key: cord-0901730-p65hhq2k
authors: Zowawi, Hosam M.; Alenazi, Thamer H.; AlOmaim, Waleed S.; Wazzan, Ahmad; Alsufayan, Abdullah; Hasanain, Rihab A.; Aldibasi, Omar S.; Althawadi, Sahar; Altamimi, Sarah A.; Mutabagani, Maysoon; Alamri, Maha; Almaghrabi, Reem S.; Al-Abdely, Hail M.; Memish, Ziad A.; Alqahtani, Saleh A.
title: Portable RT-PCR System: a Rapid and Scalable Diagnostic Tool for COVID-19 Testing
date: 2021-04-20
journal: J Clin Microbiol
DOI: 10.1128/jcm.03004-20
sha: 64210d67a86ea93b4cc8f2564abc501dd1a35d67
doc_id: 901730
cord_uid: p65hhq2k

Combating the ongoing coronavirus disease 2019 (COVID-19) pandemic demands accurate, rapid, and point-of-care testing with fast results to triage cases for isolation and treatment. The current testing relies on reverse transcriptase PCR (RT-PCR), which is routinely performed in well-equipped laboratories by trained professionals at specific locations. However, during busy periods, high numbers of samples queued for testing can delay the test results, impacting efforts to reduce the infection risk. Besides, the absence of well-established laboratories at remote sites and low-resourced environments can contribute to a silent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These reasons compel the need to accommodate point-of-care testing for COVID-19 that meets the ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable). This study assessed the agreement and accuracy of the portable Biomeme SARS-CoV-2 system against the gold standard tests. Nasopharyngeal and nasal swabs were used. Of the 192 samples tested using the Biomeme SARS-CoV-2 system, the results from 189 samples (98.4%) were in agreement with the reference standard-of-care RT-PCR testing for SARS-CoV-2. The portable system generated simultaneous results for nine samples in 80 min with high positive and negative percent agreements of 99.0% and 97.8%, respectively. We performed separate testing in a sealed glove box, offering complete biosafety containment. Thus, the Biomeme SARS-CoV-2 system can help decentralize COVID-19 testing and offer rapid test results for patients in remote and low-resourced settings.

isolate" is the cornerstone of breaking the transmission chain and controlling the outbreak. Rapid mass testing at a global level is critical, as stated by the Director-General of the World Health Organization (WHO), "You cannot fight a fire blindfolded, and we cannot stop this pandemic if we do not know who is infected. We have a simple message for all countries: test, test, test. Test every suspected case" (1) .

However, only well-established laboratories with sophisticated infrastructure, standard biosafety, and trained professionals can currently perform the RNA extraction and molecular testing with a high throughput or at point of care for COVID-19, and the number of positive cases is greatly influenced by the availability of these tests (2, 3) . Lack of infrastructure in remote or low-resourced localities and war or conflict zones impacts the overall positive cases and also increases the burden on major central laboratories testing for COVID-19, leading to delayed test results.

SARS-CoV-2 has a high infection rate and is easily transmitted among individuals, with a long incubation period. In addition, patients infected with SARS-CoV-2 can transmit the virus 2 days prior to the development of symptoms, and the virus is transmissible among many asymptomatic patients. So, people can unknowingly and rapidly transmit the infection among communities (3, 4) . Rapid testing and isolating infected cases, including asymptomatic or paucisymptomatic ones, is one of the most effective strategies to control the global spread of COVID-19 (5, 6) . However, the current testing structure has a lengthy turnaround time that can reach up to 48 to 72 h (7) . This delay can increase viral spread by infected individuals and, more specifically, asymptomatic individuals in communities. It can also increase viral exposure to health care workers and other patients in a hospital setting (8) . Results with a short turnaround time can help to immediately allocate resources that might be limited, such as health care resources, including isolation rooms and personal protective equipment (PPE) (9) . Thus, an affordable testing system for COVID-19 that is rapid, portable, and easily accessible in remote and low-resourced environments can significantly mitigate the spread of the virus in many communities.

To tackle the current crisis, portable point-of-care molecular, antigen, and antibody tests are becoming available to diagnose COVID-19. For example, the Accula SARS-CoV-2 C380/443/42). The portable RT-PCR system from Biomeme was assessed for its positive and negative percent agreements on a total of 192 samples to detect SARS-CoV-2 from nasopharyngeal and nasal swabs (mid-turbinate) using flocked swabs paired with 2 ml UTM virus transport medium (Mantacc, Miraclean Technology Co., Ltd.) (Fig. 1A) . The nasopharyngeal samples (n = 173) were collected in viral transport medium and tested for SARS-CoV-2 using the standard-of-care RT-PCR diagnostic tools. The nasopharyngeal samples in viral transport medium were stored at 270°C before testing. Of the 173 nasopharyngeal samples in viral transport medium that were assessed using the portable RT-PCR system, 92 samples were positive for SARS-CoV-2, while the remaining 81 were negative. The results from the Biomeme's portable RT-PCR system were compared with the routine standard-of-care RT-PCR for COVID-19 diagnosis.

The fresh nasal swabs (mid-turbinate) from one nostril were collected from 5 patients from the COVID-19 ward and 14 patients from the emergency ward at KFSHRC. All the samples were tested immediately for SARS-CoV-2 using the portable RT-PCR system at the point of care. Nasopharyngeal swabs were also collected from the same patients and transported on the same day in viral transport medium to the molecular virology laboratory for COVID-19 testing using the routine standard-of-care RT-PCR.

To achieve maximum biosafety while handling COVID-19 samples, two separate runs were tested inside a sealed portable glove box, which acted like a small biosafety hood (Fig. 1B) . The inner surface of the portable glove box was decontaminated with 70% ethanol before and after the tests.

Biomeme SARS-CoV-2 system. The portable, ultracompact, and user-friendly system from Biomeme Inc., (PA, USA) to identify SARS-CoV-2-positive patients was granted emergency use authorization (EUA) by the Food and Drug Administration (FDA) to on the 11th of August 2020. The system consists of four main components. The first component is the manual and equipment-free M1 sample prep cartridges for rapid nucleic acid extraction (Fig. 1A) . RNA was extracted in 1 to 2 min/sample with 750 ml of eluted extract. Biomeme RNA process/internal control (RPC) was introduced into each M1 sample prep cartridge at the beginning of the sample preparation process to demonstrate that the entire process was completed correctly for each specimen and control.

The second component includes Biomeme SARS-CoV-2 Go-Strips (Fig. 1A) . Each Go-Strip consists of three connected PCR tubes containing lyophilized master mix, multiplexed primers, and probes for the simultaneous qualitative detection of SARS-CoV-2-specific 1ab open reading frame gene (Orf1ab) and SARS-CoV-2-specific spike gene (S). The tubes also include the RPC (MS2) in a triplex reaction mixture. The RT-PCR was prepared according to the manufacturer's instructions by adding 20 ml of the eluted extract directly to the lyophilized master mix. The M1 sample prep cartridge and lyophilized Go-Strip pouches can be stored in a dry place, at room temperature (15 to 30°C), and they can remain stable for up to 18 months if unopened.

The third component is the Franklin three9 real-time PCR thermocycler, which runs on a chargeable battery for up to five PCR runs (Fig. 1A) . A unit can detect three different fluorophores per sample simultaneously but is limited to nine samples per run. The SARS-CoV-2 Orf1ab gene was detected by the 6carboxyfluorescein (FAM) fluorophore in the green channel, while the ATTO647N fluorophore detected the SARS-CoV-2 S gene in the red channel, and the TexasRedX fluorophore detected the RPC in the amber channel. Reactions were amplified with an initial reverse transcriptase (RT) step at 55°C for a 2min hold, followed by an initial denaturation step at 95°C for a 1-min hold and 45 cycles at 95°C for 3 s and 60°C for 30 s.

Results were considered positive when one or both SARS-CoV-2-specific Orf1ab or S genes were detected in the same sample, regardless of the amplification or failure of the RPC. However, any negative results due to the absence of both SARS-CoV-2-specific genes would require positive amplification of the RPC to rule out false-negative results that might occur due to inactive reaction or PCR inhibitors in the sample. Thus, any sample with negative RT-PCR results on both SARS-CoV-2-specific genes as well as RPC would be considered invalid and subjective to reextraction and retesting.

The fourth and last component is a smartphone with Biomeme Go mobile app for thermocycler operation, data entry, and visualizing the test results (Fig. 1A) .

Portability of the Biomeme SARS-CoV-2 system. Each M1 sample prep cartridge weighed 20 g, and the cartridges used for the patient testing in the emergency and COVID-19 wards weighed 380 g in total, which were carried in a medium-sized backpack (TUMI Inc.; height, 19 in.; width, 14 in.; depth, 6 in.) weighing 1.6 kg in total. It also included a pouch containing 96 Go-Strips with the lyophilized master mix for SARS-CoV-2 weighing 62 g, a 20-to 200-ml adjustable-volume micropipette (Amplyus, MA) weighing 84 g, a bag with 150 1.5-ml tubes (Eppendorf, Germany) weighing 160 g, a polypropylene microcentrifuge PCR 80-well tube rack weighing 150 g, and two boxes of 300-ml pipette tips (Eppendorf, Germany) weighing 120 g each. The entire package weighed around 3 kg, and all items fitted nicely inside the backpack.

The battery-powered Franklin three9 real-time PCR thermocycler alongside a smartphone and their chargers were carried inside a small waterproof case with inside foam (Seahorse, CA; length, 13.6 in.; width, 10.7 in.; height, 6.3 in.) weighing 3.6 kg in total. The portable glove box weighs 11.3 kg, with overall dimensions of 27-in. width, 13-in. depth, and 22-in. height. All components needed to perform a complete run using the Biomeme SARS-CoV-2 system fit inside the portable glove box (Fig. 1B) .

Standard-of-care RT-PCR testing for SARS-CoV-2. Nasopharyngeal swabs transported in viral transport medium were processed at the molecular virology laboratory at KFSHRC. Four platforms were used for the routine diagnosis of SARS-CoV-2. The first platform used the Abbott m2000 system (Abbott, IL) for high-throughput RNA extraction, and elutions were tested using the RealStar SARS-CoV-2 RT-PCR kit (Altona Diagnostics, Germany) and amplified on the Rotor-Gene Q real-time PCR cycler (Qiagen, Germany). The second platform used the Applied Biosystems MagMAX viral RNA kit on the KingFisher instrument (Thermo Fisher Scientific) for high-throughput RNA extraction, and elutions were tested using the DiaPlexQ novel coronavirus detection kit (2019-nCoV) (SolGent, South Korea) and amplified on Applied Biosystem 7500 real-time PCR system (Thermo Fisher Scientific). The third platform is the sample-to-result Abbott m2000 RNA extraction system combined with the Abbott RealTime SARS-CoV-2 assay, and the fourth platform is the point-of-care GeneXpert using the Xpert Xpress SARS-CoV-2 test (Cephid, CA). The samples were tested according to the manufacturer's protocols.

Statistical analysis. The sample size for this project was calculated based on the following parameters: anticipated sensitivity of 93%, precision level of 0.05, an alpha level of 0.05, and proportion of positive samples of 52%. Thus, the sample size of this project was 192 samples, of which 100 were positive by standard-of-care RT-PCR tests. We assessed the agreement and accuracy of the portable Biomeme SARS-CoV-2 system against the gold standard tests using various diagnostic test evaluation measures, including positive percent agreement, negative percent agreement, positive predictive value, and negative predictive value. Furthermore, we assessed the correlation between the two tests according to positive cycle threshold (C T ) values using the Pearson's correlation coefficient. The values used for the correlation analysis are the C T value of the S gene generated by the deployable RT-PCR Biomeme SARS-CoV-2 system and the minimum C T values generated by the standard-of-care RT-PCR systems. Diagnostic test evaluation was applied using SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and correlation analysis was applied using R v3.5.

Samples. The majority (51%) of the tested samples originated from female patients. The average age of all patients was 39.3 6 19 years. Only 12% of total patients were asymptomatic, but 55% of the patients who tested positive were hospitalized, of which 14 received intensive care unit (ICU) care and four patients died. One patient was reinfected recently after reporting a full recovery. More on the clinical data, demographics, symptoms, and comorbidities are summarized in Table S1 in the supplemental material.

Of the 192 samples tested using the portable Biomeme SARS-CoV-2 system, the results from 189 samples (98.4%) were in agreement with the reference standardof-care RT-PCR testing for COVID-19. The results are summarized in Table S1 in the supplemental material. Of these 189 concordant samples, 99 samples were confirmed positive for SARS-CoV-2, and 90 samples were confirmed negative for SARS-CoV-2 (Table 1) .

Of the 173 previously tested samples using the portable Biomeme SARS-CoV-2 system, 91 samples were correctly detected as SARS-CoV-2 positive and 80 samples as SARS-CoV-2 negative, which matched the reference results from the standard-of-care RT-PCR testing. Only one sample tested negative in the portable Biomeme SARS-CoV-2 system but tested positive in the standard-of-care RT-PCR method using Abbott RealTime SARS-CoV-2 with a CN value (Abbott readout value) of 6.5 (Table S1 ). One sample provided a positive result by the portable Biomeme SARS-CoV-2 system, with high C T values of 39.55 for Orf1ab and 37.55 for the S gene, but was reported negative by the standard-of-care RT-PCR method using the Xpert Xpress SARS-CoV-2 platform (Table S1) .

Of all tested samples, 10 samples (5.2%) initially provided invalid results and had to be reextracted and retested. Only one positive sample (10%) for SARS-CoV-2 and nine negative samples for SARS-CoV-2 (90%) provided invalid results. Valid results were successfully achieved after one repeat of reextraction and retesting the same samples. On the other hand, 94.8% of the samples provided valid results from the first run ( Table 2) .

The results of the five patients from the COVID-19 ward and 14 COVID-19 suspected patients from the emergency ward had a 95% agreement with the reference standardof-care RT-PCR testing for SARS-CoV-2. Only one sample from an emergency department patient with a suspected case showed a discrepant result. The portable Biomeme SARS-CoV-2 system reported positive RT-PCR with a high C T value (42.27) of the S gene, but the reference standard-of-care RT-PCR method for SARS-CoV-2 (Abbott RealTime SARS-CoV-2) showed a negative result (Table 1) . Test performance. The portable Biomeme SARS-CoV-2 system had positive percent agreement and negative percent agreement of 99.0% and 97.8%, respectively, compared to the reference results from the standard-of-care RT-PCR system for COVID-19. The positive predictive value was 98.0%, and the negative predictive value was 98.9%. The overall agreement of the portable Biomeme SARS-CoV-2 system versus the reference tests was 98.4% (Table 1) .

The portable Biomeme SARS-CoV-2 system was used at the point of care at the emergency department and COVID-19 ward versus the standard-of-care tests, including the Xpert Xpress SARS-CoV-2 platform, which had to be conducted at the molecular virology laboratory. The overall turnaround time for the portable RT-PCR system was only approximately 80 min, which included sample preparation (RNA extraction), sample loading, data entry, and assay run time for a total of nine samples (Table 3 ). This turnaround time helped to rapidly identify four emergency department patients who tested positive for COVID-19, and the results were later confirmed by the standard-of-care tests.

C T values. The S gene was amplified by the Biomeme SARS-CoV-2 system from all positive samples (n = 101), and the Orf1ab gene was not amplified from 16 (15.4%) (Fig. 2) . The RPC was detected in 43 of 101 (42.57%) samples with positive SARS-CoV-2 results. Due to the amplification of at least one of the SARS-CoV-2-specific genes, the absent amplifications of the RPC did not impact the interpretation of the positive results, which aligned with the results of the standard-of-care tests (Table S1 ). Most importantly, the RPC was detected in all (100%) negative samples, including the nine samples that were initially associated with invalid results and were later confirmed as COVID-19 negative after reextraction and retesting. The negative results aligned with the results of the standard-of-care tests (Table S1 ). The mean C T value of the RPC was 32.57 6 5.1 (9.2 to 44.76) (n = 134) (Fig. 2) .

We assessed the correlation of the deployable Biomeme SARS-CoV-2 system with the standard-of-care tests using the Pearson's correlation coefficient. Our result indicates a positive correlation between the C T values of the S gene and the minimum C T value of the matching samples generated by the standard-of-care systems with R coefficient of 0.583 and a P value of ,0.0001 (Fig. 3) . Thus, we excluded samples with CN values, because these values are specific values reported by the Abbott RealTime SARS-CoV-2 assay and not directly comparable to C T values (17) .

Given the current challenges of the COVID-19 pandemic, a rapid testing system is critical to mitigating the further spread of the disease (18, 19) . Despite multiple tests being available widely, RT-PCR remains the current gold-standard for SARS-CoV-2 detection (3). However, limitations such as the requirement of large laboratory equipment with continuous power supply, available trained professionals, sample throughput restrictions, and specific biosafety requirements impact the accessibility of the tests in remote and low-resourced environments and restrict point-of-care testing.

Our study independently evaluated a completely portable solution to test COVID-19 that can be easily made available in remote locations. The portable Biomeme SARS-CoV-2 system combined with a portable glove box could rapidly test samples within 80 min while adhering to strict biosafety measures. We tested clinical samples and compared the results against the results from the routine standard-of-care testing for COVID-19. The Biomeme SARS-CoV-2 system demonstrated superior diagnostic performance with 99.0% positive percent agreement and 97.8% negative percent agreement. The overall percent agreement between the two systems was excellent (98.4%). Also, having only around 5.2% of samples that provided invalid results demonstrated the test's efficiency to mostly (94.79%) deliver valid and reliable results from the first run. The observed high performance, correlated C T values between the Biomeme SARS-CoV-2 system and the reference methods, and their comparable characteristics demonstrate the reliable clinical testing of COVID-19. Three discordant samples were identified in our study using the Biomeme system. First was a false negative, which might have occurred due to the use of a previous sample that was subjected to initial testing and several thawing and freezing cycles. It is well known that freeze-thaw cycles can affect the nucleic acid stability leading to RNA degradation, which may affect the sensitivity of RT-PCR (20) . In an independent run, we tried to retest the same sample using the deployable Biomeme SARS-CoV-2 system, and it tested positive for both genes. The two other discrepant results were false positives, which might have resulted from accidental contamination, viral load below the detection limit of the reference test, or unspecific amplification due to primer dimers. The latter is suspected due to the exceptionally high C T values, including one sample with only one S gene amplification. It is worth mentioning that the manufacturer has reported the limit of detection of the Biomeme SARS-CoV-2 assay as 1.8 genome equivalents per ml, but no further validation for the limit of detection of the assay was conducted as part of this study.

Due to the increasing global concerns related to the emergence and spread of new SARS-CoV-2 variants, the U.S. FDA issued a letter to health care providers on 8 January 2021 warning that the genetic visitants of SARS-CoV-2 may lead to false-negative results with molecular tests for the detection of SARS-CoV-2 (21) . Biomeme has later confirmed, through in silico analysis of the genetic sequences, that the SARS-CoV-2 assay detects the new variants reported by the United Kingdom, lineage B.1.1.7, South Africa, lineage B.1.351, and Brazil, lineage B.1.1.28 (22) .

Also, our study validates the portable Biomeme SARS-CoV-2 system to be suitable for molecular point-of-care testing with high accuracy for the detection of SARS-CoV-2 compared to that of the routine standard-of-care testing. The total sample-to-result time, including hands-on time and run time, was 80 min for a batch of nine samples and was comparable to the Xpert Xpress SARS-CoV-2 platform, which could test several samples at once within 45 min in a multimodule configuration instrument (such as GX-XVI) (23) . However, multimodule GenXpert models are large, are not easily affordable, require a continuous power supply, and cannot be easily deployed at point-of-care settings.

In addition to diagnostic performance, sample throughput, simplicity of use, and biosafety measures are also critical factors to consider. The M1 sample prep cartridge has several benefits besides being a quick, user-friendly, and equipment-free method. It offers an immediate virus deactivation by placing the UTM directly into the lysis buffer. This alternative step to heat deactivation, as described by Harrington et al. (24) , adds at least 30 min to the testing turnaround-time, which could affect the RNA stability (24) (25) (26) . Also, rapid RNA extraction can be performed directly on fresh nasal swabs without collecting in viral transport medium, which could minimize sample dilution. Most importantly, this protocol is timely, as it will not be affected by the global limitation of transport media (27) (28) (29) .

Collecting nasopharyngeal swabs for COVID-19 testing can be associated with stress, anxiety, and hazardous exposure for health care workers (30, 31) . Hence, samples from nasal swabs (mid-turbinate) and saliva have been studied as alternative specimens for detecting SARS-CoV-2 using RT-PCR (32) (33) (34) (35) (36) (37) . Here, we demonstrated an agreement in SARS-CoV-2 testing between the fresh nasal swabs (mid-turbinate) versus the nasopharyngeal swabs collected in viral transport medium from patients at the emergency ward and COVID-19 ward. However, more nasal swabs will need to be tested using the Biomeme SARS-CoV-2 system to ensure their robustness as an alternative to nasopharyngeal swabs, which may later allow patients to self-collect their samples and thereby minimize exposure to health care workers.

Handling COVID-19 samples can place laboratory staff at significant risk for laboratory-acquired infections by SARS-CoV-2. Hence, biosafety prevention and control measures have been placed to limit any risks. These include performing procedures that may entail potential exposure to aerosols or droplets (such as vortexing) in a certified class II biological safety cabinet placed inside a negative pressure environment (38) . However, these procedures require large equipment and expensive infrastructure that can be absent in low-resourced settings, which can centralize the testing to be limited only to well-established infrastructures such as major public health laboratories. The use of the RNA extraction protocol and performing the entire testing process inside a portable and fully enclosed glove box can provide a safe solution to conduct field-testing and at the point of care for COVID-19 anywhere in the world.

The Biomeme master mixes combined with primers and probes are lyophilized into beads that are prepackaged in quantitative PCR (qPCR) tube "Go-Strips" and are shelf stable for up to 18 months. This feature eliminates the need for a cold supply chain and further enhances this platform's field utility to conduct testing for infectious diseases in the community and low-resourced settings. The ultracompact system is even flexible enough to use other RNA extraction kits and test in-house assays using the Franklin thermocycler (validated by our team, but data not shown in this study). Also, the use of a smartphone-enabled geolocation feature can even help in the epidemiological surveillance and tracking of pathogens, including SARS-CoV-2. Lastly, if an Internet connection is available, Biomeme Cloud offers a platform for remote access to the results and raw data (e.g., amplification curves) for each run.

The main limitation of this study is the restriction to a single site. A multicenter evaluation using several deployable Biomeme SARS-CoV-2 systems would provide a comprehensive analysis of the system's reproductivity for COVID-19 testing. A multicenter evaluation would also allow testing for more samples, allowing for a lower margin of error for the performance matrix. This is particularly important when testing the nasal swabs (mid-turbinate) to fully evaluate their suitability to be used routinely for COVID-19 testing. Another limitation of our study is to evaluate the possibility for sample pooling on the Biomeme SARS-CoV-2 system to test several samples at the same time. The evaluation of pooled sample data would require a separate study to test a large number of samples, at separate runs and sites, to ensure pooling's suitability to assess the testing capacity efficiently and confidently. Our study was limited to the use of the Biomeme SARS-CoV-2 system for point-ofcare testing at a clinical setting. Independent studies would need to be carried out at fields with limited resources/trainings to mimic real-life scenarios that may associate with clinical challenges. Yet, it is important to note that the EUA for the Biomeme SARS-CoV-2 system is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a that meet requirements to perform high-complexity tests. Lastly, the Franklin thermocycler has only nine sample wells. This integral feature can limit the number of samples tested per run to nine samples, except if more Franklin thermocyclers are used simultaneously. Another integral feature is the Go-Strips, which requires batching at least three samples per run.

In summary, we have evaluated a highly sensitive, specific, and deployable system that offers point-of-care testing for SARS-CoV-2 anywhere in the world, allowing for the decentralization of infectious disease testing. We have demonstrated the system's ability to rapidly perform RNA extraction with limited equipment to test nine samples at once. To eliminate any biosafety concerns, we also incorporated the fully sealed portable glove box containing the entire testing process. Given the lengthy testing times for standard-of-care tests, our results showed significantly reduced testing times to 80 min, allowing for rapid triaging decisions.

Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.3 MB. We declare no competing interests.

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