key: cord-0846197-jrmf1xgd authors: Li, Z.; Bruce, J. L.; Cohen, B.; Cunningham, C. V.; Jack, W. E.; Kunin, K.; Langhorst, B. W.; Miller, J.; Moncion, R. A.; Poole, C. B.; Premsrirut, P. K.; Ren, G.; Roberts, R. J.; Tanner, N. A.; Zhang, Y.; Carlow, C. K. S. title: Development and Implementation of a Simple and Rapid Extraction-Free Saliva SARS-CoV-2 RT-LAMP Workflow for Workplace Surveillance date: 2022-03-13 journal: nan DOI: 10.1101/2022.03.11.22272282 sha: ece14b774ea01abbbd94fecf47b3602f84b8f381 doc_id: 846197 cord_uid: jrmf1xgd Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/L of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection comparable to commercially available RT-qPCR-based diagnostics. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infections rates in Massachusetts and nationwide. The rapid identification and isolation of infected individuals with trace viral loads before symptom onset minimized viral spread in the workplace. Initial studies exploring the potential use of saliva primarily used RT-qPCR for 84 SARS-CoV-2 detection [17] [18] [19] . More recently, reverse-transcription loop-mediated 85 isothermal amplification (RT-LAMP) has emerged as an attractive and affordable 86 alternative to RT-qPCR. RT-LAMP permits the rapid detection of pathogens without 87 sophisticated equipment while retaining high levels of specificity and sensitivity [20, 21] . can obviate the need for a nucleic acid extraction/purification step, reducing both the time 98 and cost to process samples. In the case of saliva, a more biologically complex sample 99 [33] than nasal fluid, additional care should be taken when a minimal or extraction-free 100 method is being considered. Saliva pH, color, viscosity, and RNAse activities can vary 101 widely and potentially impact the ability to detect viral RNA. 102 In the present study, we report an extraction-free, saliva-based RT-LAMP 103 workflow for SARS-CoV-2 detection with the option of a simple colorimetric endpoint 104 and/or a semi-quantitative fluorescence readout. We demonstrate the robustness of the 105 method using a large cohort of contrived human samples and successful implementation 106 6 for frequent surveillance testing in the workplace. This has enabled us to identify and 107 isolate infected individuals with trace viral loads before symptom onset and limit viral 108 CoV-2 reference positive and negative saliva samples, previously tested following an 126 RNA extraction and RT-qPCR procedure, were kindly provided by Mirimus Clinical 127 Labs (Brooklyn, NY). 128 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 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272282 doi: medRxiv preprint Prior to saliva collection, donors were requested to refrain from drinking anything 129 but water, eating, chewing gum or tobacco, or smoking for at least 30 minutes prior to 130 collection. Saliva was self-collected by passive drooling through a 1.0 mL unfiltered 131 pipette tip into 1.5 mL tubes, each pre-applied with a pair of QR Codes, one on the top 132 and one on the side, for accurate specimen identification. Unless specified, samples were 133 stored at room temperature for less than 4 hours or overnight at 4 o C prior to testing. 134 and Nasopharyngeal Swabs. RNA was eluted with 80 µL of nuclease-free water, 150 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. 167 RT-qPCR was performed using the Luna® SARS-CoV-2 RT-qPCR Multiplex Assay Kit 168 (NEB Cat # E3019) following the manufacturer's instructions. Each 20 μL reaction 169 contained 2 μL RNA purified from saliva. N1 (HEX), N2 (FAM) and RNase P (Cy5) 170 targets were simultaneously detected using the following cycling conditions: carryover 171 prevention (25°C for 30 sec), cDNA synthesis (55°C for 10 min), initial denaturation 172 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. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. To achieve highly reliable detection of SARS-CoV-2 in crude saliva, several buffer 238 formulations including both commercial and previously published protocols were 239 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 evaluate the performance of SLB at various temperatures, contrived samples 258 spiked with 2-10,000 viral copies/µL, were processed in triplicate using either 75 o C for 259 15 min, 85 o C for 10 min or 95 o C for 5 min (Figure 1B-C) . It was apparent that heat 260 treatment was essential with the highest levels of sensitivity achieved after heating at 261 85 o C for 10 min or 95 o C for 5 min, both detecting 39 copies/µL in all triplicate samples. 262 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 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272282 doi: medRxiv preprint Heating samples at 95 o C for 5 min resulted in the best performance and greatest 263 reproducibility across a range of viral loads and was selected as the optimal temperature 264 to rapidly release and stabilize viral RNA. This protocol also proved optimal for actin 265 detection (Supplementary Figure S1) . optimal for testing and no pre-mixing is required. Viral and actin detection was least 279 efficient when crude sediment was used as input. For comparison, RNA was also purified 280 from supernatant and sediment. This resulted in overall faster amplification for both 281 COVID and actin assays than with crude lysate, likely due to the elimination of inhibitors 282 or interfering substances present in crude saliva. These results demonstrate that saliva 283 supernatant is suitable for viral detection, obviating the need for an additional re-284 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 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272282 doi: medRxiv preprint suspension step, which is particularly helpful when dealing with large numbers of 285 samples simultaneously. 286 To evaluate the tolerance of the LAMP assay to increasing amounts of saliva, 287 amounts ranging from 0.25-6 µL SARS-CoV-2 positive saliva were used as input in a 20 288 µL reaction (Figure 2B) . At all volumes of saliva input, virus was detected, but the best 289 overall performance was observed with 1 µL of a saliva sample. Lower than 1 µL, more 290 variation was observed in triplicate samples. Increasing saliva input volume did not 291 increase sensitivity and delayed the reaction time for viral detection, with a difference as 292 much as 4 min when using 1 µL versus 6 µL saliva. A similar trend, though not as 293 pronounced, was also observed with respect to actin detection following volumetric 294 adjustments in saliva input ( Figure 2B) . correlation coefficient R 2 value of 0.63, P-value < 0.0001) was observed ( Figure 3C) . 307 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. positive (100% sensitivity) within 10 (pool 1) or 11 (pool 2) minutes. When spiked with 324 fewer copies of virus, the viral RNA was also detected but at a reduced frequency. At 20 325 copies/µL or 10 copies/µL, sensitivities of 82% (49/60) and 63% (38/60) were obtained, 326 respectively. Therefore, the LOD for this direct saliva RT-LAMP assay is 40 copies of 327 virus/µL of saliva. 328 To evaluate the diagnostic capabilities of the workflow using pre-defined clinical 329 samples, a total of 30 positive and 30 negative saliva specimens were tested at least three 330 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 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272282 doi: medRxiv preprint times by two different operators in a blinded manner (Supplementary Figure S3) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 2) . A strong agreement between 372 the two readout methods was observed with a yellow color (positive) corresponding to a 373 fast amplification and pink/orange shaded as negative (Tt > 26 minutes or N/A). The six 374 samples that were not detected by eye, were orange shaded and corresponded with late Tt 375 values, representing low viral loads or at the limit of detection of the test. This 376 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. This is likely due to the high level of endogenous RNases in human saliva [46, 47] . In the 396 extraction-free method described in this study, 95 o C heating is used to disassemble the 397 viral particle and release RNA as well as denature and inactivate some of the RNases in 398 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. RNase activity is lower. 420 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. and validation of the corresponding COVID test (data not shown). Since low pH saliva 435 was usually associated with a particular individual, we found it simpler to inform the 436 donor and request an adjustment to their collection method, such as providing a sample at 437 a different time of the day or rinsing the mouth with water briefly before collection. This 438 behavioral change improved the quality of the sample and substantially decreased the 439 inconclusive rate. We also discovered that pooling saliva prior to testing can be beneficial 440 in cases where a particular specimen is problematic due to low pH, color, or viscosity, or 441 has substances that interfere with nucleic acid amplification. Pooling saliva for large-442 scale surveillance programs has proven to be a highly cost-effective strategy [12, 17, 38, 443 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. positive. This likely reflected a low viral load present. We also noted more variance in 466 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. In an analysis of almost 100,000 individuals in the United States, more males 479 tested positive for SARS-CoV-2 than females [57] . Viral loads have also been reported to 480 be ~ 10 times higher in males compared to females, as well as a slower viral clearance in 481 males [30] . In our study, involving more than 30,000 saliva samples from 406 males, 482 341females and 8 gender not indicated, we did not observe a significant difference in 483 positivity rates between genders, likely due to the small number of cases identified (47 484 males and 35 females). Interestingly, we found a higher viral load in samples from males, 485 however, clearance rates did not differ between males and females. At 10 days after the 486 initial positive test, we found 13% of positive individuals still testing positive with most 487 of them testing negative for viral RNA by day 14. This may be a general trend for saliva-488 based COVID diagnostics since in a mass screening program in Slovenia, the viral load 489 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. Roitman for participation in the blinded reading of colorimetric data, and Nicole Nichols 505 for helpful discussions, Lori Tonello for organizing the many volunteers involved in 506 saliva kit assembly, Tasha José for the diagrammatic representation of the workflow, Dr. 507 Gyorgy Abel for medical oversight, Lea Antonopoulos for reagents, and Tom Evans for 508 guidance and feedback on the manuscript. 509 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. 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. 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 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272282 doi: medRxiv preprint plotted. All samples were tested in triplicate. 539 540 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 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272282 doi: medRxiv preprint (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 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272282 doi: medRxiv preprint (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 13, 2022. ; https://doi.org/10.1101/2022.03.11.22272282 doi: medRxiv preprint Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-571 Exploring salivary diagnostics in COVID-19: a scoping review and research suggestions. 576 BDJ Open Saliva as a gold-standard 579 sample for SARS-CoV-2 detection Saliva as a 583 Noninvasive Specimen for Detection of SARS-CoV-2 Saliva sample as a non-invasive specimen for the diagnosis of 588 coronavirus disease 2019: a cross-sectional study Comparison of Saliva and Nasopharyngeal Swab Nucleic Acid Amplification Testing 593 for Detection of SARS-CoV-2: A Systematic Review and Meta-analysis The Sensitivity and 597 Costs of Testing for SARS-CoV-2 Infection With Saliva Versus Nasopharyngeal Swabs : 598 A Systematic Review and Meta-analysis Saliva 606 is a reliable tool to detect SARS-CoV-2 Asymptomatic Persons for Severe Acute Respiratory Syndrome Coronavirus 2 Using 611 Saliva Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers Saliva Pooling Strategy for the Large-Scale 620 Detection of SARS-CoV-2, Through Working-Groups Testing of Asymptomatic Subjects 621 for Potential Applications in Different Workplaces Sample-627 to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, 628 nasal, and saliva samples: Implications and use for surveillance testing Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal 634 Amplification, and a Rapid Antigen Test To Diagnose COVID-19 COVID-19 638 screening test by using random oropharyngeal saliva Temporal dynamics in 642 viral shedding and transmissibility of COVID-19 No reuse allowed without permission. 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