivity of the modern world, which many would prefer to resume, strategies to prevent the establishment of persistent reservoirs in resource-limited settings would also be highly desirable. We consider feasible and affordable strategies for high-frequency, low-latency population-wide testing to intercept transmission, and show that in combination with other health measures (masks, contact tracing, isolation of infected individuals), these can suppress contagion and substantially eliminate a pathogen from a population in advance of earliest possible availability of a successfully trialed vaccine. We note that essential personnel (e.g. healthcare workers, law enforcement) can feasibly be screened multiple times per shift. We further show costs of these measures to be far lower in cost to each of: avoided medical costs, immediate economic losses consequent to a pandemic, and immediate daily decreases in tax-revenues associated with reduced economic activity (each of which are in turn smaller than consequent long-term losses associated with morbidity and mortality, and a lower economic-growth basis [including for future tax revenues absent increased taxation rates]). Finally, we note that universal testing significantly reduces the need for labor-intensive contact-tracing, which has also faced capacity limitations.

Abstract: We consider various strategies to enable universal high-frequency (multiple times per week) low- latency (90 minutes-7 hours for positive individual sample identification) screening, suitable for saliva samples, to intercept contagion for pandemic containment. Specifically, we show that it is feasible to obtain negative results to exclude infection of most samples in under one hour, and that assay costs can be reduced such that sample collection likely becomes the dominant cost. We analyze different strategies to reduce testing cost, with an emphasis on sample pooling strategies, including ranges of applicability and advantages of different pooling strategies for different situations. We introduce combined strategy unequal-split quaternary pooling (C+USQP) trees and show that different degrees of

1 An earlier related manuscript on this topic is at https://www.nanotech.international/COVID19/Rabani2020__BinaryPoolingPathogenSurveillance__v0.3.6.8alpha5__20 2003311134.pdf © 2020, Eli Rabani; CC BY-NC-ND 4.0

1 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 pooling of this type minimize required number of assays for prevalence p<0.025 (e.g. 77.6- and 211.7- fold reductions at p=0.001 and 0.0001, respectively), while for p<0.05 matrix pooling can rapidly exclude most samples as negative with large but smaller (usually around a factor of 2 relative to C+USQP) reductions in required assays, and up to 0.0750.1. We consider particular implementation cases such as universities and workplaces, and situations such as large events and air-travel, where efficient, rapid exclusion of samples from positivity can, in combination with other preventive measures, facilitate earlier resumption of near-normal operations with risks of transmission significantly reduced compared to physical public-health measures alone.

Introduction:

Because transmission can occur before symptom onset, because some contagious individuals remain entirely asymptomatic, and because a respiratory pandemic poses particular problems for physically preventing transmission, absent adequately resourced, well managed, high-adherence public health measures, control of the COVID-19 pandemic has proven an overwhelming challenge in numerous countries. Here, we propose various strategies to enable minimized cost universal high-frequency low- latency screening with applicability in low-resource settings. We show implementation costs for these to be far below losses to tax revenues incurred by governments due to the pandemic, such that implementation is a direct economic benefit to the short-term balance sheets of governments.

We emphasize sample pooling as a force-multiplier which can utilize existing equipment, standard reagents and materials, and be implemented with standard assays which are either patent- unencumbered or available under royalty-free COVID-19 licensing, such that there are no barriers to implementation; although further developments [Rab20c] may provide additional advantages in the relevant foreseeable future, here we focus on strategies and assays which may be widely implemented immediately and independently. Hierarchical pooling is treated in general in [Hou17] and an information theoretical treatment is given in [Ald19]. Here, we focus on specific implementations which account for practical factors including some specific to the COVID-19 pandemic, and suggest various strategies for various situations or to optimize for different considerations.

Although well-established in various analytical applications, pooling is currently somewhat against- paradigm in medical testing of individuals (see also introductory discussion in [Lit94]), however, for the dual task of identifying individual carriers and protecting a population, we argue that pooling is most appropriate, and can additionally focus use of scarce certified diagnostics for confirmation of individuals who screen positive. We show these strategies give advantages in positive-predictive value over individual testing.

[Wyl20] showed that saliva samples can enable more sensitive detection of SARS-CoV-2 than NP swabs, facilitating sample self-collection, including by many differently-abled persons. Multiple

2 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 protocols with varying degrees of complexity have been developed for saliva samples, e.g. [Vog20b]; see also Table VII.

This paper is organized as follows: first we summarize C+USQP and matrix pooling strategies; then we discuss the efficacy of population-wide testing for pandemic control, including discussion of the effect universal high-frequency low-latency testing can have reducing the replicative number Re and the consequent prospect of pathogen elimination; we discuss high-efficiency screening strategies with emphasis on various sample-pooling strategies as well as their implementation with well established assays; we briefly review tagging strategies for higher prevalence settings, where untagged pooling strategies become inefficient, and discuss their limitations; finally we consider costs, including relative to various costs posed by the ongoing pandemic, as well as some ethical-legal-societal implications (ELSI).

Summary of Pooling Strategies:

Tree-based pooling strategies have been used extensively [Lit94], and have also been proposed for SARS-CoV-2 testing. [Rab20a],[Men20] Here we detail a novel variation which minimizes assays for multistep pooling for individual sample identification, and in the case of quaternary pooling also minimizes steps.

C+US{B,Q}P:

Trees are created by successive pooling of samples and then subpools culminating in the ultimate pool of a tree; assays are minimized in the case m⊂{2,4} {2,4}(BP&QP);residualmaterialisretainedineach (BP & QP); residual material is retained in each succeeding subpool for subsequent conditional testing (obviating recreation of subpools from samples; unequal-split (US) ensures sufficient constituent target material from positive samples remains in each subpool). The ultimate pool is assayed, and only if the result is positive, the immediate subpools are assayed (otherwise all samples of the tree are negative), in a recursive process akin to a following a decision tree. In cases of very low incidence, e.g. where n⩾64 64or128isadvantageous,themixed or 128 is advantageous, the mixed strategy of of combining the classical (C) pooling strategy with tree pooling done only conditionally (C+USBP, C+USQP) by instead directly creating and testing ultimate pools of all n samples, can conserve materials, at the cost of the slight additional delay for later creating trees for pools penultimate to ultimate pools found positive.

Pools of n=mj for different values of m and j are possible; pooling with m=2 is binary pooling, m=4 gives quaternary pooling; successive or hierarchical pooling with j⩾642 or128isadvantageous,themixed creates trees of pools to trace back to samples to effect efficient screening; the portion aj of a sample or pool aliquoted into a subsequent rank pool is 0.5 for the intuitive binary-pooling by equal-splitting (ESBP), but in USBP 2 sufficient material from each sample is pooled with at least some aj>0.5. Since 4 = 2 , quaternary- and binary-pooling yield equal numbers of required assays, but quaternary-pooling can reduce time required by up to a factor of 2. We note that assays may be done in the quaternary pattern on pooling trees of the binary pattern; for highly sensitive assays on unpurified samples, in some instances this may .

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To convey the concept, binary pooling is most intuitive. In binary-pooling, sample portions from individuals are mixed pairwise, mixtures are preserved, sub-sampled and again combined pairwise recursively creating a convergent binary tree culminating in a single pool, which is then assayed. The immediate precursor mixtures for pools testing positive can then be tested; this is then repeated to work back to individual positive samples. For pools of n = 2j samples which test positive, j additional testing cycles are required to identify positive individual samples (also effectively providing further internal controls); for pools with only one infected individual, 2(j) additional assays are consumed in individual identification (with rapid assay cycles enabling identification in 1-2 days). Where active infection rate p remains low, for population size N, approximately (N/n)(1 + (p)(2j)) tests will be required to comprehensively screen the population once, so that in early stages of spread, fractionally more than (N/n) tests must be performed per population surveillance cycle (accounting for ultimate-pool false- positives occurring at rate fP, this becomes ~(N/n)(1 + (p + fP)(2j))). For quaternary pooling, this becomes ~(N/n)(1 + (p + fP)(4j))) but j is ~1.6-2 times smaller.

We note that false-negatives can be cumulative and are most significant for assays on ultimate pools, so where possible, assays minimizing false-negatives are highly preferred for at least ultimate pools. In particular, ddPCR has been found to be highly sensitive. [Rač14], [Suo20], [Romeo20]

Figure 1 shows pooling trees for binary and quaternary pooling. S denotes samples and Jm,j denotes pools of rank j for pooling degree m. Figure 2 illustrates a path along which screening identifies a positive sample in an n=64 quaternary pooling tree. Figure 3 shows plate-to-plate pooling steps for binary pooling suitable for electronic pipettors, while Box 1 shows a pattern for manual intraplate pooling (requiring an additional transfer to a PCR plate for any subpools to be assayed. Figure 4 shows a flowchart for tree-based penultimate pooling, magnetic extraction, ultimate pooling and assay, recursive assays of subpools of positive ultimate pools or positive subpools while concurrently extracting Jm,j-2 subpools as these are constrained by available data leading to positive sample identification.

The mixed-strategy pooling (C+USBP) which yields large savings relative to single-strategy pooling (either classical screening or binary-pool screening by themselves) where negative pools, occurring at ~(1-p)n are frequent. Here classical pooling is done for population surveillance (which also gives an indication of of p, and possibly also regional distribution where pooling is done with samples from specific regions). A smaller proportion of initial individual sample volume VS, e.g 0.1 or 0.2 volume (50-100μl each); these are combined up to practical limits, so, e.g. (128)(0.05ml) = 6.4ml total volume l each);thesearecombineduptopracticallimits,so,e.g.(128)(0.05ml)=6.4mltotalvolume is mixed, and RNA extraction is performed (e.g. divided across seven 1.5ml or five to six 2ml microcentrifuge tubes or in a single polycarbonate centrifuge tube used with a lid); resuspension after isopropanol extraction and combining back into one sample yields a concentrated n=128 pool [this is reasonable because samples resuspended from swabs are far more dilute than typical cell cultures], for reverse transcription and conventional RT-PCR. Alternatively, a pair of n=64 pools are similarly made with 0.2 or 0.4 VS which are RNA extracted and half of the resulting volume of each of the pair is pooled to yield the two n=64 pools and an ultimate n=128 pool. Only for n=128 pools that test positive (e.g. expected frequency shown in Table II), are n=64 two RNA pools assayed (if necessary, preparing binary trees in the first alterenative).

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Real-time evaluation of the false-negative rate (fN), presently unknown, is discussed. In comparison to classical pooling strategies [Dor43] retesting all individual samples from pools which test positive, for j>2 and low fP, retesting is further reduced compared to the (N/n)(1 + (pn)) tests required to go beyond population-wide surveillance to comprehensive preliminary screening for infected individuals. The underlying concept can be applied with various types of assays, including but not limited to nucleic acid assays such as RT-PCR, multiplex-PCR, immunoassays (antibody assays, antigen assays, pathogen assays), and single-molecule fluorescence microscopy-based assays, etc., although each will entail its own critical implementation details, particularly concerning dilution and sensitivity, nonspecific-background/noise, and susceptibility to contamination or interference. Combinations of different assay types (e.g. immunoassays [Li20] plus RT-PCR [Won20],[Lee20] for SARS-CoV-2 on different pools along positive paths or on the same) are also considered, noting that this adds further constrains, e.g. on the choice of biocides which can be used on samples to further protect workers to those compatible with more than one class of assays where resuspension medium is pooled.

Compared to equal-split pooling, in unequal-split variations, pooling proportion aj such as 0.7 in successive steps j increase material in ultimate (e.g. j5 for j=5) pools from each sample (e.g. j4 gets 4 (0.7) = 0.24 of material from each sample, in a volume ((2)(0.7) = 1.4 of that of original samples for binary-pooling or (4)(0.7) = 2.8 volumes for quaternary pooling), which is then extracted or precipitated with resuspension into a convenient total volume for both j5 and, where positive, j4 assays; j=6 and j=7 suitable for p<0.0001 appear feasible with a concentration step and proportions 0.75 and 0.8 with automated pooling); intermediate steps effecting increase in concentration (e.g. precipitation) such as RNA extraction, offsets dilution resulting from pooling.

Some assays feature limit-of-detection (LOD) as low as one molecule, but both for applicability to a wider range of assays and for better reliability with the more sensitive assays, we consider LOD=100. If we employ an assay with a LOD=100 and wish to detect 103 copies/ml of each RNA target, material equivalent to 100μl each); these are combined up to practical limits, so, e.g. (128)(0.05ml) = 6.4ml totalvolumelsamplemustreachtheassayreaction;theseobjectivesentailextractionor precipitation to effect required increases in target concentration. Samples may be pooled and then extracted, or extracted and then pooled, but to balance reduction of material consumption and labor with minimization of time to positive individual sample identification, we suggest a strategy of extraction of penultimate pools and concurrent extraction of potentially positive precursor pools as assay results increase our knowledge such that total time required is the time for required assays and only the first extraction (as we will now show, this only approximately doubles required extractions at p=0.005 while saving an hour for positive sample identification). For C+USQP we suggest that samples are pooled to penultimate pools for magnetic extraction, and a portion of extracted material is pooled into ultimate pools and assayed. In this case, once any ultimate pool yields a positive result, the corresponding precursor pools may be immediately assayed and, optionally if time is to be minimized, the 16 j4,1 precursor pools concurrently extracted. For n=64 quaternary pooling, in this case 4 (i.e. N/16) extractions are required for negative pools. At p=0.005 (where n=64 yields 훼=25.856), for the 27.4% of ultimate pools which test positive, the 16 samples for any positive j4,2 pools are extracted while the j4,1 pools are assayed, so each positive n=64 pools requires 4+16+16 extractions, with required time for individual positive sample identification being:

(purification time) + (4)(assay time),

5 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 so for 20 minute magnetic nanoparticle extractions and 80 minute RT-PCR assays, individual positive samples are identified in under 6 hours, while for 30 minute RT-LAMP total time is reduced to 2.33 hours. At p=0.001, (4)(0.726)+(36)(0.274) = 5.014 extractions and 2.475 assays on average per n=64 pool.

For BP/QP values tabulated in Tables I-II, 훼 is calculated as the sum of tests required for Poisson weighted occupancy classes; tests required for each occupancy class were determined by comprehensive enumeration up to k=5 and the heuristic that upon finding k>4 or 5, e.g. due to that many positive subpools being found in the same rank (which should be rare), individual samples are immediately instead assayed. Note that these values are in close agreement with corresponding values among results tabulated in [Men20] based on simulation of binary trees based on Hwang's generalized binary splitting (GBS) with j = ln2[(1/p) – 1] using our present notation and on fixed degree pooling.

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Table I.a: Binary-pooling & classical pooling savings factors 훼 versus direct individual sample testing, n⩽32. 32. p 0.0001 0.00025 0.0005 0.001 0.005 0.01 0.025 0.05 0.075 0.1 0.11

TbinPool32 30.91 29.42 27.23 23.73 11.96 7.62 3.805 2.416 1.75 1.33 1.202

TDor32 29.03 25.498 21.22 15.94 5.58 3.277 1.718 1.205 1.063 1.009 0.998

TDor8 7.949 7.87 7.752 7.52 6.089 4.953 3.265 2.199 1.735 1.480 1.408

TDor4 3.993 3.984 3.968 3.94 3.706 3.458 2.897 2.319 1.964 1.725 1.650

R32 1.0648 1.1537 1.283 1.49 2.14 2.326 2.309 2.0 1.646 1.317 1.204

RX is ratio of n=X TbinPoolX to n=32 TDorX classical pooling Bold values denote recommendation; italicized values denote usefulness with automation, underlined values denote values to consider without automation.

Table I.b: bin/quad n=32 versus mixed strategy n=64-256 advantage factors 훼 over individual testing p 0.0001 0.00025 0.0005 0.001 0.005 0.01 0.02 0.025 0.03 0.04 0.05 n bin/quad 32 30.9127 29.4165 27.227 23.73 11.96 7.62 4.4998 3.8054 3.3206 2.6862 2.416 mixed 64 63.1657 61.8636 59.5887 54.8278 25.8560 12.7151 5.6796 4.4375 3.6681 2.7845 2.2990 128 121.5968 112.5732 99.1132 77.6499 21.0078 9.6094 256 211.6905 166.2858 120.2063 74.4644

Table II, Poisson statistics expectation frequency of positive pools for n=128 and 256 at low p p 0.0001 0.00025 0.0005 0.00075 0.001 n=64 P(⩾1) 1) 0.0064 0.01587 0.0315 0.04687 0.062 n=128 P(⩾1) 1) 0.0127 0.03149 0.0619 0.09153 0.1201 n=256 P(⩾1) 1) 0.0252 0.061995 0.12014 0.17469 0.226 P(⩾1) 2) 0.0003 0.001962 0.00752 0.01623 0.027 P(⩾1) 3) 2.7e-06 4.165e-05 0.00032 0.00102 0.0023

Table III Classical pooling T as a function of p 0.0001, 0.00025, 0.0005, 0.001, 0.005, 0.010, 0.025, 0.050, 0.075, 0.100, 0.110 64 45.44, 31.7483, 21.223, 12.88, 3.454, 2.048, 1.229, 1.026, 0.992, 0.986, 0.995 32 29.03, 25.4984, 21.221, 15.94, 5.583, 3.277, 1.718, 1.205, 1.063, 1.009, 0.998 16 15.60, 15.0394, 14.190, 12.76, 7.174, 4.754, 2.550, 1.631, 1.313, 1.162, 1.123 8 7.949, 7.87414, 7.752, 7.52, 6.089, 4.953, 3.265, 2.199, 1.735, 1.480, 1.408 4 3.993, 3.98407, 3.968, 3.94, 3.706, 3.458, 2.897, 2.319, 1.964, 1.725, 1.650

0.125, 0.150, 0.175, 0.200, 0.2125, 0.2250, 0.2500 8 1.320, 1.214, 1.138, 1.083, 1.061 4 1.554, 1.426, 1.327, 1.249, 1.215, 1.1856, 1.1336

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Figure 1. Binary n=32 and Quaternary n=64 Pooling Trees.

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Figure 2. Example of tracing positive pools back to positive samples for n=64 USQP.

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Figure 3: Illustration of j=5 Binary pooling with 96-well PCR microwell plates & electronic pipettors

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Figure 4: Example flowchart for USQP screening to identify positive samples, look-ahead extraction of lower-rank subpools on constraint by available data.

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Adaptive Matrix Pooling:

Matrix-pooling has been considered for SARS-CoV-2 testing [Sin20], but there for non-square matrices (which are suboptimal if convenient) of lower maximal extent.

For adaptive matrix testing on batches size b, tests are minimized in the case of square matrices, with samples pooled by row and column. We expect h = (p)(b) infected individuals in the batch, with up to h row pools and h column pools testing positive, so 훼 is maximized while still detecting positive samples for p and b such that on average h⩽2, 2, b ⩽2, 2/p (We may neglect row or column pool containing multiple positive samples here because we are interested in the worst case cost.) Matrix testing excludes proportion 2 E1 ⩾64 or128isadvantageous,themixed 1 – (h )/b ⩾64 or128isadvantageous,themixed 1 - p2(b) of individuals from positivity in the first round of assays, but holds H ⩽2, 1 - (1/h) negative individuals whose samples were pooled with positive samples for the time to retest the ⩽2, h2 samples that made up positive pools. A batch is tested with 2(b)0.5 tests and ⩽2, h2 second-pass tests are needed to identify positive individual samples, so for direct second-round testing, T ⩽2, 2b0.5 + p2b2 tests are required to identify positive individuals within two rounds, so 훼 ⩾64 or128isadvantageous,themixed b/(2b0.5 + p2b2)

A more thorough treatment of relative cost (here, cost reduction 훼) is given by [Ben20]; an online calculator implementing that is found at [Bil], although there the sensitivity analysis is for uncorrelated errors, whereas practical reliability will depend mainly on adequacy of samples.

Table IV shows worst-case 훼 and E1 as a function of p and b. Comparing these values to those for US{B,Q}P or C+US{B,Q}P in Tables I-II, it can be seen that the speed advantage of AMP comes at the cost of roughly halving 훼 at a given 0.005⩽2, p 0.025⩽2, and larger reductions at lower p. Table V shows the number of samples which may be tested by AMP in various formats of multiwell plates.

Samples can be prepared in 5-30 minutes and assayed in 80 by RT-PCR, or assayed in under 30 minutes with microfluidic RT-LAMP, so first-pass exclusion can be accomplished in <40-110 minues and two-pass screening to identify positive individuals can be completed in <70-190 minutes.

Sparse-coding/Error-correcting/Compressed-Sensing Approaches:

Various pooling schemes based on sparse codes, algorithms deriving from digital compression with error correction, or compressed sensing have been suggested or implemented experimentally. Some theoretically improve accuracy via reducing the effects of random errors on results because each

12 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 sample is tested in multiple pools, however such random errors are outweighed by practical sample variability such that the added costs (time, steps and pipette tips) appear unjustified, especially at low p.

For example, 384 samples are pooled sparsely in 48 pools in P-BEST [She20], which includes redundant detection and maintains 훼=8 up to p=0.015. These approaches do offer the advantage of single-pass positive sample identification within their ranges of applicability. All suffer declines of 훼 with increasing p, although Tapestry [Gho20] performs adequately up to at least p=0.075 (p=0.1 suggested to be the limit but with graceful failure), providing redundancy and also permitting approximate quantitation in a single pass, although 훼=2.5 in cases shown; Tapestry appears potentially most useful for present purposes in the range 0.05

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Table IV: Square-matrix batch size advantage & exclusion rates at different incidences p Batch Size Advantage Exclusion

0.0001 256 7.999836 0.999997 400 9.999600 0.999996 576 11.999171 0.999994 1024 15.997379 0.999990 2304 23.986736 0.999977 4096 31.958112 0.999959 0.00025 256 7.998976 0.999984 400 9.997501 0.999975 576 11.994818 0.999964 1024 15.983633 0.999936 2304 23.917342 0.999856 4096 31.739986 0.999744 0.0005 256 7.995906 0.999936 400 9.990010 0.999900 576 11.979300 0.999856 1024 15.934731 0.999744 2304 23.672748 0.999424 4096 30.984694 0.998976 0.001 256 7.983649 0.999744 400 9.960159 0.999600 576 11.917625 0.999424 1024 15.742082 0.998976 2304 22.742434 0.997696 4096 28.291744 0.995904 0.002 256 7.934997 0.998976 400 9.842520 0.998400 576 11.677150 0.997696 1024 15.015917 0.995904 2304 19.653058 0.990784 4096 20.993408 0.983616 0.0025 256 7.898894 0.998400 400 9.756098 0.997500 576 11.503067 0.996400 1024 14.513788 0.993600 2304 17.835910 0.985600 4096 17.590150 0.974400 0.003 256 7.855213 0.997696 400 9.652510 0.996400 576 11.297222 0.994816 1024 13.943890 0.990784 2304 16.024956 0.979264 4096 14.681270 0.963136 0.004 256 7.746173 0.995904 400 9.398496 0.993600 576 10.805048 0.990784 1024 12.676842 0.983616 2304 12.733879 0.963136

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Table IV (continued) p Batch Size Advantage Exclusion

0.005 256 7.610350 0.993600 400 9.090909 0.990000 576 10.231924 0.985600 1024 11.350738 0.974400 2304 10.073875 0.942400 4096 7.482230 0.897600 0.0075 144 5.721915 0.991900 256 7.173601 0.985600 400 8.163265 0.977500 576 8.640553 0.967600 1024 8.326395 0.942400 2304 5.838848 0.870400 4096 3.821899 0.769600 0.01 64 3.900156 0.993600 144 5.522828 0.985600 256 6.640106 0.974400 400 7.142857 0.960000 576 7.095553 0.942400 1024 6.064281 0.897600 0.02 25 2.439024 0.990000 36 2.875767 0.985600 49 3.275313 0.980400 64 3.628447 0.974400 144 4.458977 0.942400 256 4.397537 0.897600 400 3.846154 0.840000 576 3.187420 0.769600 0.025 25 2.406015 0.984375 36 2.810304 0.977500 49 3.161163 0.969375 64 3.448276 0.960000 144 3.896104 0.910000 256 3.508772 0.840000 0.03 25 2.366864 0.977500 36 2.734233 0.967600 49 3.032009 0.955900 64 3.250975 0.942400 144 3.375338 0.870400 256 2.813731 0.769600 0.04 25 2.272727 0.960000 36 2.557981 0.942400 49 2.746390 0.921600 64 2.837684 0.897600 144 2.518469 0.769600 0.05 25 2.162162 0.937500 36 2.362205 0.910000 49 2.449694 0.877500 15 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020

Table V: Square-matrix batch row & column pools per plate at different formats, and corresponding number of batches & pooled samples per plate.

b r+c plate batches/plate samples/plate

64 16 96 6 384 384 24 1536 1536 96 6144

144 24 96 4 576 384 16 2304 1536 64 9216

256 32 96 3 768 384 12 3072 1536 48 12288

576 48 96 2 1152 384 8 4608 1536 32 18432

1024 64 96 1.5 1536 3 batches/2 plates 384 6 6144 1536 24 24576

2304 96 96 1 2304 384 4 9216 1536 16 36864

4096 128 96 0.75 3072 3 batches/4 plates 384 3 12288 1536 12 49152

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Box 1, intra-plate pooling: j=5 example with 96-well microtiter plate binary-tree pooling pattern (pooling 32 samples takes 4 columns, so 3 pools of 32 samples per plate, by repeating pattern again in columns 5-8 and 9-12)

col1 col2 col3 col4 A 1+ 2 17+18 1+ 2+17+18 1+ 2+17+18+ 3+ 4+19+20 B 3+ 4 19+20 3+ 4+19+20 5+ 6+21+22+ 7+ 8+23+24 C 5+ 6 21+22 5+ 6+21+22 9+10+25+26+11+12+27+28 D 7+ 8 23+24 7+ 8+23+24 13+14+29+30+15+16+31+32 E 9+10 25+26 9+10+25+26 1+ 2+17+18+ 3+ 4+19+20+ 5+ 6+21+22+ 7+ 8+23+24 F 11+12 27+28 11+12+27+28 9+10+25+26+11+12+27+28+13+14+29+30+15+16+31+32 G 13+14 29+30 13+14+29+30 All32 H 15+16 31+32 15+16+31+32

Pipettor at a fixed volume; deposit-mix-deposit doubles and halves volume in a well.

Advance once well complete manual mixing pattern, uses single channel pipettor, 1 tip/sample to form binary tree pools without back-contamination.

Steps fresh tip, deposit 1 to well A1 fresh tip, deposit 2 to well A1, mix & deposit mixture to well A3 fresh tip, deposit 17 to well A2 fresh tip, deposit 18 to well A2, M&DM to well A3, M&DM to well A4 fresh tip, deposit 3 to well B1 fresh tip, deposit 4 to well B1, M&DM to well B3 fresh tip, deposit 19 to well B2 fresh tip, deposit 20 to well B2, M&DM to well B3, M&DM to well A4, M&DM to well E4 fresh tip, deposit 5 to well C1 fresh tip, deposit 6 to well C1, M&DM to well C3 fresh tip, deposit 21 to well C2 fresh tip, deposit 22 to well C2, M&DM to well C3, M&DM to well B4

17 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020

Population Testing:

First we consider universal testing, and then testing in specific cases or settings which could serve as prototype programs for universal testing in addition to in themselves providing protection in those settings and contributing to attenuation of the pandemic. We then show that two concerns raised against universal testing to not be applicable to pandemic containment via high-efficiency screening with certified diagnostic confirmation (and that positive predictive value is actually improved compared to individual testing with only the certified assay). Application to specific settings or cases is then discussed.

Universal or Full-Organization Testing:

Since asymptomatic and presymptomatic transmission is estimated at 40-70%, asymptomatic testing to intercept contagion can facilitate shortening the duration of outbreaks or the entire pandemic, effectively reducing Re. Combined with other public health measures which also reduce Re, situations of exponential growth in active infection can be changed to unsustained chains of transmission.

Universities present a favorable implementation for population-wide screening and can validate this approach for full populations. Similarly, congregate settings such as care facilities and prisons, or high- contact or personnel-dense workplaces such as food- or meat-processing facilities or shipping centers, all of which have proven to be problematic settings of transmission, can be tested at high-frequency. Further, essential personnel with high exposure risks such as healthcare workers and public-safety personnel (e.g. law enforcement, firefighters), as well as delivery personnel, can be tested multiple times per shift, both avoiding spread among coworkers and transmission to the public, including members of the public they may interact with whom might otherwise be reducing potential exposure.

Contagion Interception & Pandemic Limitation:

Others have also considered universal testing, either as an exit-strategy for lockdowns or as independent measures to reduce Re. Universal weekly testing (in that case, antigen screening) was proposed as an exit strategy for the UK's lockdown. [Pet20], [Romer20]

[Mog20] analyzed the effect of silent transmission and found that with suppression of 45% of asymptomatic and presymtomatic transmission in addition to isolation of symptomatic individuals, the attack rate could be reduced below 1%.

Very recently, [Lar20] analyzed the effect of assay limits-of-detection, testing interval and delays in result reporting on reductions of Re, and found that reducing delays and increasing testing frequency could significantly outweigh reductions in LOD. Note that most of the pooled assays considered here are actually below or near the lowest LODs (103) considered in that work. Using their online simulator ([Lar20b]), ranges of interest were calculated and are listed in Table VI; significant reductions are seen for all values with LOD⩽2, 105, frequency⩽2, 2 days and reporting delays 1⩽2, day (all same-day reported results yield Rrel⩽2,0.05), but delays have the greatest effect on diminishing the relative reduction in R.

18 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020

Although various assumptions (e.g. concerning the rate of increase of viral load) underlie their calculations, considering the incubation period, window period and importance of asymptomatic transmission, the qualitative trends seem reasonable and the resulting effects on R underline both the prospect of efficacy of universal testing in driving Re to levels which no longer sustain presence of a pathogen in the population, but also reasons why many testing efforts in the U.S., which have notoriously been plagued by delays and limited availability, have contributed less than they might have to containment efforts.

Considering that the serial interval, I, for SARS-CoV-2 infection is 4-6 d and the demonstrated effectiveness of public health measures such as masks and other restrictions reducing spread at achieving R ⩽2, 1, and considering the present number of active cases, C, in the U.S. to be 2×106 and estimating this to be a 10-fold undercount, a universal testing regime yielding a sustained Rrel ~ 0.05 could in combination with other measures yield elimination of the virus from the U.S. population in under 1.5 months: t/I 7 7 (C)(Rrel) ~ (2×10 )(0.05) = 0.00781 which would occur well in advance of the earliest possible vaccine, the arrival of which is uncertain. With LOD < 1000 and prompt results, testing every other day yielding Rrel=0.006 could reduce time to elimination to 16 to 24 days. While uptake rates would likely make the task more difficult than this simplified analysis might suggest, additional measures such as sewage surveillance already being implemented could mitigate non-ideal implementation.

In regions with low p, and where low p has been achieved after effective control measures, continued cycles of universal testing at high n enjoy high 훼 and hence favorable economies, and prevent reemergence until elimination can be confidently declared.

19 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020

Table VI: Effects of LOD, testing frequency & reporting delay on relative reduction of Reproductive Number (calculated using [Lar20b]) log10LOD frequency delay Rrel 3 1 0 0 1 0.023 2 0 0.006 1 0.087 4 1 0 0 1 0.045 2 0 0.015 1 0.114 5 1 0 0 1 0.073 2 0 0.033 1 0.158

Positive Predictive Value and False Positives for Screening Followed by Confirmatory Tests:

First, we address two related concerns raised against universal testing, positive predictive value at low prevalence, and false-positves. Then we discuss efficacy of universal testing for reduction of contagion. Finally, we discuss concerns raised about pooling, and show that these either do not apply or can be avoided in specific implementations.

Reluctance to adopt universal or full-population testing may be informed by the fact that at low incidence, imperfect diagnostics yield low positive predictive values (PPVs), which are lower than for symptomatic testing. In the context of decisions to treat incurring any significant treatment-risks, low positive predictive value is a matter of obvious concern, since more members of the tested population could be harmed through adverse effects of unnecessary treatment than helped through correct identification and treatment of true positives. Thus conventionally, testing is done upon specific indications such as symptomaticity or risk-group, increasing positive predictive value. Universal testing for pandemic control differs in two important ways: first, the consequence of false positives (rates of which which are reduced in the case of screening plus individual confirmatory testing) are temporary quarantine till retesting negative, and second, the benefit is not only to the subject but to the entire population, i.e. containment and ultimate elimination of the pathogen by reduction of Re to values below those which maintain its presence. Thus, we properly compare the burden of erroneous temporary quarantines to the long-term morbidity and mortality due to permanent establishment of the pathogen), leading to clear net benefit from universal testing.

One concern which has been raised concerning increased asymptomatic testing is the possibility that false-positives will increase with more extensive testing. Here we show that screening of pooled samples with positive sample identification followed by confirmatory testing reduces the false-positive rate compared to either the confirmatory test or screening assays alone. [Hog20] found false-positives

20 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 to be low (0.03463% or 1/2888) in their implementation of rtPCR on pooled samples at n=9-10. Here, we show that even at higher false-positive rates, screening pooled samples with individual positive sample identification followed by confirmatory testing with a certified assay can outperform either on its own, leading to significant improvements in combined PPV. If asymptomatic testing is conducted at e.g. p=0.025, even a 0.8% assay false positive rate (FPR) would yield a PPV of only 75.75%, but confirmatory testing (e.g. with FPRc=0.5%) of the subset of individuals who screen positive, e.g. via sample pooling in universal high-frequency low-latency screening, would yield a final PPVs+c=99.34% (0.7575/0.7625), whereas simply using the test used for confirmation directly would have yielded a PPV of 83.3% (0.025/0.030), such that screening via pooling, even if imperfect, can both conserve available individual assays of higher quality and improve resulting overall accuracy. This analysis assumes the assays used for screening and pooling are different, i.e. do not share any systematic false- positive errors. In general,

PPV = p / [p + (1-p)(FPR)]

Because confirmatory testing test samples which screened positive, the probability that these samples are positive is the PPVs from screening, so for confirmatory test false-positive rate FPRc,

PPVs+c = PPVs / [PPVs + (1-PPVs)(FPRc)]

= p / [p + (1-p)(FPRs)(FPRc)], and, PPVrel = PPVs+c / PPVs = [p + (1-p)(FPRc)] / [p + (1-p)(FPRs)(FPRc)].

Positive-predictive error PPErr = 1 – PPV = (1-p)(FPR)/[p + (1-p)(FPR)] for screening with confirmatory testing is reduced relative to only testing with the confirmatory assay as: PPErrrel = PPErrs+c / PPErrc,

= (1 - PPVs+c) / (1 - PPVc) relative to the performance of the assay used for confirmation instead used alone, 0.03934 in the above example, or a 25.417-fold improvement. Thus, the same number of false-positives would result from screening the entire population as would result from testing 4% of the population with the certified assay used here for confirmation.

A meta-analysis [Kuc20] of diagnostic accuracy found significant false-negative rates (FNRs) even at the optimal delay post-infection (~20%, and that only 3 d post-onset of symptoms), but we note that this included data from assays from early in the pandemic of potentially lower reliability. This result was used by [Chi20] to arrive at a lower estimation of effectiveness of workplace screening, e.g. 48% reduction in infection rate for screening every two days with a reporting delay of one day, but ~75% reduction with daily testing. Due to the uncertainties in these estimations, empirical data on

21 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 implementation would be needed for greater certainty, but even these lower estimates support the proposition that high-frequency universal or enterprise-wide testing can meaningfully reduce contagion and associated disruptions, with avoided economic losses and medical costs far outweighing testing costs.

Specific applications: Schools & universities, workplaces, air-travel and events:

Situations involving extended proximity in enclosed settings entail higher risk of spread and can become super-spreader events, which notoriously can undermine hard-won progress in pandemic control. Rapid, economical testing of participants shortly in advance of congregation can drastically reduce transmission risks by steeply reducing prevalence among those in attendance. Here we note that for this purpose in particular, lower LOD, associated with detection at lower viral loads and hence earlier in advance of transmissibility, increases the margin of safety by reducing the likelihood that anyone below the threshold of detectability could become infectious within a few hours.

Adaptive matrix testing can clear high proportions E1 of participants in a first pass (35-100 minutes), and testing of all individual contained in positive pools can be completed with test numbers below or near those of classical pooling for 0.01

As an example, consider p=0.025 and b=144 AMP with epRT-PCR. 96-well formats can process first- pass pooled tests for 4 batches or 576 individuals at once, and would yield E1 of 91%, so 524 individuals are cleared in under 90 minutes at materials costs <$330 (including sample collection tubes, the dominant material cost). Individual identification in a second pass will on average take ~16 more assays per batch, costing ~$64 for four batches, identifying >14 individuals as screening positive, whose samples may then be subjected to confirmatory testing. Using 384-well formats, Even at these lower 훼, cost per individual is <$1, and cost per identified infected individual is <$30 (exclusive of subsequent confirmatory testing), which compares favorably to costs for contact tracing in e.g. HIV control efforts. p=0.025 would otherwise warrant restrictions precluding many non-essential congregate activities.

Universities:

There are at present concerns over the wisdom of holding regular university sessions for Fall 2020 in light of recent increases in prevalence [Hua20]. [Ber20] has discussed the role batch testing can play in safely reopening universities. Universal testing twice weekly for universities has also been proposed by [Lan20] utilizing pooling (although with lower n and higher costs than attainable with what is presented here); such efforts are being prepared (e.g. Cornell University, [Cas20]), but screening strategies, pooling strategies and assays considered here may reduce costs while increasing effectiveness.

22 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020

Some modifications to the general strategy may be applied. Returning students could bring samples from immediate close contacts (ICC samples) to be pooled with their own for an initial round of batch testing (or these could be provided and tested within a few days in advance of return, or multiple times in the week prior to return), which gives the advantage that if any close contacts would be their index case and they are not yet past their window period, this strategy will reveal that they are exposed.

Where it is suspected that p may be high (>2.5-5%) incoming individuals would ideally provide individual and ICC samples (I+ICCS) on return (similar to the door-to-door testing strategy [Dec20]) or where possible 1-2 days before return. These would be pooled and I+ICCS pools tested individually (1 I+ICCS pool per reaction) unless capacity precludes this. Where capacity is limiting, matrix pooling at b=16 can still yield 훼=1.333 at p=0.125. Using RT-LAMP ($1/reaction, [LLKP20]), for with I+ICCS requiring 5 sample tubes, a round of intake testing can still cost $3-4/returning individual; using magnetic nanoparticle extraction [Zha20] and precipitation and smartphone- or digital-camera detection, there should be few issues of capacity limitation.

Where p is not excessive (>5-10%), initial testing is done by C+USQP (vide infra), such that ultimate pools provide estimates of p. Individuals are asked or required to limit their contact number prior to exclusion as negative. If p is low, samples can then be tested in the AMP format, so that within ~1.5 hours most individuals can be excluded from positivity and within ~3 hours, individuals testing positive can be identified, and import of the pathogen is significantly reduced, although until a full window period and at least one subsequent round of testing, it should be assumed that the initial p still obtains in terms of any heightened distancing measures. With this approach, however, plocal can be reduced within 1.5 weeks such that 훼 becomes high. This approach will also provide realtime comprehensive information if p is unacceptably high to commence or continue classes, or classes above some threshold, without extensive infection occurring.

Optimizing the advantage 훼 of pooling involves an estimate of p. This can be done simply by starting pooling on an initial batch of samples and running assays on the full binary or quaternary tree, or alternate levels of a tree. This serves the further purpose of providing a quality control as pooled testing begins, and yields a real-time estimate of p pertinent to the population being tested while still delivering initial results. Since it is likely that efforts may be expedited by utilizing used or locally available thermocyclers, such a procedure including both individual samples and the pooling tree can provide a secondary validation of equipment after running a full plate of positive control reactions to detect any intraplate variation associated with a particular piece of equipment. Once an estimate of p has been determined, the appropriate degree of pooling or batch size can be chosen.

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Assays:

Any type of pathogent assay could conveniently be used. The most immediately useful are those which are already in widespread use for general purposes, nucleic acid amplification assays and antigen assays, although with further development, single-molecule assays may soon be practical [Rab20b].

In addition to ddPCR (vide supra), we review pertinent RT-PCR and RT-LAMP-based assays. For the former, we note evidence that endpoint-RT-PCR can substitute for realtime-RT-PCR [Ma18], significantly reducing equipment costs, and permitting utilization of more widely available instruments. For RT-LAMP, we note that a regulated temperature water bath can suffice to incubate reactions, and that inexpensive domestic appliances (sous vide) have been shown adequate. [Jou20] Both in general, and in particular for pooled samples, assays can be made more sensitive for diagnostic purposes by increasing the effective concentration of pathogen derived targets through precipitation, whether direct (e.g. isoporopanol precipitation) or in the course of extraction (silica [Rabe20], [LLKP20] or magnetic nanoparticle [Zha20], [Obe19] based extractions; these references provide protocols for preparation of respective extraction media from readily available materials).

Table VII shows high sensitivity multiplexed assays likely to be useful with pooling and also for individual samples; also vide infra.

Table VII: Sensitive assays useful for pooled-sample screening (non-exhaustive). Assays are multiplex-RT-PCR (either realtime or endpoint) unless otherwise indicated.

Ref Targets Sample LOD (copies/reaction) Notes

[Byr20] N1, N2, hRP NPS, column: 10 NPS, extraction-free: >270

[Wag20] N2, E, hRP NPS, extracted

[Ran20] ORF1ab, NC, S * Saliva, extraction-free 5 Heat treatment (95 for 30 min.)

[Kud20] N1, N2, hRP NPS and saliva, 500 MagMax extraction

[Sun20] simplex Nasal swab (horse) 18 Microfluidic LAMP, 30 minutes

24 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 hRP = human RnaseP positive control

* TaqPath RT-PCR COVID-19 kit, Thermo Fisher Scientific.

RT-PCR:

Detection of amplification products may be conventiently be via intercallating fluorescent dyes, colorimetric detection or, especially for multiplex RT-PCR, fluorescently labeled probes. In [Bha20], detection was via oligonucleotide strand displacement, so signal is highly specific to the probed sequence, and with multiple fluorescent dyes, multiplex probing for multiple viral target sequences would be feasible. [Bha20] provides a buffer for Taq DNA Polymerse which is optimized to yield adequate reverse-transcriptase activity for this enzyme so that single-enzyme RT-PCR is possible. Additionally, [Bha20] provide a crude preparation of BstLF polymerase expressed in E. coli which can reduce costs or address shortages.

[Won20] screened for asymptomatic SARS-CoV-2 carriers, performing two-step rtPCR assays on RNA samples obtained from subject self-collected throat swabs (released into 0.5ml medium, of which 0.3ml was purified with Guanidinium thiocyanate-Acid Phenol-Chloroform [Trizol™], [Cho87] [Cho06][Ope09] by the receiving technician, isopropanol precipitated and resuspended in 10μl each); thesearecombineduptopracticallimits,so,e.g.(128)(0.05ml)=6.4mltotalvolumelRNase- free H2O, yielding on average 287ng/μl each); these are combined up to practical limits, so, e.g. (128)(0.05ml)=6.4mltotalvolumelRNA)usingfourpairsofvirus-specificPCRprimersin individual PCR reactions (0.5μl each); these are combined up to practical limits, so, e.g. (128)(0.05ml) =6.4mltotalvolumeMofeachprimertherespectivepair),alongwiththreepositive controls (human RNase P specific primer pairs) and a negative control (no template). Samples are deemed non-infectious upon treatment with Trizol™, the toxicity of which is noted, early in the protocol. The reverse-transcription step was performed using SuperScript™ III First-Strand Synthesis System (Invitrogen) in a 20μl each); these are combined uptopracticallimits,so,e.g.(128)(0.05ml)=6.4mltotalvolumelreactionusingoligo-(dT)20 primers (2.5μl each); these are combined up topracticallimits,so,e.g.(128)(0.05ml)=6.4mltotalvolumeM)and8μleach);thesearecombineduplofpurifiedsample RNA. Detection of PCR products in 20μl each); these are combined up to practical limits, so, e.g. (128)(0.05ml)=6.4mltotalvolumelreactionsfrom2μleach);thesearecombineduptopracticallimits,so,e.g.cDNAtemplatewasviafluorescenceof SYBR Green binding to duplex-DNA during amplification, with fluorescence below threshold for Ct > 37 deemed negative for target sequences. The overall materials costs to test each sample for four viral target sequences were under $15 per subject (with materials for each PCR reaction costing < $0.75); the full protocol required < 4 hours; the only specialized equipment required was the rtPCR machine. By reference to a calibration curve, [Won20] estimate sensitivity of ~1-10 virus particles for their assay for Ct = 37. Note that each PCR reaction receives cDNA equivalent to (0.3/0.5ml sample)(8μl each); thesearecombineduptopracticallimits,so,e.g.(128)(0.05ml)=6.4mltotalvolumel/10μleach); extracted RNA)(2μl each); these are combined up to practical limits,so,e.g.(128)(0.05ml)=6.4mltotalvolumel/20μleach);thesearecombineduptopracticalcDNA)0.048ofsampleRNA.

Unfortunately, neither [Won20] nor much other data available as of this writing provide reliable indication of expected false-negative rates (fN), particularly for asymptomatic carriers (fN:A) ([Xie20] finds a 3% fN (5/167), while another finds fN~10% [] for a different RT-PCR assay; to the extent possible, fN must be minimized to enable ultimate containment). [Vog20] did preliminary comparisons of different widely used primer sets under matched conditions; while the sample-count was too low to establish accuracies, it was clear that ~100 copies were needed for amplification by most primer sets, and some had high levels of background. Preliminary operations of screening could attempt to validate

25 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 against gold-standard cell-culture assays to the extent feasible. It is critical that, particularly in settings with lockdowns concurrent to population-wide surveillance efforts, all individuals with close contact to new COVID-19 cases be screened by both these and other distinct methods to evaluate, in real time, the putative fN and fN:A for a given implementation, so that this information can be used to evaluate the expected efficacy of containment with two population-wide surveillance cycles, and make any necessary adjustments. This is critical to understanding and reducing any post-surveillance transmission or outbreaks.

We note that [Won20] suggested sputum as an alternative sample; saliva has been shown to contain SARS-CoV-2 particles [To20]; saliva is preferable both because this is easier for subjects (which in large populations will include people with disabilities) and obviates swabs and tongue depressors, which are presently in short supply in various regions, also reducing cost per subject. However, should it prove necessary to improve accuracy in actual implementation, the contrasting suggestion that combining different intrasubject sampling modes, “both nasopharyngeal and oropharyngeal swabs test of SARS-CoV-2 RNA should be performed to reduce the false negative rate,” [Che20] should also be considered and evaluated empirically. Present evidence shows that saliva samples enable improved detection. [Wyl20]

[Won20] designed their assay to avoid false-negatives (which is the overriding concern in screening for asymptomatic carriers at low prevalences) and selected SYBR Green dye for detection of dsDNA amplification products rather than probe-based detection (which is appropriate for avoiding false- positives by virtue of specifically detecting non-primer and non-host viral sequences, i.e. not detecting PCR artifacts). In our case, it might similarly be preferable to bias against false-negatives for assays of ultimate pools (and also avoids the cost of FRET-based fluorescent probes such as used in TaqMan assays [where target amplification yields probe-target hybrids which are substrates of the 5’-3’ exonuclease activity of the native Taq DNA polymerase which are then degraded, relieving quenching of the fluorophore label to yield a signal] or molecular beacon probes [where probe-target hybrid structure enforces a greater separation of fluorophore and quencher relieving quenching to yield a signal {5’-3’ exo– polymerase used}]). We note here that multiplex-RT-PCR reactions where multiple primer pairs are included in the same mixture to yield multiple target fragments; each primer pair costs less than $0.02-$0.04 per PCR reaction, although labeled probes are more costly. [Won20] did not discuss the suitability of their primer pairs for use in multiplex, although even if none are suitable, other primer pairs targeting different virus sequences could be designed for use in multiplex. We suggested that primer-pairs validated for multiplex-PCR would be preferable for our case since (a) this further biases against false-negatives in ultimate pools (e.g. with SYBR Green detection) and (b) enables use of target-specific probes with different fluorophore labels and detection in multiplex for assays of precursor pools, where detection of all or at least most target fragments serves as a check on internal consistency and also balances the initial designed bias against false-negatives at points where the proposed strategy is availed for preliminary screening, and automatically highlights ambiguous precursor pools or samples. Similarly, positive-control tracer RNA could added to samples and be detected in ultimate-pool multiplex RT-PCR, as a rigorous check against RNase contamination.

We suggest the addition of a biocidal disinfectant to whatever fluid used to suspend sample materials from swabs, as this will further protect technicians, where biocides compatible with necessary processing steps & assays . The Armed Forces Institute of Pathology tested [Kra05] the effect of

26 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 extended storage of nasal swab samples in 100% ethanol on subsequent RT-PCR detection of influenza and other febrile respiratory viruses. While detection relative to culture of frozen specimens was incomplete (likely due to mismatch between PCR primers based on expected sequences and actual isolates) this shows that there exist biocides compatible with subsequent RNA extraction and RT-PCR steps, and suggests that ethanol (also shown to be an effective biocide at 62-71% concentration for surface decontaminiation of SARS-CoV-2 [Kam20]) should be validated experimentally for this purpose in our application (particularly since [Kra05] used Phenol-Chloroform extraction of resuspended pellets and isopropanol precipitation of RNA with a glycogen carrier [Kra97] rather than Trizol™). We note that rendering samples non-infective as early as possible is highly desirable (although note that tube exteriors handled by subjects could still constitute fomites unless also disinfected), and may enable pooling to be conducted by technicians engaged in sample collection (e.g. using tuberculin syringes [provided these are adequately chemically compatible with whatever biocide used/do not introduce interfering agents – these factors would need to be checked experimentally; all- crosslinked-polypropylene syringes might be preferable if available]), reducing required laboratory personel if field technicians can accomplish pooling concurrent with other activities without consuming additional time.

We also suggest that in the context of reverse transcription of pooled samples it is worth evaluating use of one or more viral specific primers to initiate reverse transcription rather than oligo-(dT) as this is expected to reduce the cDNA in pools subject to amplification (which otherwise would include abundant host poly-A tailed mRNA), provided that viral RNA secondary structure (often regarded to be less problematic for priming with oligo-(dT) than with internal primers at the lower incubation temperatures for reverse transcription reactions) does not pose and additional problems and this variation can be experimentally validated (various agents added to reduce polynucleotide Tm [which hence would destabilize secondary structure] such as proline [Iak99], DMSO [] (possibly in conjuction with an 3’-5’ exonuclease [Haw03]), betaine [], trehalose [], bacteriophage T4 gp32 ssDNA binding protein [], or we further suggest glycerol, sorbitol, urea and DMF, could be evaluated in the context of our present application for mitigation of viral template secondary-structure and non-interference with RT-PCR). The reverse primers for amplifying target sequences may be suitable for this purpose. Similarly, there are various one-step RT-PCR methods and formulations (e.g. using Tth polymerase + Mn++ which reverse transcribes at higher temperature) addressing template secondary structure which could be evaluated for our purposes.

We now consider modifications of [Won20] to facilitate pooling and reduce costs further, and issues relating to fast implementation of a surveillance program at scale.

Switching from real-time/quantitative PCR to post-amplification detection of PCR products, i.e. using endpoint-RT-PCR, significantly reduces equipment costs (this has been shown equivalent for detection of a porcine coronavirus [Ma18]); thermocyclers without on-line fluorescence detectors are common as are microplate fluorescence readers. Both are widely available for 96-well (and 384-well) microplate/tube formats. There are likely enough available for diversion or on the used equipment market at affordable prices to quickly scale to population-relevant capacity, albeit at the cost of additional labor and materials for calibration and quality control; voluntary loans of equipment (especially if idled by lockdowns) could also facilitate rapid implementation, and may be the most

27 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 preferable option for quickly obtaining equipment (particularly due to better provenence). Two initial calibration runs of alternated checkerboards of positive and negative controls would be appropriate. Capacity-building should also factor aggregate down-time/failure rates of 10-25%

Considering implementation with [Won20] as a point of departure, but relying on simpler equipment we take an exemplary case, a 96-well microplate thermocycler with 32-sample pooling can amplify material from ~3000 samples (plus a few positive and negative controls and retest samples) in ~1.3 hours for a throughput of > 55,000/day; 25 such machines would be adequate to process samples for complete surveillance of the population of a state like Minnesota (population 5.6×106) within 6 days at 80% utilization; a single microplate reader can detect products from several thermocyclers. The dominant materials cost (53%) in [Won20] was reverse transcription using the SuperScript™ III First- Strand Synthesis System at $8 per sample.

The sample resuspension medium volume must be reduced before formats such as microtiter plates can be utilized to facilitate processing of multiple samples simultaneously. However, it is preferable to avail the advantage of pooling as early in the processing steps as possible to reduce the overall number of manipulations and costs. This particularly affects the applicability of simple lab automation which could greatly reduce labor required for pooling, such as electronic 24-, 96- or 384-channel pipettes (e.g. Viaflo96/384 [Ura20]). Although costs are not insubstantial (e.g. $6-24K), where such equipment is available considerable labor savings are possible where such equipment can be procured. 24- and 48- well culture plates feature 3.4 and 1.6ml wells, respectively, so these can be used for initial pools, and a plate-to-plate pooling format or mixed manual and plate-to-plate pooling (see Box 2) can be used instead, with an intermediate concentration step enabling shift to 96-well formats (or 384- or 1536-well formats where corresponding thermocyclers are also available, increasing productivity by factors of 4 or 16 respectively for RT-PCR steps).

RT-LAMP:

For RT-LAMP, a regulated temperature water bath can suffice to incubate reactions; inexpensive domestic appliances (sous vide) have been shown adequate. [Jou20]

[Rabe20] used a 100X inactivation reagent (250mM TCEP-HCl, 250mM EDTA, 1.15N NaOH) and inactivated saliva samples samples by 5 minute incubation at 95C or in a boiling water bath, and demonstrated 40-minute detection of SARS-CoV-2 in 5μl each); these are combined up to practical limits,so,e.g.(128)(0.05ml)=6.4mltotalvolumelinactivatedsalivaorresuspendedfrom swabs in 25μl each); these are combined up to practical limits, so, e.g. (128)(0.05ml) =6.4mltotalvolumelRT-LAMPreactions(usinga1.25Xreactionmix)withsensitivityof250 copies/reaction. For saliva, this would correspond to 5x104 copies/ml. [Rabe20] further improved on this to better than 103 copies/ml by glassmilk (silica nanoparticle) extraction of 0.5ml saliva, and provide low-cost preparation for this matrix and necessary reagents from readily available materials, so that this approach is suitable for low resource settings, and estimate costs of this extraction to be $0.07 per sample. [Ana20] evaluated this assay on clinical samples and found a sensitivity of 87.5% and 100% specificity. For crude saliva samples, centrifugation to precifitate flocculants was necessary in [Rabe20] , and gravity settling of glassmilk was ineffective, so a centrifuge is required, but [LLKP20] demonstrated simple construction of a suitable centrifuge capable of 500 RCF found adequate for this

28 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 task for $5, and estimate this assay will cost $1 per sample, making this assay fully suitable for low- resource settings. However, where electronic pipettors are utilized, we recommend magnetic nanoparticles prepared according to [Zha20] because this is readily done with multiwell PCR plates and electronic pipetting in high volume.

[Bha20] also show that sensitive multiplex RT-LAMP on crude saliva samples can be done using crude preparations of Bst-LF DNA Polymerase can be used for (including for reverse-transcription, enabling one-enzyme reactions), with a sensitivity of 100 copies/reaction using the 6-primer Lamb oligonucleotides [Lam20] and fluorescent oligonucleotide strand-displacement (OSD) detection, however, this level of sensitivity required 90 minute incubations. These assays are this highly suitable for low-resource settings or in shortage conditions. Allowing 3μl each); these are combined up topracticallimits,so,e.g.(128)(0.05ml)=6.4mltotalvolumelforcrudeenzymeand6.25μleach);thesearecombinedup for a 4x-master mix of all other reagents and using the same 25μl each); these are combined up to practical limits,so,e.g.(128)(0.05ml)=6.4mltotalvolumelvolume,theseassayswouldthus be useful, for example, for use in matrix-pooling of unpurified saliva samples diluted 1:0.97 saliva:TE buffer would allow 8 samples to be pooled and hence batches of 64 without extraction or precipitation, partially mitigating for the longer incubation time, and the corresponding LOD calculates to 105 copies/ ml per sample, which is still useful for universal screening (especially when results are returned on the same day, see Table VI). With magnetic or silica extraction, more samples could be pooled

[Sun20] demonstrated LAMP with sensitivity of 18 copies (1ul sample) per reaction in 30 minutes in microfluidic channels with detection via smartphone. If such devices can be produced at population- relavent scale, reduced reagents and enzymes usage per reaction will be facilitated and equipment requirments reduced, while offering more rapid and sensitive results, all of which are significant advantages for universal screening. Here, individual LAMP reactions are simplex but applied samples flow into multiple channels each of which performs a LAMP reaction with different targets. [Sun20] note that this can readily be adapted to perform RT-LAMP reactions for detection of SARS-CoV-2.

Tagging-based methods:

A different approach to increasing the throughput and efficiency with which samples may be tested involves associating a known identifying sequence tag with target material in an individual sample, combining samples into a large pool, determining the identity of tags associated with pathogen targets, thus revealing which samples contained pathogen targets. Tags are often referred to as barcodes or indices, the essential feature being that they have a unique sequence which in this use is known and through association identifies the individual sample from which target material derives. Two such approaches have been disclosed thus far for large-scale SARS-CoV-2 testing.

It is essential to note that unlike other pooling methods described herein, the efficiency gained by pooling is not lost with tagging-based methods as prevalence p increases, so despite the limitations of these methods (vide infra), tagging can be useful in high prevalence conditions.

LAMP-Seq [Sch20] performs RT-LAMP reactions on individual samples to add a known DNA barcode to any target material in each sample; these isothermal reactions may be done remotely. Then barcoded samples are subjected to pooling RT-PCR and barcodes from amplified products (having target

29 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 sequences) are determined via next-generation sequencing, exploiting the high throughput of NGS so that, for example, the authors expect that 2x108 75 base-pair NGS reads per day may enable throughput of >106 samples per day, although at a cost of $2 per individual sample with economies of scale. It was found that 160 template molecules could be reliably detected in a sample; dynamic range compression due to LAMP saturation reduces the number of reads per sample over a wide range of possible viral loads, so at most only limited quantitative data are yielded about each sample.

SwabSeq [Jon20] places a lysate of each sample in a microwell having a multiple unique identifying indices associated with primers, and does one-step RT-PCR yielding amplified sample material with a known index corresponding to the microwell and hence the original sample; following amplification, all samples are pooled and index sequences associated with amplified target material are determined via next-generation sequencing, revealing which samples are positive for the pathogen due to the known relationship between the sequences of indices and individual samples. A 1-6 molecules/sample LOD is reported, giving this method a very high analytic sensitivity. Samples are spiked with an internal positive control sharing target priming sequences but which have distinct sequences to discriminate these from the actual target., although at an expected cost of $1 per individual sample and with 12-hours required for processing.

Although tagging methods do not suffer from steep reduction and ultimate loss of efficiency as p exceedes 10-20% as is the case for other pooling methods discussed herein, both LAMP-Seq and SwabSeq depend on individual reactions for tagging, so at least initial amplifications are not reduced relative to individual testing. The fact that pathogen-derived target sequences are read directly by these methods stringently prevents false positives due to pathological amplifications, so false positives would only result from erroneous association of tag sequences with pathogen sequences from different samples, which might occur via carry-over of unextended tagged primers from initial tagging reactions and/or strand-switching.

We suggest that these methods may instead be useful for confirmatory purposes using material from individual tests (done with necessary known identifying tag sequences associated with primers) which are found to be positive (e.g. colorimetric detetion or fluorescent probes, e.g. OSD probes specific for target sequences, described for assays supra), where NGS reads determine both tag sequences and pathogen sequences not included in primer and probe sequences in the original individual assay.

Discussion:

Sample collection considerations:

The largest logistical and labor requirements for population-wide pathogen surveillance may be sample collection.

Both biosafety and procedural training of sample-collection workers would be required in advance, and this could proceed concurrent to preparations for laboratory implementation.

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Note on Devolution of Emergency Authorization of Diagnostics in the United States: https://www.fda.gov/media/135659/download

On March 13, 2020, the President issued a “Memorandum on Expanding State-Approved Diagnostic Tests” (Memorandum), which refers to the flexibility that FDA allowed New York State and states as follows: [...] In accordance with the Memorandum, FDA describes below its policy regarding States and territories that authorize laboratories within their State or territory to develop their own COVID19 tests and perform specimen testing, where the notification of SARS-CoV-2 test validation is not submitted to FDA and the laboratory does not submit an EUA request to FDA.

A State or territory choosing to authorize laboratories within that State or territory to develop and perform a test for COVID-19 would do so under authority of its own State law, and under a process that it establishes. FDA does not intend to object to the use of such tests for specimen testing where the notification of SARS-CoV-2 test validation is not submitted to FDA and the laboratory does not submit an EUA request to FDA, and where instead the State or territory takes responsibility for COVID-19 testing by laboratories in its State during the COVID-19 outbreak.

Thus an additional legal authorization for this purpose has effectively devolved to state Governors within their jurisdictions.

It is an open question whether recognized Native American Tribal Governments independently could successfully assert similar authority, should any wish to.

Note on Emergency Use Authorizations in the United States for organizations:

Wide EUAs have been made for diagnostics manufacturers and for clinical laboratories certified to perform high-complexity testing under CLIA; some hospitals are associated with the latter. http://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use- authorizations https://www.fda.gov/medical-devices/emergency-situations-medical-devices/faqs-diagnostic-testing- sars-cov-2

On 19 June 2020, the FAQ for EUA tests was updated to specifically note that testing of pooled samples would be considered eligible for EUAs and encouraged laboratories and manufacturers interested in developing pooled-sample tests to enter discussion with the FDA.

31 of 42 Pandemic Containment via Universal High-Frequency Low-Latency Screening, Draft v1 E.M. Rabani, 9 July 2020 https://www.fda.gov/medical-devices/emergency-situations-medical-devices/faqs-testing-sars-cov- 2#clinical

Q: Is any EUA-authorized SARS-CoV-2 diagnostic test authorized for use with sample pooling? (New 6/19)

A: Currently no SARS-CoV-2 diagnostic test has been authorized for use with sample pooling. If you would like to develop and offer a test for use with sample pooling, please see the updated Molecular Diagnostic Template for Commercial Manufacturers or Molecular Diagnostic Template for Laboratories for recommendations on how to validate the test for this use and submit your EUA request to the FDA.

Additionally, Deborah Birx, response coordinator for the White House coronavirus task force, solicited validation of pooled samples on different widely used diagnostic instruments in an address at ASM Microbe on 22 June 2020. [JohM20]

Costs of Universal Screening:

Costs of implementation are likely to be far below those incurred to government revenues due to economic impacts of restrictions. As an example, we may crudely estimate the economic impact of the California lockdown commencing 19 March 2020 as a 30% reduction in daily gross state product ($3.137T/yr) and proportionate reduction in state government revenue, yielding losses of $2.5B to the state economy per day and $176M in state tax receipts per day. Accordingly, even if the screening strategy proposed here only modestly shortens lockdown duration, since while p remains low, costs are below a low estimate of daily tax receipt losses, it is a revenue-positive investment even under the most fiscally conservative analysis. Even as lockdown restrictions have been lifted, economic activity remains diminished and employment has not recovered due both to remaining restrictions and limitation of activity on the part of individuals to avoid exposure to SARS-CoV-2; economic recovery and recovery of tax receipts will depend on public confidence that the ongoing risks have been adequately abated.

Implementation details could impact efficacy of the two-surveillance-cycle restriction lifting posed here (and it is noted this is a preliminary idea which demands further analysis and modeling). We note, however, that even short of the desired level of complete fixation of a pathogen from the population, other benefits are realized. In the case of SARS-CoV-2, there would be better utilization of scarce certified diagnostics; even with higher f’N:A, rates of transmission from asymptomatics could be reduced by a factor of (1 – fN:A) per surveillance cycle

Costs per life saved; avoided medical costs:

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Taking the example of MN, Governor Tim Waltz in a State Address on 25 March 2020 indicated expectation that the total population infected in MN could reach 2.4 million people. If the mortality rate with good medical care is 2.5% (and medical resources are not overwhelmed), 6×104 lives would be lost, i.e. 1 in 95 people. (In a non-action scenario, i.e. without social distancing or lockdown, Gov. Waltz projected that as many as 740,000 deaths could directly or indirectly result from COVID-19.) If costs for two cycles of population-wide pathogen surveillance and screening were $15M (including sample collection), and this only reduces total prevalence and mortality by half (saving 30,000 lives – though we envision yet better outcomes), the cost per life saved would be $500, which would be reduced further with larger effects of screening on reducing total prevalence. We note that there are lower credible estimates of the fatality rate, which would affect this crude analysis.

Hospitalization costs per case, comparable to pneumonia hospitalization, exceed $10-20K. Considering only the above cases eventually resulting in fatality, and only half were avoided through population-wide surveillance, avoided medical costs would be >> (104)(6×104)/2, i.e. >> $300M.

Advantages of soonest possible implementation: It is also noted that as p increases with exponential spread, and the relative advantages of either classical or binary pooling decline, costs would likely increase, also affecting the calculated value, further underlining the importance of rapid implementation, and implementation as early as possible in jurisdictions which still have low infection rates, p (which, due to latency period and the fact that many [likely over 66%] infected individuals remain asymptomatic, are likely higher than the prevalence of case reports suggests).

ELSI Considerations:

The completeness of surveillance across a population is an important factor affecting likelihood of elimination of a subject pathogen with latency and/or asymptomatic transmissibility, and so maintaining voluntary cooperation of individuals is essential. There already exists significant distrust of governments among disadvantaged populations (e.g. homeless, refugees, immigrant communities, impoverished communities, indigenous peoples, racial or ethnic minorities who perceive discrimination against them, those with history of criminal prosecution or perceived adverse interactions with law enforcement), associated psychological traumas are not uncommon in these communities, and many such communities would be the most problematic as reservoirs for incompletely eradicated emerging pathogens. It is highly preferable, therefore, to minimize the use of law enforcement, militia or military personnel directly in sample collection efforts if these can be avoided.

Similarly, use of samples for any purposes not strictly related to pandemic control would both be an abuse of implied patient consent, but would also undermine the trust essential for future pandemic control efforts.

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Acknowledgments:

We thank Dr. Lesley A. Perg for discussion, suggestions, editing, assistance in preparing this manuscript, support and encouragement, Dr. Carla Uranga for discussion, critique, editing of an earlier version and suggestions, Chloe Chun-Wing Lo for discussion and related maths analysis in progress, Alexis-Walid Ahmed, and Sarah Yang for discussion and evaluation of technical points, and Dr. David Odde, Ghaidan Shamsan, Dr. Douglas W. Smith, and Dr. Judith Zyskind for review and encouragement.

Funding: None

Competing Interests:

Eli Rabani has and continues to develop technologies pertinent to biological assays and diagnostics and other areas, including in commercial contexts, and has interests in private corporations engaged in technology R&D. The present disclosure is made on a pro-bono basis under emergent circumstances, for widespread implementation without any requirement for permission or compensation where respiratory pathogen pandemics have been declared, (with certain reservations; licensing terms for any pertinent intellectual property in progress; will restrict profit by manufacturers under such provisions to 6%). The author or associated organizations may in future develop devices, automated systems implementing same, and associated equipment, including for commercial purposes.

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Prior Manuscript References [to be merged]:

[] These are citations not yet incorporated or found as of this early draft; due to the exigent circumstances surrounding SARS-CoV-2, we feel it justified to circulate an incomplete manuscript.

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