key: cord-0701017-g9kinuwb authors: Sahajpal, Nikhil S.; Mondal, Ashis K.; Ananth, Sudha; Njau, Allan; Ahluwalia, Pankaj; Kota, Vamsi; Caspary, Kevin; Ross, Ted M.; Farrell, Michael; Shannon, Michael P.; Fulzele, Sadanand; Chaubey, Alka; Hegde, Madhuri; Rojiani, Amyn M.; Kolhe, Ravindra title: Clinical Validation of a Sensitive Test for Saliva Collected in Health Care and Community Settings with Pooling Utility for Severe Acute Respiratory Syndrome Coronavirus 2 Mass Surveillance date: 2021-05-04 journal: J Mol Diagn DOI: 10.1016/j.jmoldx.2021.04.005 sha: f2771bbb6ba238cfde21bdae18b0a604f895ffbf doc_id: 701017 cord_uid: g9kinuwb The clinical performance of saliva compared with nasopharyngeal swabs (NPSs) has shown conflicting results in health care and community settings. Pooled testing with saliva is also challenging, owing to the ambiguous sensitivity, limit of detection, and processing challenges. A total of 429 matched NPS and saliva sample pairs, collected in either health care or community setting, were evaluated. Phase 1 (protocol U) tested 240 matched NPS and saliva sample pairs; phase 2 (SalivaAll protocol) tested 189 matched NPS and saliva sample pairs, with an additional sample homogenization step before RNA extraction. A total of 85 saliva samples were evaluated with both protocols. In phase 1, 28.3% (68/240) samples tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from saliva, NPS, or both. The detection rate was lower in saliva compared with NPS samples (50.0% versus 89.7%). In phase 2, 50.2% (95/189) samples tested positive for SARS-CoV-2 from saliva, NPS, or both. The detection rate was higher in saliva compared with NPS samples (97.8% versus 78.9%). Of the 85 saliva samples evaluated with both protocols, the detection rate was 100% for samples tested with SalivaAll, and 36.7% with protocol U. The limit of detection with SalivaAll protocol was 20 to 60 copies/mL. The pooled testing approach demonstrated a 95% positive and 100% negative percentage agreement. This protocol for saliva samples results in higher sensitivity compared with NPS samples and breaks the barrier to using pooled saliva for SARS-CoV-2 testing. Thus, rapid and accurate detection of SARS-CoV-2 is the foremost and likely most essential component in controlling this outbreak and has immediate clinical, epidemiologic, and policy implications. The detection of the SARS-CoV-2 virus has been applied to various clinical specimens that include bronchoalveolar lavage, sputum, saliva, nasopharyngeal swabs (NPSs), oropharyngeal swabs (OPSs), feces, and blood. 2 NPS and OPS samples are the current standard upper respiratory tract specimens recommended for COVID-19 diagnostic testing. However, the collection of NPS samples poses certain challenges that include exposure risk to health care workers, supply chain constraints pertaining to swabs and personal protective equipment, and self-collection being difficult; inappropriate sampling may lead to false-negative results. 3e5 Amidst these challenges, several other sample types have been under investigation for COVID-19 testing, of which saliva samples are of significant interest owing to their ease of collection, and alleviating some of the challenges associated with NPS sampling. True saliva is defined as the naturally collecting clear liquid that accumulates in the mouth. However, saliva from patients can be confounded with the presence of mucus or blood, thereby rendering it a difficult sample to process in the laboratory. Several reports evaluating the clinical performance of saliva compared with NPS/OPS samples have demonstrated conflicting results. In a health care setting, studies have demonstrated comparable 6e9 and even higher sensitivity of saliva 10 /early morning saliva collection 11 compared with NPS samples, as well as reported higher viral titer values in saliva. 9 Conversely, deep-throat saliva 12 and typical saliva samples have also been demonstrated to be less sensitive compared with NPS samples in both health care 13 and community settings. 14 Furthermore, saliva samples are difficult to pipet by the testing personnel, which leads to increased processing time. 15 Although different collection devices, media, sample handling, extraction procedure, and RT-PCR Q7 methods may have accounted for these discrepancies, we investigated a critical facet of the saliva samples that, if accounted for, renders the saliva samples more sensitive than NPS samples. In our analysis, the saliva samples initially demonstrated a lower sensitivity compared with NPS samples. We introduced a simple processing step using the bead mill homogenizer and observed that the saliva samples showed higher sensitivity compared with NPS samples. Not only did the saliva processing become significantly easier after using the homogenizer, but we were also able to validate saliva samples with a five-sample pooling strategy. The print & web 4C=FPO Figure 1 Schematic overview of sample processing and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay workflow, depicting main steps. Matched nasopharyngeal swab (NPS) and saliva sample pairs collected in health care and community setting were tested and validated as follows. Top panel: NPS or saliva samples were processed with protocol U for nucleic acid extraction using a semi-automated instrument, followed by RT-PCR for N and ORF1ab gene targets and internal control (IC) used as extraction and RT-PCR IC. Middle panel: Saliva samples processed with SalivaAll protocol that included a saliva homogenization step using a bead mill homogenizer before RNA extraction and downstream processing. Bottom panel: Saliva samples were homogenized using a bead mill homogenizer (SalivaAll protocol) before pooling samples with a five-sample pooling strategy for SARS-CoV-2 testing. 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 SalivaAll protocol is sensitive and detects up to 20 copies/ mL, facilitating utility in saliva sample pooled testing, which will be critical in SARS-CoV-2 mass surveillance. Our single-site study identified both increased and decreased sensitivity of saliva as a diagnostic sample type, consistent with the discrepancies reported in the literature, that were largely believed to be the result of processing challenges. We contend that with appropriate management of these processing challenges, saliva samples are more sensitive compared with the NPS samples. This single-center diagnostic study was conducted at the Augusta University (Augusta, GA). This site is a Clinical Laboratory Improvement Amendmentseaccredited laboratory for high-complexity testing and is one of the main SARS-CoV-2 testing centers in the state of Georgia. The study evaluated 429 matched NPS and saliva samples (sample pairs) collected from 344 individuals in either a health care or a community setting. Of the 344 individuals, 95 matched clinical specimen pairs were collected in health care setting from individuals at either a medical nursing home or Augusta University Medical Center, both in Georgia. In the community setting, 249 matched clinical specimen pairs were collected from drive-through collection centers in different regions of Georgia that include Augusta, Albany, and Atlanta. As a standard protocol in both settings, NPS from individuals was collected by a health care worker using a sterile flocked swab placed in a sterile tube containing 3 mL of viral transport medium (Specimen Collection and Transport System BD Sterile, catalog number 220531; Becton Dickinson, Franklin Lakes, NJ). Before collecting the NPS samples, the individuals were instructed to provide saliva samples by spitting into a sterile container (DNA/RNA Shield Saliva Sputum Collection Kit e DX, The Journal of Molecular Diagnosticsjmdjournal.org 3 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 catalog number R1210-E; Zymo Research Q8 ) over which the health care worker added 2 mL of viral transport medium. All samples were stored at 4 C temperature and transported to the SARS-CoV-2 testing facility at Augusta University within 24 hours of sample collection, for further processing. The assay is based on nucleic acid extraction followed by TaqMan-based RT-PCR assay to conduct in vitro transcription of SARS-CoV-2 RNA, DNA amplification, and fluorescence detection [Food and Drug Administration (FDA) Emergency Use Authorization assay by Perki-nElmer Inc., Waltham, MA]. The assay targets specific genomic regions of SARS-CoV-2: nucleocapsid (N ) gene and ORF1ab. The TaqMan probes for the two amplicons are labeled with FAM and ROX fluorescent dyes, respectively, to generate target-specific signals. The assay includes an RNA internal control (IC; bacteriophage MS2) to monitor the processes from nucleic acid extraction to fluorescence detection. The IC probe is labeled with VIC fluorescent dye to differentiate its fluorescent signal from SARS-CoV-2 targets. The samples were resulted as positive or negative based on the Ct Q9 values specified by the manufacturer (Supplemental Table S1 ). In phase 1 of this study, 240 matched NPS and saliva sample pairs were tested prospectively for SARS-CoV-2 RNA by RT-PCR. Of the 240 samples, 95 were collected in a health care setting and 145 were collected in a community setting. In brief, all samples were vortexed, and an aliquot of 300 mL from each sample (NPS or saliva), positive and negative controls, was added to respective wells in a 96-well plate. To each well, 5 mL IC, 4 mL poly(A) RNA, 10 mL proteinase K, and 300 mL lysis buffer 1 were added. The plate was placed on a semi-automated instrument (Chemagic 360 Instrument; PerkinElmer Inc.) following the manufacturer's protocol. The nucleic acid was extracted in a 96-well plate, with an elution volume of 60 mL. From the extraction plate, 10 mL of extracted nucleic acid and 5 mL of PCR master mix were added to the respective wells in a 96well PCR plate. The PCR method was set up as per the manufacturer's protocol on Quantstudio 3 or 5 (Thermo Fisher Scientific, Waltham, MA). The samples were resulted as positive or negative, based on the Ct values specified by the manufacturer (Figure 1 ). In phase 2 of this study, 189 matched NPS and saliva sample pairs were tested for SARS-CoV-2. Of the 189 samples, 40 were collected in health care and 149 were collected in a community setting. More important, 85 samples that had been previously evaluated with protocol U were re-evaluated with SalivaAll protocol to determine the effect of bead homogenization. In SalivaAll protocol, an additional processing step was added for saliva samples that included aliquoting saliva samples Q11 from the collection tubes into Omni tubes (2 mL reinforced tubes, SKU Q10 : 19-628D; Omni International), which were then placed in Omni bead mill homogenizer (Bead Ruptor Elite, SKU: 19-040E; Omni International). The samples were homogenized at 4.5 m/ second for 30 seconds. From the Omni tubes, an aliquot of 300 mL homogenized saliva samples was added to 96-well plate and downstream processing was performed as described in protocol U. The NPS sample processing remained the same as in protocol U ( Figure 1 ). However, an additional study was done with 189 NPS samples with both protocol U and SalivaAll protocol to determine if bead homogenization would affect the clinical sensitivity in NPS samples. The limit of detection (LoD) studies were conducted as per the FDA guidelines (https://www.fda.gov/medical-devices/ coronavirus-disease-2019-covid-19-emergency-use-authori zations-medical-devices/vitro-diagnostics-euas, last accessed September 9, 2020) using two different reference materials [FDA Emergency Use Authorization assay by PerkinElmer Inc. and AccuPlex SARS-CoV-2 Molecular Controls Kit -Full Genome, Mat . number 0505-0159 (SeraCare Q13 ) ]. Briefly, SARS-CoV-2 reference control materials were spiked into the negative saliva samples to serve as positive samples at 180, 60, and 20 copies/mL concentrations, and were processed with SalivaAll protocol. The lowest concentration detected in all three triplicates was determined as the preliminary LoD. To confirm the LoD, 20 replicates of preliminary LoD were analyzed and deemed as confirmed if at least 19 of 20 replicates were detected. The PerkinElmer Inc. and SeraCare reference control materials consisted of encapsulated synthetic RNA with concentration of 1000 and 5000 copies/mL, respectively. 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 generated by aliquoting 60 mL from each of the five homogenized samples to make 300 mL for extraction. Data were analyzed for descriptive statistics and presented as number (percentage) for categorical variables and means AE SD for continuous variables. Ct values were compared using paired t-test. Regression analysis with slope and intercept along with a 95% CI was determined in the pooling sample study. Comparison of SARS-CoV-2 Detection between Saliva and NPS (Phase Of the 189 matched sample pairs tested in phase 2, 50.2% (95/189) tested positive for SARS-CoV-2 from saliva, NPS, or both. The detection rate for SARS-CoV-2 was significantly higher in saliva compared with NPS testing [97.8% Eighty-five saliva samples, of which 47% (40/85) were detected positive with NPS samples, were tested with both protocols. Of this, 57.6% (49/85) tested positive for SARS-CoV-2 with protocol U, SalivaAll, or both. The detection rate was significantly higher 100% (49/49) with samples tested with SalivaAll compared with 36.7% (18/49) with protocol U (P < 0.001). The concordance for positive The Journal of Molecular Diagnosticsjmdjournal.org 5 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 ). In the preliminary LoD study, all replicates were detected at the three tested concentrations with the PerkinElmer Inc. reference material, and two of three replicated were detected at 20 copies/mL with SeraCare material. The LoD was determined to be 20 and 60 copies/mL with PerkinElmer Inc. and SeraCare material, respectively, and all 20 replicates were detected (Table 1 ½T1 ½T1 ). The five-sample pooling strategy was evaluated by comparing the results of the 20 positive and negative pools with individual sample testing results. The pooled testing results demonstrated a 95% positive and a 100% negative agreement. The N and ORF1ab gene Ct values were compared between pooled and individual testing. The shift in Ct value was found to be significant, with pooled testing toward higher Ct values; nonetheless, the pools containing positive samples with viral loads close to the assay's LoD (ie, weak positives) were accurately detected ( Table 2 ½T2 ½T2 and Figure 4 ½F4 ½F4 ). An optimal sample type and easier collection method for the detection of SARS-CoV-2 are primary requirements in the global effort to control this pandemic. NPS and OPS samples are the currently recommended sample types for COVID-19 diagnostic testing. Testing for SARS-CoV-2 has relied primarily on these samples, but the associated challenges with NPS/OPS sampling have engendered a need to evaluate several other sample types, of which saliva has remained an attractive alternate. However, there are conflicting reports in the literature on the clinical performance of saliva compared with NPS/OPS samples. In a health care setting, initial reports from To et al 6 In phase 1 of our study, comprising 240 matched NPS and saliva sample pairs (protocol U), we observed lower sensitivity of saliva samples compared with NPS samples. The detection rate in saliva samples was significantly lower compared with NPS samples. In addition, the Ct values for N, ORF1ab, and IC (extraction and PCR control) were significantly higher in saliva compared with the NPS sample. Furthermore, a high percentage of saliva samples yielded invalid results. A significant challenge in the wet laboratory revolved around accurate pipetting of saliva samples because most of these were viscous, even after intensive vortexing. The viscous gel-like consistency not only caused issues with pipetting but also led to a high percentage of invalid results. This was demonstrated as a significantly high Ct value drift in the IC, suggesting that the lower sensitivity of saliva samples is most likely due to processing and sometimes pre-analytical issue(s). Several factors that might have led to the lower sensitivity of saliva samples were contemplated, including inaccurate pipetting of sample volume into the extraction plate for nucleic acid extraction, the viscous gel-like texture of saliva samples prevented appropriate mixing with viral transport media, which led to the pipetting of primarily saliva or media into extraction plate for nucleic acid extraction, and the likelihood that the higher viscosity of saliva might also impair sample lysis for nucleic acid extraction. 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 The underlying issue associated with these challenges emerged to be the gel-like consistency of saliva samples. We addressed this significant pre-analytic concern by adding a simple step before processing samples for nucleic acid extraction. The saliva samples were homogenized using a bead mill homogenizer at a speed of 4.5 m/second for 30 seconds (SalivaAll). The homogenization rendered the saliva samples to uniform viscosity and consistency, making it easier to pipet for the downstream assay. Thus, in phase 2 of the study, with 189 matched NPS and saliva sample pairs, adding this single preprocessing step rendered saliva samples more sensitive compared with NPS samples. The detection rate was higher in saliva samples, with significantly lower Ct value for IC, compared with NPS samples. Also, on comparing the 85 saliva samples that were processed both in phase 1 and phase 2 studies, the detection rate was significantly higher in samples processed with the homogenization step (SalivaAll) compared with the sample processed without it (protocol U). The Ct values in the same saliva samples were lower for the N gene, and significantly lower for ORF1ab and IC with samples processed with SalivaAll compared with protocol U. These results demonstrate that the lower sensitivity of the saliva samples observed in the phase 1 study was because of inadequate sample processing. The saliva samples were found to be more sensitive compared with NPS samples, with the addition of this simple processing step. Furthermore, the LoD of 20 copies/mL with the PerkinElmer Inc. material and 60 copies/mL with the SeraCare material confirmed the sensitivity of saliva samples as the LoD was determined to be the similar as that for the FDA Emergency Use Authorization comparator test for NPS samples. The LoD for the assay in NPS samples was determined to be 20 copies/mL with PerkinElmer Inc. positive control and Ser-aCare reference material. In addition, to depict the effect of homogenization on saliva samples, we performed viscosity measurement studies, and identified that the unprocessed saliva samples (Saliva-U) had the viscosity ranging from 176 cP Q14 to 677 cP (between the viscosity of olive oil and honey), compared with homogenized samples (SalivaAll) with 2.1 cP to 3.1 cP, a viscosity close to that of water (1 cP). These findings highlight and explain the difficulty the saliva samples would pose during pipetting and in the extraction procedure, where the uniform mixing of reagents would be challenging. The homogenization of saliva samples leads to a uniform and less viscous sample that would allow adequate extraction of the nucleic acids in the extraction procedure, leading to higher sensitivity of the assay. 16 Furthermore, saliva processing became significantly easier, and we were also able to successfully validate saliva samples with a five-sample pooling strategy. The pooled testing results demonstrated a positive percentage agreement of 95% (19/20 pools showing positive results), with one pool that contained the sample with high Ct (N: 38.4; ORF1ab: undetermined) being undetectable. The negative percentage agreement was found to be 100%. We have previously demonstrated the feasibility and accuracy of a sample pooling approach with NPS samples for wide-scale population screening for COVID-19. 17 Herein, we extend the utility and potential benefits of the sample pooling approach for population screening with saliva samples. Considering the evolving epidemiology of COVID-19 and the reopening of educational and professional institutions, travel, tourism, and social activities, monitoring SARS-CoV-2 will remain a critical public health need. Therefore, the use of a noninvasive collection method and easily accessible sample, such as saliva, will enhance screening and surveillance activities. In conclusion, this study has demonstrated that the saliva samples are more sensitive than NPS samples if collected and processed appropriately before extraction and PCR. The study evaluated matched NPS and saliva sample pairs collected in both health care and community settings. In the community setting, no control was exercised regarding food or drink restriction and time of sample collection as most of the community-collected samples came from drive-through facilities that operate from morning until evening. The The Journal of Molecular Diagnosticsjmdjournal. org 7 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 spectrum of disease also varied from asymptomatic to severely ill patients, and the study was not biased for a particular group. However, it must be highlighted that homogenization is an additional step in the workflow and will increase the processing time compared with NPS samples. In addition, similar approaches to homogenize the saliva samples should be evaluated by laboratories intending to implement saliva testing in different regions of the world, where this particular homogenizer might not be available. Furthermore, the study has two limitations: the Ct value in this study reflects viral load but not the viral copies per mL, and the sample collection was not controlled for a specific day of illness. Nonetheless, despite these limitations, this study presents a significant and clinically validated approach to the utilization of saliva samples for COVID-19 testing, individually or as pooled samples. COVID-19: towards controlling of a pandemic Detection of SARS-CoV-2 in different types of clinical specimens Inappropriate nasopharyngeal sampling for SARS-CoV-2 detection is a relevant cause of false-negative reports No one likes a stick up their nose: making the case for saliva-based testing for COVID-19 Self-collected anterior nasal and saliva specimens versus health care worker-collected nasopharyngeal swabs for the molecular detection of SARS-CoV-2 Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study Consistent detection of 2019 novel coronavirus in saliva Saliva is a reliable tool to detect SARS-CoV-2 Clinical significance of a high SARS-CoV-2 viral load in the saliva Saliva or nasopharyngeal swab specimens for detection of SARS-CoV-2 Comparing nasopharyngeal swab and early morning saliva for the identification of SARS-CoV-2 Prospective study comparing deep-throat saliva with other respiratory tract specimens in the diagnosis of novel coronavirus disease (COVID-19) Sensitivity of nasopharyngeal swabs and saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Saliva is less sensitive than nasopharyngeal swabs for COVID-19 detection in the community setting Challenges in use of saliva for detection of SARS-CoV-2 RNA in symptomatic outpatients SalivaSTAT: direct-PCR and pooling of saliva samples collected in healthcare and community setting for SARS-CoV-2 mass surveillance Proposal of reverse transcription-PCRebased mass population screening for SARS-CoV-2 (COVID-19) We thank Dr. Brooks Keel, President, Augusta University and AUHS , for strategic, science-based, data-driven leadership in all matters COVID-19, and constant support and encouragement at every step of this work. Supplemental material for this article can be found at http://doi.org/10.1016/j.jmoldx.2021.04.005. 8 jmdjournal.org -The Journal of Molecular Diagnostics 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937