key: cord-1054741-5dt5a0sc authors: Banada, P. P.; Park, C.; Elson, D.; Daivaa, N.; Desind, S.; Kwiatkowski, R.; Chakravorty, S.; Alland, D.; Xie, Y. L. title: Evaluation of sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing date: 2021-03-05 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2021.03.03.21251172 sha: 67551b87917449d9a3f62f550b576c120f88df2c doc_id: 1054741 cord_uid: 5dt5a0sc Sensitive, accessible, and biosafe sampling methods for COVID-19 reverse-transcriptase polymerase chain reaction (RT-PCR) assays are needed for frequent and widespread testing. We systematically evaluated diagnostic yield across different sample collection and transport workflows, including the incorporation of a viral inactivation buffer. We prospectively collected nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal and oral swabs were placed in both viral transport media (VTM) and eNAT, a sterilizing transport buffer, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert) test. The sensitivity of each sampling strategy was compared using a composite positive standard. Overall, swab specimens collected in eNAT showed superior sensitivity compared to swabs in VTM (70% vs 57%, P=0.0022). Direct saliva 90.5%, (95% CI: 82%, 95%), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50%, P<0.001) or eNAT (67.8%, P=0.0012) and oral swabs in VTM (50%, P<0.0001) or eNAT (56%, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert test; however, no single sample matrix identified all positive cases. Accurate, efficient, and biosafe detection of SARS-CoV-2 in both symptomatic and asymptomatic 41 individuals with active COVID-19 infection is an essential public health strategy for preventing 42 transmission and controlling the COVID-19 pandemic. Although nasopharyngeal (NP) swabs are 43 a preferred specimen type, the invasiveness of this procedure, potential for variable collection 44 quality, need for supervised collection with biohazard risks have hindered the scalability of this 45 testing method [1] [2] [3] [4] . Rather, there has been progress in establishing utility of non-invasive sampling 46 methods that use saliva 5-7 or self-collected nasal or oral swabs 8, 9 which can enable broader testing 47 of at-risk populations and decrease exposure risk to healthcare workers 10 . When combined with 48 self-testing, wider implementation of rapid, CLIA-waived COVID-19 assays 11,12 could 49 dramatically increase public access to tests for SARS-COV-2 infection by expanding testing in at-50 risk locations including school and the workplace. However, the feasibility of scaling these non-51 invasive samples for widespread testing outside of carefully controlled environments remains 52 limited by the operational and biosafety challenges. 53 Our group has recently demonstrated that a guanidine-thiocyanate transport buffer (eNAT™, 54 Copan diagnostics Murrieta, CA) leads to viral inactivation, with at least 5-log reduction in viable 55 SARS-COV-2, and stabilization of viral RNA in a sample 13 . With the premise that eNAT could 56 optimize testing yield while simplifying sample transport and handling, we evaluated and 57 compared the yield of eNAT versus standard transport media, across different non-invasive 58 samples, using the Cepheid Xpert Xpress SARS-COV-2 test ('Xpert'). Xpert is a FDA-EUA 59 approved rapid, integrated, cartridge-based RT-PCR test that can be run on widely existing 60 GeneXpert instruments used in over 130 countries. To our knowledge, this is the first study to 61 demonstrate the use of a viral inactivating buffer across various non-invasive samples in a point-62 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Patients that could not or did not consent, were pregnant or breastfeeding, prisoners, or who were 74 unable to provide any respiratory specimens were excluded. Trained study personnel collected 1 75 NP swab (baseline only), 2 oral swabs, 2 nasal swabs, and a saliva sample from all participants 76 who consented to all sample types. A subset of participants being evaluated for hospital outcomes 77 in the parent study continued to be sampled longitudinally by oral swabs, nasal swabs, and saliva 78 every 2-3 days until discharge. All swab types were immediately placed into 3mL of sterile 79 Universal Viral Transport Medium (VTM; Labscoop, Little Rock, AR) whereas a second nasal 80 and oral swab was collected and immediately placed into 3mL of eNAT (COPAN Diagnostics, 81 Murrieta, CA, USA). A thinner nylon tip swab designed for nasopharyngeal (NP) sampling was 82 used to obtain the NP swab (baseline only), and a thicker nylon tip swab designed for oral and 83 nasal samples (Copan diagnostic, Murrieta, CA) was used for these sample types. NP swab 84 collection was performed in accordance with CDC guidelines 14 ; oral swab collection was 85 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 5, 2021. ; https://doi.org/10.1101/2021.03.03.21251172 doi: medRxiv preprint performed by swabbing both buccal surfaces and tongue with an alternating order of collection for 86 each media; nasal (anterior nares) swab collection was performed by rotating the swab 1 cm inside 87 the nostril for 10-15 seconds, alternating nostrils for each media. Additionally, participants were 88 instructed to self-collect a posterior saliva sample by clearing the back of their throat, then 89 collecting 4 mL of saliva into a marked, empty, sterile wide-mouth cup (though any volume over 90 0.5 mL was accepted). All specimens were transported at room temperature and stored in a 2-4°C 91 prior to testing, which occurred within 48 hours of sample collection. Testing by Xpert Xpress SARS-Cov-2 ('Xpert'). NP, nasal, and oral swabs were tested by 93 adding 300µl of the sample (either in VTM or eNAT) directly to the Xpert SARS-Cov-2 test 94 cartridges and the test was run in the GeneXpert system as per the manufacturer's instructions. The saliva sample was tested using three different methods. First, 300µl of the saliva sample was 96 directly added into the Xpert test cartridge ("saliva direct" sample). Additionally, the same saliva 97 sample was swabbed with two separate swabs (thicker nylon tip swabs) for 10 twirls followed by 98 incubating each swab in the saliva for ~10-20 seconds ("saliva swab" sample). Each saliva swab 99 sample was then transferred into test tubes containing 3mls of either VTM or 2 ml of eNAT buffer 100 and mixed well. From each of these mixtures, 300µl were added directly to the sample chamber 101 of Xpert cartridges. Saliva samples <300 µl were tested only by swabbing in eNAT and VTM. 102 We also compared saliva samples directly diluted in 1:1, 1:2 and 1:4 ratios of saliva to eNAT. A 103 minimum volume of 700µl of saliva was needed to test all saliva processing methods: 'saliva 104 direct', saliva swab in eNAT and all three dilutions. For saliva samples with volumes less than 105 700µl, we prioritized saliva direct and saliva swab testing. Out of the 44-saliva direct positive 106 samples tested with eNAT ratios, 1:1 dilution was not performed for one saliva sample due to 107 insufficient volume. One each of the sample types had an error either due to pressure aborts (Error 108 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. were also not suitable as true negative comparators due to known COVID status of these 118 individuals. To confirm the discordancy that negative comparison sets was not due to difference 119 in the in-hospital versus Xpert PCR tests, we obtained leftover media from positive NP swabs of 120 a random subset of six participants. We then tested this archived sample as validation samples on 121 Xpert. Xpert correctly detected SARS-CoV-2 in all six of these archived samples. 129 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. indications, had no respiratory symptoms, and were incidentally found to be COVID-positive. Average participant age was 55, 37% were female, and the most common comorbidities were 142 hypertension and diabetes. On average, the baseline collection took place 2 days after the last positive in-hospital NP swab 144 PCR test for participants in the analysis group, and 3 days for participants with no positive samples. The biologic variability of PCR positivity from samples collected several days apart was evident 146 in the discordancy of longitudinal in-hospital NP swab PCR testing results even when the same 147 test was used. Nineteen (38%) of the 52 participants in the analysis group had at least one 148 subsequent negative in-hospital NP swab PCR test during their hospital admission (Table 1) . participants. These observations support that positive-negative discordancy across time was likely 152 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. to saliva direct (Fig. 2B ). There were three negative NP specimens that were detected in saliva, 175 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Fig. 2A and 2B ). Saliva direct, the only sample type analyzed as is without (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 5, 2021. We found that saliva is an excellent test matrix for the Xpert Xpress SARS-CoV-2 test, providing 223 a sensitivity that is comparable to NP swabs and better than nasal and oral swabs. This finding is load. This suggests that testing with multiple samples and perhaps multiple sample types when 236 clinical suspicion is high may provide the highest sensitivity and negative predictive value for 237 SARS-CoV-2. 238 We additionally found that eNAT, a buffer we have previously determined to be effective at 239 inactivating SARS-CoV-2 in-vitro (Banik et al., submitted) , increased the test positivity rates 240 across all sample types compared to VTM (P=0.0032), with a saliva to eNAT ratio of 1:2 being 241 optimal in our sample set. We also found that adding eNAT to saliva possibly mitigates the PCR 242 interference from saliva with lower pressure values and recovery of otherwise delayed SPC Ct 243 values seen with direct saliva. These findings suggest that the application of eNAT as a sample 244 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 5, 2021. ; https://doi.org/10.1101/2021.03.03.21251172 doi: medRxiv preprint buffer may be advantageous not only in safe handling and transport, but also in improving yield 245 and processing capability on the Cepheid system. There were several limitations in this study. First, there were less contemporaneous NP swabs 247 collected with saliva, thereby reducing the number of direct comparisons between these two 248 sample types, although they were found to be comparable. An underlying reason for this -249 participants declining NP swab collection due to its discomfort -also demonstrates the real-world 250 limitations that would be magnified with larger scale testing such as in schools or the workplace. Secondly, we added eNAT to saliva in the laboratory, whereas the benefit of eNAT would be to 252 sterilize samples immediately after collection and before transport and test set up. However, this 253 allowed us to evaluate the combination of eNAT and saliva under different conditions and inform 254 optimal design of kits to add eNAT immediately to saliva upon collection. Finally, our participants 255 were patients who had either been admitted to the hospital or seen in the emergency department. This population may not be generalizable to ambulatory individuals who would benefit the most 257 from self-collection. However, we captured a diverse patient group in our cohort including those 258 who were never admitted, as well as patients who were detected by universal screening but 259 reported no COVID symptoms. 260 Altogether, our findings support the use of saliva and eNAT sterilizing buffer to enhance effective, 261 safe, and accessible COVID testing and screening in the many health care systems worldwide 262 already using GeneXpert instruments. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 388 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 5, 2021. ; https://doi.org/10.1101/2021.03.03.21251172 doi: medRxiv preprint Safety management of nasopharyngeal 287 specimen collection from suspected cases of coronavirus disease 2019 289 SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and 290 commercial response with associated advantages and limitations Suboptimal Biological Sampling as a Probable 294 Cause of False-Negative COVID-19 Diagnostic Test Results False-positive COVID-19 results: hidden problems and 297 costs. 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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Acknowledgements: We thank Dr. Jason H. Yang (Rutgers New Jersey Medical School) for