RADx Submission
July 2020

FloodLAMP Biotechnologies is a Deleware Public Benefit Corporation that was spun out from a STEM education 501(c)3 non-profit Focus on Foundations. While still at the non-profit, founder Randy True and a small team applied for a RADx. Unfortunately, we did not make the cut and it seems that most or all of the RADx funding was allocated shortly after we applied. Despite this, FloodLAMP has achieved several of our milestones such as successful development of a full plate based, high throughput assay SOP, establishing high sensitivity with an LOD at 2 cp/ul,  and building the app for streamlined sample pooling. Additionally, the filing of our pre-EUA and organizing Open EUA Consortium are significant achievements. There have been some changes to the technical details of our plans, but the main goal of building out an ultra-low-cost, instrument-free molecular test for mass screening has remained the same.

The COVID-19 crisis is the worst public health disaster of our time, and the U.S. does not have a comprehensive testing strategy to contain the pandemic. New mass screening needs to be scaled quickly and coordinated with the current testing capacity. Fortunately, no new breakthroughs are needed to achieve this. A major cooperative effort, however, is urgently needed. We propose a startup-like nonprofit to build the mass screening program, supported by government and academic institutions. The assay/program:
  • flexibly pools to hundreds of samples per tube
  • has already demonstrated 10 sample pools on real saliva;
  • uses standard lab equipment, no complex instrumentation;
  • uses only readily available chemicals for front end inactivation and RNA purification;
  • uses colorimetric LAMP for fast, easy, binary readout;
  • is 100% additive to current testing;
  • is extremely inexpensive ($1-2/reaction);
  • uses saliva or swabs;
  • avoids commercial RNA extraction kits;
  • avoids supply chain problems;
  • has already been clinically validated;
  • was independently chosen for large scale screening by several groups.
$2.4M covers equipment for 600 stations (nominally 5 stations/lab but flexible). Each station runs 1,000 tubes/day. Central production and distribution of ready-to-use reagents, coupled with extensive training and support, achieve a massive economy of scale. This new program, in coordination with current testing, enables us to scale to identify almost all active infections, containing outbreaks. At this testing volume, we can effectively end the COVID crisis, first in the U.S., then in the rest of the world.

Manufacturer
New England Biolabs

Product name and model number
WarmStart Colorimetric LAMP 2X master mix M1800B-1L

CLIA status
Waived

Sampling
Saliva, Nasal swab, Oral swab

PPE/Biosafety Requirements
Safety is a top priority for the program, as are sample integrity and system efficiency. The primary mode of sampling is self collection, either at-home or on-site (i.e. a school or workplace). Routine COVID safety precautions will be practiced: masks, hand washing, and physical distance. A viral inactivation buffer (TCEP/EDTA) and heat step are used, greatly reducing risk. Lab
personnel will follow BLS2 level requirements when possible. For on-site collection, personnel will typically inactivate samples prior to transport. If so, they will utilize a gown, N95 mask, and face shield while doing the inactivation, assuming they are available.

Level of Detection (LoD)
10 genomes = 2 copies per μl x 5μl

Sensitivity
80%

Specificity
100%

Test Time (from sample handling to readout)
70 minutes

Suitable for asymptomatic testing?
Yes

Current capacity
450

Expected increased capacity
6,800,000

Scenario
The above throughputs are for a single station and for 616 stations, both running in a pool-of-pools format with 450 samples per tube. The relevant metric for the large scale program we are building is the population level that can be screened per day, at what upfront capital cost and running cost. With 5 stations per lab, we can screen approximately 1.3M people per day by adding $20K of readily available, standard equipment to existing labs, at a labor+materials cost of about $30K/day. That would require staffing at each 5-station lab of about 50 technicians. The staffing and preparation of materials is done at a centralized facility for distribution to 100 such labs across the U.S. For the sample collection program, we anticipate large numbers of volunteers helping with coordination of screening their immediate communities.

The above scenario is for “green” populations, where the region or demographic has a prevalence of 1 in 30,000 (or less). Here, the labs receive pre-pooled tubes of 10 samples (0.5ml x 10 = 5ml in a 15ml centrifuge tube). They pull .67ml (13%) from 45 such tubes to make a pool of pools containing samples from 450 individuals. These 30ml are processed in a scaled up version of the Rabe Cepko assay protocol [1], in a 50ml tube. The volumes, pool sizes, and labware are all chosen to optimize lab efficiency.

A cornerstone of the screening program is the pooling flexibility. The same lab station can process tubes screening for the entire range of currently infectious prevalence:

Green: 1 in 30,0000 – 450/10 pooling, 266K people/day
Yellow: 1 in 1,000 – 100/10 pooling, 36K people/day
Orange: 1 in 100 – 10/1 pooling, 5K people/day
Red: 1 in 33 – 5/1 pooling, 3K people/day

The key to achieving this pooling flexibility is the liquid phase nucleic acid binding step, currently accomplished with ultra inexpensive prepared silica microparticles. Alternatively the same can be accomplished with magnetic capture beads, and New Zealand has developed a low cost version of this that may be investigated.

Sampling is done very efficiently with at-home and on-site pooled self collection schemes. For both saliva and swab sample types, participants place their samples into the common pooled tube immediately. Under some high prevalence scenarios and for FDA EUA submission, participants will simultaneously collect individual samples, which will be bagged (barcode matching pooled tube), stored, and then only accessed if the pool is positive.

Samples are inactivated with TCEP/EDTA at a drop-off location or on-site. Pooled, inactivated tubes are delivered to the lab where processing proceeds with secondary pooling, followed by the assay protocol consisting of silica “glass milk” RNA purification and concentration, then the RT-LAMP reaction. Importantly, the highly pooled concentrated sample can go into a PCR reaction instead of LAMP, making the whole front end infrastructure plug-and-play with current PCR capacity.

[1] Rabe B, Cepko C. medXriv 4-23-20 https://www.medrxiv.org/content/10.1101/2020.04.23.20076877v1

Sample Collection Coordinator

Description of duties: Oversees the sample collection at staffed drop off locations. Provides tubes and consumables. Ensure safety procedures are followed. Prepares collected tubes for safe transport back to the lab by courier.
Expected level of training required: Can be certified with short on-line course
Number required: 4,400

Courier

Description of duties: Transports sample tubes in proper containment vessels (biosafety case), from sample collection site to lab. Using a separate courier to do this allows the Site Coordinator to continue to supervising the next batch of samples from the same site or to drive to a new site.
Expected level of training required: No training
Number required: 900

Lab Technician

Description of duties: Perform assay in the lab. Pipetting and standard incubation steps. Cleanliness and attention to detail a must. Since it’s a single protocol with a small number of
step, training of new technicians can be accomplished in one week.
Expected level of training required: Requires professional training
Number required: 5,000/p>

$10,000,000

$1 per individual result

Overall this proposal has very favorable supply chain characteristics.

“Since the required reagents are easily manufactured by multiple manufacturers, access to this test does not rely on traditional commercial diagnostic supply chains that have hindered the broad distribution of SARS-CoV-2 testing. In addition, there is no need for de novo manufacturing. The reagents needed for this assay can be purchased by any laboratory from a number of sources, except for the colorimetric RT-LAMP master mix where the manufacturer has large-scale production in place with millions of reactions worth of product available.” [2].

“A single lab can prepare enough for tens of millions of purifications in a day at a cost of less than $45 per 1 million purifications.” [1]

Primary bulk reagents needed are: TCEP, EDTA, Tris, PBS, EtOH, NaI, NaCL.

The immediate pooling of 10 samples greatly saves on plastic tubes and tips. A wide variety of tubes may be used for the sample collection. For saliva, straws and widemouth tubes are used for collection, and transfer pipettes for self pooling. For nasal swabs, a variety of products can be validated. LAMP reactions are carried out in readily available 0.2ml PCR strip tubes. The supply chain for these will be investigated and dedicated manufacturing secured if necessary. 

[2] Anahtar M. Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification. medXriv preprint 5-12-20.

https://www.medrxiv.org/content/10.1101/2020.05.12.20095638v1

Data will be handled in the standard channels for clinical diagnostic testing, adding sample pooling information. Custom software will be used for sample registration, lab tracking, and results communication. Existing LIMS will be used for the lab.

The primary data generated will be name and contact information of the participants and the day/time of sample collection. Data sharing will be the subject of agreements and may vary by jurisdiction or group. Typically, transparency will be desirable and opt-in disclosure urged for members of groups that coordinate to be tested together because they belong to the same school or workplace. We will facilitate classification of participants into risk groups for optimal pooling through the use of transparent software tools, potentially including an anonymous version.

The important non-PII data from the screening program will be the geographic regions, participating organizations, and frequency of screening. The program will strongly encourage all participants to share this information in a legal way, and will create data visualizations and API access. This high degree of transparency will facilitate public trust in the program and encourage widespread adoption. Further, it will encourage volunteer efforts.

What is crucially needed to realize this program is immediate support, in the form of 1) funding to rapidly grow the team and 2) connections to help the program integrate with existing public health efforts. Because there are no technical or scientific barriers, our efforts will primarily be focused on execution: scaling through sample pooling and launching an ambitious communications and sample collection scheme. The full Rabe Cepko assay protocol has been clinically validated [2 Anahtar reference above]. The RT-LAMP step is well proven, with an EUA by Color Genomics on 5/18. More recently, on 7/9 NEB launched an improved formulation of the master mix along with a new RUO product – SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit. LAMP is exploding, being used at universities across the country and around the world (i.e. Univ of SC, Cornell, Univ of Vienna, Columbia).

Funding
Proper funding will dramatically accelerate all parts of the program in parallel: the pooling level LOD characterization, the clinical validation with partners toward IVD and LDT EUA’s, the central lab production scale up of ready-to-go kits for distribution, the component stability studies, the pilot programs, the app and website development, the user testing for sample collection modes, the recruitment of labs across the country, the production of training materials, and the outreach to ready the public for participation.

Connections and Integration
Connections with public health stakeholders and government decision makers will enable us to lead an awareness campaign of the program’s capability and create a distributed pull for its success. LAMP-based screening will likely have a significant impact on testing over the next 6 months, however with RADx support of this program, it can have a massive impact over the next 2 months, in time for school openings in the fall. We believe that a coordinated, realistic effort for identifying current infections and catching new ones will sway public sentiment and change the national dialogue from one of confusion and fear to one of collective effort and optimism. The vaccine is the long term solution. This mass screening program is the near term solution.

Acceptance of Sample Pooling
China tested 9M people in 10 days in May. Amazon is building a central mega lab with 50K/day capacity, intending to test 1M employees. Both China and Amazon are doing pooling. They understand that pooling is critical to achieve the scale needed to identify active infections among a large population. The importance of pooling as a means for effectively screening large populations has been understood by some in the U.S. at least since early April. Stanford demonstrated it and published a preprint. However, they have not used it and recently stated they have no plans to because of their current excess unused capacity, running at the 20-25% utilization of their 5K/day. Other high throughput academic labs have had similar low utilization of their capacity (CZI/UCSF).

This low utilization is because these labs’ capacities have not been mated to ambitious, scalable sample collection programs and also because, in many cases, these labs are running expensive tests or have components with supply chain bottlenecks. Some testing companies are looking into pooling (Color) but the financial incentives are not in place. CMS high throughput reimbursement is $100/individual test, which is what companies and labs have invested in setting up. They are not incentivized to deviate from their current business plans and invest in modifying their protocols for pooling, especially with insistence from the FDA in their June 26th pooled guidance that pooled tests have sensitivities close to the gold standard PCR level.

The U.S. currently has a testing capacity of 600K/day, but because that capacity is fractured, uncoordinated, and poorly prioritized, it has been far less effective at stopping the spread than it otherwise could be. Moreover, we have not deployed pooling except in a few small instances (Nebraska). Pooling could be used to great effect to screen large metro areas or hot spot states. The costs that American society is paying for our failures in COVID testing is immense. Emergency action is overdue.

One path forward, probably the best one, is top down control of all clinical diagnostic testing, via the War Production Act. Assuming that’s not likely, the best alternative is quickly building new testing capacity that is totally additive and does not cannibalizes the current diagnostic supply chain. The proposed program does that.

Program Staffing and Support
The sooner this program is brought online, the greater the impact. What is needed to make this happen quickly is to bring other complete teams onboard. Those teams need to have the experience and cohesion to execute on discrete chunks, and strong leadership to integrate and coordinate with the overall program. A good source of such teams is other RADx applicants who are passionate about the cause and hungry for funding for their teams. It is likely that many of them have the expertise needed and could be financially incentivized to pivot for a discrete time frame.

We seek assistance from other major biomedical nonprofits and research institutes, such as CZI, Gates, and the Broad, who could greatly accelerate progress. These institutions already have funding, facilities, and very knowledgeable staff. The hope is that by making the details of this program public, along with strong support from the NIH and BARDA through RADx, such groups would be galvanized into action. Those organizations alone could catalyze this plan, but collectively we can reach the goal much faster. The U.S. needs a viable plan out of this crisis, one that calls upon government, scientific, and philanthropic institutions to collaborate in the service of our return to a thriving, open society. If we act now, we can conceivably end COVID-19 in the U.S. and reopen our schools safely this Fall. This proposal, with RADx support, can achieve this goal.

Budget: $1M

Duration: 4 weeks

Deliverable: Standard operating procedure of inactivation and assay protocol

  • Domain: Technical/Science
  • % of Budget: 5%
  • Success: The SOP is a success if newly trained lab techs can routinely run it successfully after just a day of training (or few days depending on previous wet lab experience). The protocol is currently being optimized for throughput, robustness and sensitivity. Success of the SOP further means it meets
    the target specs of 1hr pipelined batches, < 2hr sample to answer turnaround, <5% failure rate, and < 1 copy/μL LoD.

Deliverable: Pilot mobile app for registration, sample collection, results

  •  Domain: Commercial
  • % of Budget: 5%
  • Success: The initial pilot app will be created on a secure and rapid, low-code development platform with a current collaborator, Appivo. Research and design have already started, including the user journey story boards and mock-up prototypes. Success involves implementing an iterative process starting with a basic app for pilot studies, obtaining valuable user feedback, and making changes as necessary. The mobile app will support a wide variety of Apple and Android devices to maximize outreach/penetration.

Deliverable: LDT EUA submitted with clinical partner

  • Domain: Regulatory
  • % of Budget: 10%
  • Success: Success is meeting the criteria described in the FDA guidance for pooled SARS-CoV-2 testing (July 6) and filing and LDT EUA with a clinical partner. Specifically, that is identifying a single positive sample in a pool with N-1 negative samples in 30 different pools, and also getting a negative result for 30 pools with all negative samples. Measuring an LoD capable of identifying weak positive samples, i.e. an analytical sensitivity close to the gold standard RT-PCR. Screening can being right away by the CLIA lab of the clinical partner with an accompanying LDT EUA submission.

Deliverable: Large pilots using multiple pooling strategies

  • Domain: Clinical/Workflow
  • % of Budget: 78%
  • Success: Success for the pilots involve smooth execution, devoid of hiccups such as running out of key supplies, errors with reagents that result in scrapped samples, and problems with the website or software. Ultimately success means the participants are satisfied with the experience, and both they and us have confidence in the results they receive. Some of the pilots will be run under IRB’s and we will investigate the performance at various pool levels, including 2 stage pooling for populations with very low prevalence (< 1:30,000 “green” threshold).

Deliverable: Scaling Plan

  • Domain: Commercial
  • % of Budget: 2%
  • Success: Success here means that independent experts have thoroughly vetted the scaling plan. The plan involves expanding lab capacity to run this assay in 3 ways: 1) utilizing existing equipment already in place in a lab, augmenting parts if necessary; 2) the installation of new equipment sets; and 3) creating an equipment loaner program for other labs to place equipment into a pool that can be accessed by those labs running this assay. Success is a clear description of those 3 ways which outsiders judge to be a viable plan.

Budget: $10M

Duration: 6 weeks

Deliverable: Product App and Website

  • Domain: Commercial
  • % of Budget: 5%
  • Success: The app will be further enhanced with features and additional cloud compute resources. Success means capturing customer data, ongoing sample collection data and displaying results. We will also have a sharing component which will be successful if used by 30% of participants.

Deliverable: IVD EUA Submission

  • Domain: Regulatory
  • % of Budget: 5%
  • Success: The additional requirements for an IVD EUA submission will be determined and provided, such as the Fact Sheet for Healthcare Providers, Fact Sheet for Patients, and the Instructions For Use. The FDA’s “Molecular Diagnostic Template for Commercial Manufacturers” will be closely followed to produce an IVD EUA submission.

Deliverable: 4M Reactions

  • Domain: Commercial
  • % of Budget: 50%
  • Success: Pilot manufacturing of assay components such as the inactivation solution, binding solution, glass milk, and LAMP+primer reaction plate will already be underway. This next phase entails the scale up under good manufacturing practices. Consultants with experience in IVD manufacturing will be utilized and we will engage a CMO for the volume production runs.

Deliverable: 600 Stations Buildout

  • Domain: Commercial
  • % of Budget: 30%
  • Success: Many labs will already have the necessary equipment for running this assay (pipettors, heaters, centrifuges). Regardless, we will budget for and procure all of the equipment necessary to run at the designed throughput. In additional, we’ll launch an equipment donation and loaner program for biotech companies and academic labs to help the cause.

Deliverable: Training and support program

  • Domain: Commercial
  • % of Budget: 10%
  • Success: A suite of training materials will be produced for all aspects of the program. These will be used by our own staff and made publicly available for groups for use in standing up their own labs, whether in the U.S. or international. Videographers, animators, and web graphic designers will create material to be incorporated into active learning, mastery based modules. 24/7 phone and web support will be put in place with experienced staff on hand to help troubleshoot problems labs may be having. Success is thousands of lab techs trained and successfully running the assay.