key: cord-1007763-od2mat8o authors: Brotons, P.; Perez-Arguello, A.; Launes, C.; Torrents, F.; Saucedo, J.; Claverol, J.; Garcia-Garcia, J. J.; Rodas, G.; Fumado, V.; Jordan, I.; Gratacos, E.; Bassat, Q.; Munoz-Almagro, C. title: Validation and implementation of a direct RT-qPCR method for rapid screening of SARS-CoV-2 infection by using non-invasive saliva samples date: 2020-11-27 journal: nan DOI: 10.1101/2020.11.19.20234245 sha: 0fa68b4a299089c06eeae6ccdd9a4f6917ac4a06 doc_id: 1007763 cord_uid: od2mat8o Objective To validate and implement an optimized screening method for detection of SARS-CoV-2 RNA combining use of self-collected raw saliva samples, single-step heat-treated virus inactivation and RNA extraction, and direct RT-qPCR. Design Study conducted in three successive phases including: i) method analytical validation against standard RT-qPCR in saliva samples; ii), method diagnostic validation against standard RT-qPCR in nasopharyngeal samples; and iii), method implementation through pilot screening in a reference hospital. Setting Sant Joan de Deu University Hospital (Barcelona, Spain). Participants Phase 2, a prospective cohort of asymptomatic teenagers and young adult players and staff in a youth sports academy followed up during 9 to 12 weeks; Phase 3, asymptomatic health workers, students, aid volunteers, and other staff of the setting. Main outcome measures Method diagnostic sensitivity and specificity. Method performance in a pilot screening. Results Diagnostic validation included 173 participants. At week 0, all saliva and nasopharyngeal samples were negative. In the following weeks, standard RT-qPCR yielded 23 positive results in nasopharyngeal samples. Paired saliva specimens yielded 22 positive and one inconclusive result. Method diagnostic sensitivity and specificity values were 95.7% (95% CI, 79.0-99.2%) and 100.0% (95% CI, 98.6-100.0 %), respectively. A total of 2,709 participants engaged in the pilot screening, with a high rate of participation (83.4% among health workers). Only 17 (0.6%) of saliva samples self-collected by participants in an unsupervised manner were invalid. Saliva was positive in 24 (0.9%) out of 2,692 valid specimens and inconclusive in 27 (1.0%). All 24 saliva-positive and 4 saliva-inconclusive participants were positive by standard RT-PCR in nasopharyngeal samples. Use of a high throughput system allowed fast screening workflow (up to 384 samples in <2 hours). Conclusion Direct RT-qPCT on self-collected raw saliva is a simple, rapid, and accurate method with potential to be scaled up for enhanced SARS-CoV-2 community-wide screening. The burden and health, educational, and economic implications of the Coronavirus Infectious Disease 19 pandemic have underlined an urgent need for rapid and accurate diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1, 2] . Early identification of SARS-CoV-2 is challenging since a proportion of infected individuals can show little or no symptoms for an indeterminate period of time [3, 4] . Noticeable rates of asymptomatic and pre-symptomatic infection have been observed across different studies, ranging from 3 to 67% [5] . Asymptomatic or presymptomatic individuals are nevertheless likely to be infectious [6, 7] . Upper respiratory tract (URT) samples are the specimens currently recommended for diagnosis of COVID-19 [8] . Reverse transcription real-time polymerase chain reaction (RT-qPCR) constitutes the preferred method for detection of SARS-CoV-2, given its high sensitivity and specificity [9] . RT-qPCR accuracy may vary depending on URT sample quality and time elapsed since virus acquisition [10] . Standard RT-qPCR protocols for SARS-CoV-2 typically follow three sequential phases: i) URT sample swabing and sample transport in viral inactivation transport medium (VITM) to the laboratory for analysis or, alternatively, sample transport in viral transport medium (VTM) and inactivation in the laboratory; ii) RNA extraction, purification, and concentration with use of targeted reagents and automated robots; and iii) viral RNA amplification and detection in thermal cyclers. RNA extraction, purification and concentration are slow and cumbersome activities that take from 40 minutes to 3 hours, . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020. 11.19.20234245 doi: medRxiv preprint 4 depending on the type of RNA extraction robot utilised and the number of samples batched together. During the first wave of the pandemic, shortage of personal protection equipment (PPE), swabs, VITM, and RT-PCR reagent supplies created serious bottlenecks in the diagnostic workflow of clinical and epidemiological surveillance laboratories [11] . Saliva appears to be a promising URT specimen type for screening, diagnosis, follow up, and infection control of SARS-CoV-2 in all age groups. Diverse studies have reported consistent detection of SARS-CoV-2 RNA in saliva of symptomatic COVID-19 patients and sensitivities of saliva-based RT-qPCR ranging from 84 to100% compared to paired positive nasopharyngeal (NP) samples [12] [13] [14] . While collection of NP or oropharyngeal samples is inconvenient for patients and exposes health care workers to infection risk, saliva specimens can be repeatedly collected or self-collected in a simple, safe, and inexpensive manner without specific training or use of PPEs. In addition, good saliva stability at room temperature can simplify sample transport, avoiding cold-chain conditions [15] . Recently, the U.S. Food and Drug Administration granted accelerated emergency use authorization for the use of saliva, in addition to other respiratory specimen types, to facilitate mass screening of SARS-CoV-2 [16] . However, there is scarce evidence on the implementation of saliva-based screening approaches to identify asymptomatic subjects. We have developed a novel screening method for SARS-CoV-2 that combines use of self-collected raw saliva samples, heat-treated virus inactivation and RNA extraction in a single step, and RT-qPCR, herein referred as direct RT-qPCR. This simple, safe, and rapid method circumvents use of collection swabs, VITM, and RNA extraction reagents, as well as RNA purification and concentration steps, allows utilisation of different commercial RT-qPCR kits, and minimises dependence on the supply chain of reagents and consumables. The objective of the study was to validate and implement . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint 5 direct RT-qPCR on self-collected saliva for first-line screening of SARS-CoV-2 infection. The study was conducted in the Molecular Microbiology Department of Sant Joan de Déu Hospital (SJDH), a university reference maternal and child health medical centre located in Barcelona (Spain), in several successive phases: Phase 1. Analytical validation. SARS-CoV-2 RNA detection yield was assessed in saliva samples by direct RT-qPCR and a 3-phased standard RT-qPCR protocol. Additionally, intra-and inter-assay precision and effect of saliva storage under different conditions on performance were evaluated. Phase 2. Diagnostic validation. Performance of direct RT-qPCR was compared against standard RT-pPCR on paired saliva-NP samples serially obtained from asymptomatic young and adult participants included in a prospective cohort. Outcomes sought were diagnostic sensitivity, specificity, and predictive values of saliva-based direct RT-qPCR against standard RT-qPCR in NP samples. Phase 3. Pilot screening programme. Once validated, saliva-based direct RT-qPCR was deployed in SJDH to screen volunteer health workers and other staff. Planned outcomes were rate of participation (as a proxy for pilot acceptance), identification of positive cases for prevention of COVID-19 nosocomial outbreaks in the setting, and feasibility of unsupervised saliva self-collection by end-users. Comparative performance of direct RT-qPCR and standard RT-qPCR in saliva. Samples required for analytical validation were voluntarily provided by healthy adult researchers involved in the study or obtained from SJDH's Biobank, a research . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Direct RT-qPCR intra-and inter-assay precision. A set of saliva specimens including one sample with high SARS-CoV-2 RNA load, one sample with low RNA load, one negative sample, and a negative control (water) were tested by triplicate in the same run to assess intra-assay precision. Three sets of saliva specimens including each of them one SARS-CoV-2 high positive sample, one low positive sample, one negative sample, and a negative control were tested in different runs in different days to evaluate inter-assay precision. Effect of saliva storage conditions on direct RT-qPCR performance . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint SARS-CoV-2 RNA detection yield by direct RT-qPCR was determined for different conditions of saliva storage: at room temperature for a maximum period of 24 hours, refrigerated at 4ºC for 24 hours, or frozen -80ºC for longer than 24 hours. Diagnostic validation was conducted using samples collected from participants in the ongoing "Kids Corona Study of SARS-CoV-2 transmission at Football Club Barcelona Academy "La Masia", run by SJDH. In brief, that study entailed self-collection of saliva by teen and young adult soccer, basketball, handball, futsal, and roller hockey players, as well as adult acompanying coaches, teachers, physiotherapists, and staff residing at is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint 8 least two target genes of SARS-CoV-2 were detected and the amplification curves were adequate; and inconclusive if either only one gene was detected or amplification curves were unusual. Saliva self-collection coaching. Health workers, students, aid volunteers, and other professionals of the study setting were invited to participate in a pilot screening programme for SARS-CoV-2 based on the validated method. Instructions were disseminated to participants so that they could collect their own saliva in an unsupervised but safe manner. Participants were recommended to collect their own saliva in the first morning hours or after a fasting period of 2 hours to avoid food remains, according to recent evidence [17] . They were instructed to spit their saliva into tube collectors, transfer samples to sterile Eppendorf tubes with disposable Pasteur pipettes, close tubes with screw caps, decontaminate external surfaces of tubes with a hydroalcoholic solution, and identify them with heat resistant barcode labels before delivery to the SJDH Molecular Microbiology Department. All the information about the adequate pre-analytical procedure was gathered in an explanatory video and a brochure. This training material was made accessible on line to the participants through SJDH's intranet web site. Pilot screening programme. Eppendorf tubes received in the laboratory were not opened until the virus had been inactivated with heat, for safety reasons. A high throughput system was put into service for rapid screening workflow utilising an aliquot robot (Microlab ® STAR M, Hamilton Robotics, US) and a thermal cycler (QuantStudio 7 ® , Thermofisher, US). Up to 384 batched RNA extracts, positive, and negative controls were dispensed by the aliquot robot to the PCR plate of the thermal cycler for performance of direct RT-qPCR reaction with TaqPath COVID-19 RT-PCR kit reagents. This process workflow can be completed in less than 2 hours. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint Statistical analysis SARS-CoV-2 detection yields in saliva by direct and standard RT-qPCR, measured in cycle threshold (Ct) values, were compared using the Student t-test or the Mann-Whitney U test. Mean Ct values were determined from the SARS-CoV-2 genetic loci targeted by the two commercial RT-PCR kits used. Differences between Ct values obtained for SARS-CoV-2 targeted genes in different replicates and runs were analysed to assess precision. Associations between Ct values of direct RT-qPCR in saliva samples exposed to different storage temperature conditions were evaluated using the Pearson coefficient of correlation. Diagnostic sensitivity, specificity, positive and negative predictive values were determined as reported elsewhere [18] . Statistical significance was set at a p-value of <0.05 and confidence intervals (CI) at 95% level. All statistical analyses were performed using Stata v.15 software (Stata Corp., TX, US). A higher mean SARS-CoV-2 Ct value was obtained in saliva using GeneFinder amplification reagents for direct RT-qPCR (28.2, SD 6.0) compared to standard RT-qPCR (24.9, SD 5.4, p=0.13). In contrast, mean Ct values yielded by direct RT-qPCR when utilising TaqPath reagents were lower for direct RT-qPCR (21.4, SD. 5.2) than for standard RT-qPCR ( GeneFinder, 24.9, p=0.34) ( Table 1) . Saliva-based direct RT-qPCR showed differences in Ct value in a range of -0.99 to 2.84 within a run of replicates (Table 2 ) and in a range of -5.57 to 4.28 between diferent runs (Table 3) . Strong correlations were found . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020. 11.19.20234245 doi: medRxiv preprint between Ct values for samples stored at room temperature, in refrigerator for 24 hours or frozen at -80ºC, either for direct RT-PCR using GeneFinder kit (Pearson coefficient of correlation r>0.99) or TaqPath kit (r>0.94) (Table 4) . A total of 183 out of 230 participants in the "Kids Corona Study of SARS-CoV-2 transmission at Barça" (185 children, 45 adults) met inclusion criteria. Ten participants were excluded from the validation process because they were positive for SARS-CoV-2 antibodies by ELISA at baseline. The remaining 173 participants yielded negative results in both paired saliva and NP samples at baseline and were followed up during 9 to 12 weeks. A positive RT-PCR in NP sample was detected in 23 participants in weeks 4 (n=1), 6 (n=1), 9 (n=4), 10 (n=7), 11 (n=2), and 12 (n=8). SARS-CoV-2 positivity was confirmed by direct RT-qPCR in 22 paired saliva samples and one was inconclusive. Of note, viral RNA was detected in the saliva samples of three participants one week earlier than being detected for the first time in NP specimens (Fig 1) . An inconclusive result in NP specimen by standard RT-qPCR was obtained for seven participants. All serial paired (n=100) and non-paired preceeding saliva samples obtained from these subjects were found negative by direct RT-qPCR, except for one individual whose paired saliva yielded a positive result. Sensitivity and specificity values were 95.7% (95% CI, 79.0-99.2%) and 100.0% (95% CI, 98.6-100.0 %), respectively (Table 5 ). Positive predictive value was 100.0% (95% CI, 85.1-100.0%) and negative predictive value was 99.6% (95% CI, 98.0-99.9%). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint When the method was implemented for pilot screening of SARS-CoV-2 in a reference hospital, all saliva-positive results (0.9%) agreed with positive results in paired NP samples. In addition, a few inconclusive results in saliva (1.0%) raised the need for confirmatory testing and uncovered a minor proportion of additional NP positive samples. Overall, these results indicate that the proposed method performs adequately in a real-life scenario for its intended use of screening. It is worthwhile to highlight that no significant usability issues occurred during the pre-analytical phase, as shown by the negligible proportion of invalid results obtained in saliva (0.6%). Moreover, pilot screening gained high participation among health workers in the study site, suggestive of their willingness to self-collect and dispense saliva samples according to a simple set of instructions. In operational terms, use of a high throughput system allowed fast analytical workflow for close surveillance and timely control of potential SARS-CoV-2 nosocomial infection in the setting. Overall, we speculate that method implementation may result in savings both in consumables (swabs, PPEs, VITM, RNA extraction reagents) and health workforce before RNA amplification step. Research on SARS-CoV-2 RNA detection in pre-heated URT specimens other than saliva has been addressed by diverse groups, with a primary focus on diagnosis of symptomatic patients [19] [20] [21] . A pre-print study by Fernández-Pittol and colleagues specifically compared accuracy of direct RT-qPCR on saliva against standard RT-PCR on NP or oropharyngeal swabs of adult patients with SARS-CoV-2 symptoms [22] . While we observed 95.7% sensitivity and 100% specificity of saliva-based direct RT-qPCR in asymtomatic individuals, that group reported sensitivity and specificity values of 90.0% and 87.5%, respectively, in a cohort of adults who had experienced symptoms onset within the preceding 9 days. Being the protocols of heat treatment identical in both studies, our hypothesis is that after SARS-CoV-2 acquisition, viral load in saliva may progressively decrease across the pre-symptomatic stage, a declining trend that is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint 13 could continue after onset of symptoms, as already observed in symptomatic patients [23, 24] . Few studies have analysed performance of saliva-based direct RT-qPCR on individuals without SARS-CoV-2 symptoms. In this regard, our results were consistent with those of a study by Wyllie and colleagues with 495 asymptomatic health workers tested by a RT-qPCR protocol that included use of VTM and previous RNA extraction [23] . This is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint values in 9 paired positive and 91 paired negative saliva-NP samples. Similarly, Vogels et al. developed a protocol that consists of mixing saliva without preservative buffers with a proteinase K before performing heat inactivation at 95ºC during 5 minutes and a dualplex RT-qPCR [27] . High positive (97.1%) as well as negative agreement (100.0%) was found in 37 paired positive and 91 paired negative saliva-NP samples. Comparatively, our optimized method did not require addition of specific buffers to saliva for optimal performance while maintaining process workflow as safe and simple as possible. The main strengths of this study were diagnostic validation of the proposed method in a diverse cohort of asymptomatic teenagers and young and older adults, as well as extensive method implementation for screening SARS-CoV-2 in a hospital environment. A limitation was that significant differences in Ct values were observed for direct RT-qPCR depending on the use of GeneFinder or TaqPath amplification reagents in the analytical validation process. To be noted, GeneFinder kit is designed for performance of 45 amplification cycles whereas TaqPath kit entails 40 cycles, and each of them sets different threshold values set for a positive result (GeneFinder, 40; TaqPath 37). Therefore, we were not able to provide insights into the significance of saliva viral load or Ct values obtained from these two commercial reagent kits. In conclusion, this study showed that a novel direct RT-qPCT on self-collected raw saliva is a simple, safe, and accurate method for first-line screening of SARS-CoV-2. High throughput pilot implementation proved to be feasible, allowed fast analytical workflow, and gained high levels of voluntary participation in a sensitive hospital scenario. Self-collection of saliva by end-users had negligible effects on validity of results. Evidence generated by this study supports the potential scale up of selfcollected, saliva-based direct RT-qPCR for enhanced community-wide screening of SARS-CoV-2. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint Author summary Why dis study was done?  Saliva is a promising non-invasive specimen type for screening, diagnosis, follow up, and infection control of SARS-CoV-2 in a safe and convenient manner.  Diverse studies have addressed performance of RT-qPCR in saliva for clinical diagnosis of symptomatic patients. However, there is scarce evidence about salivabased RT-qPCR protocols aimed to screen asymptomatic subjects. What did the researchers do and find?  We validated and implemented an optimised screening method that combines use of self-collected raw saliva samples, single-step heat-treated virus inactivation and RNA extraction, and direct RT-qPCR.  Saliva-based RT-qPCR showed high sensitivity (95.7%) and specificity (100.0%) to identify asymptomatic individuals in a validation cohort of 173 teenagers and young and older adults, compared to standard RT-qPCR in nasopharyngeal sample. What do these findings mean?  The present study serves as a demonstration that direct RT-qPCT on self-collected raw saliva is a simple, rapid, and accurate method that can be scaled up for enhanced community-wide screening of SARS-CoV-2. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 27, 2020. ; https://doi.org/10.1101/2020.11.19.20234245 doi: medRxiv preprint . 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