key: cord-0696911-920mxq7f
authors: Brotons, Pedro; Perez-Argüello, Amaresh; Launes, Cristian; Torrents, Francesc; Subirats, Maria Pilar; Saucedo, Jesica; Claverol, Joana; Garcia-Garcia, Juan Jose; Rodas, Gil; Fumado, Vicky; Jordan, Iolanda; Gratacos, Eduard; Bassat, Quique; Muñoz-Almagro, Carmen
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: 2021-07-25
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.07.054
sha: fde9ffe82eebd5da0daa289a8b962138bb1de685
doc_id: 696911
cord_uid: 920mxq7f

Objective: To validate and implement an optimised 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. Methods: 3-phased study conducted in Barcelona (Spain) in June-October, 2020, including: i) analytical validation against standard RT-qPCR in saliva samples; ii) diagnostic validation against standard RT-qPCR using paired saliva-nasopharyngeal samples obtained from asymptomatic teenagers and adults in a sports academy; and iii) pilot screening of asymptomatic health workers in a tertiary hospital. Results: Phase i) Detection yield of the new method was comparable to that of standard RT-qPCR. Phase ii) Diagnostic sensitivity and specificity values in 303 self-collected saliva samples were 95.7% (95% CI, 79.0-99.2%) and 100.0% (95% CI, 98.6-100.0 %), respectively. Phase iii) Only 17 (0.6%) of saliva samples self-collected by 2,709 participants without supervision were invalid. Rapid analytical workflow by the new method (up to 384 batched samples processable in <2 hours) yielded 24 (0.9%) positive results in the remainder 2,692 saliva samples. Paired nasopharyngeal specimens were all positive by standard RT-qPCR.. Conclusions: Direct RT-qPCR 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.

constitutes the preferred method for detection of SARS-CoV-2, given its high sensitivity and specificity (Tang et al., 2020) . RT-qPCR accuracy may vary depending on URT sample quality and time elapsed since virus acquisition (Kucirka et al., 2020) . 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, depending on the type of RNA extraction robot utilised and the number of samples batched together. During the first pandemic wave, 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 (Ranney et al., 2020 ). Saliva appears to be a promising URT specimen type for screening, diagnosis, follow up, and infection control of SARS-CoV-2. 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 Procop et al., 2020; Pasomsub et al., 2020) . 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 maintenance of cold-chain conditions (Ott et al., 2020) .

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 (U.S. FDA, 2020). 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 this study was to validate and implement 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 three successive phases.

SARS-CoV-2 RNA detection yield was assessed in saliva samples by direct RT-qPCR and a standard RT-qPCR protocol.

Samples required for analytical validation were voluntarily provided by healthy adult researchers involved in the study or obtained from SJDH's Biobank, a research biorepository integrated into the Spanish Biobank Network of Instituto de Salud Carlos III. We used positive saliva samples of known Ct values to produce quantified standard concentrations of SARS-CoV-2 RNA load to be tested by direct and standard RT-qPCR. Additionally, volumes of 90 μL of SARS-CoV-2 RNAnegative saliva samples were spiked with 10 μL of positive NP samples to increase the range of standard concentrations available for analytical validation. Direct RT-PCR workflow involved saliva incubation in block heater for 15 minutes at 96ºC to maximise virus inactivation and RNA extraction. RNA amplification was performed using two RT-qPCR kits (GeneFinder ® COVID-19 Plus RealAmp kit, Elitech, France; TaqPath ® COVID-19 RT-PCR kit, Thermofisher, US) and two thermal cycler platforms (Applied Biosystems ® QuantStudio 7 and Applied Biosystems ® Prism 7500, Thermofisher, US). Standard RT-qPCR workflow included viral chemical inactivation and RNA extraction, purification, and concentration using NucliSense ® easyMAG ® platform and reagents (bioMérieux, The Netherlands) or viral inactivation with 2 mL of sample preservation solution (Mole BioScience, China) and RNA extraction, purification and concentration using an aliquot robot (Microlab ® STAR M, Hamilton Robotics, US) and reagents (MagMAX ® Viral/Pathogen Nucleic Acid Isolation kit, Thermofisher, US). RNA amplification was performed following the same procedure as direct RT-PCR.

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.

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 hoursPhase 2. Diagnostic validation Diagnostic validation was conducted using samples collected prospectively 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 or attending the Football Club Barcelona Academy "La Masia" (Barcelona, Spain). A team of SJDH research nurses supervised saliva selfcollection by participants on site and simultaneously collected paired NP swabs from them for comparative testing. Inclusion criteria in the diagnostic validation process were participant recruitment during August 2020 and follow up for at least 9 weeks. Collected saliva and NP samples were transferred to sterile Eppendorf tubes (0.5 mL) and NP VITM tubes respectively, labelled, and transported by the nurses in ambient temperature to SJDH's Biobank for storage or to SJDH's Molecular Microbiology Department (NP samples) for standard RT-qPCR. Saliva was selfcollected at baseline and on a weekly basis whereas NP samples were collected at baseline and every second week. Serum-based enzyme-linked immunoassays (ELISA) were also performed at baseline. All baseline saliva, NP, and serum samples were tested at study start and any saliva and NP samples paired with ELISA-positive specimens were excluded from the validation. In case of a positive RT-qPCR result in a NP sample, both the paired biobanked saliva sample collected at the same time point and the series of saliva samples obtained previously from the same participant were retrieved and retrospectively analysed by direct RT-qPCR using GeneFinder COVID-19 Plus RealAmp kit. Results by any RT-PCR method were interpreted as positive if at 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.

.

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 rate of inhibitions due to unsupervised saliva self-collection by end-users.

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 (Hung et al., 2020) . 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.

Eppendorf tubes received in the laboratory were not opened until the virus had been inactivated with heat, for safety reasons. A high productivity 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. In case of positive detection of SARS-CoV-2 RNA in a saliva sample, a paired nasopharyngeal sample was obtained from the infected individual and a confirmatory standard RT-qPCR was performed within 24 hours.

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. Ct values obtained for the SARS-CoV-2 genes targeted by the two commercial RT-PCR kits across samples were summarized as mean and standard deviation or median and interquartile (IQR) range values.

Differences between Ct values obtained for SARS-CoV-2 targeted genes in different replicates and runs were analysed to assess precision and effect of saliva storage conditions. Diagnostic sensitivity and specificity values were determined as reported elsewhere (Altman and Bland, 1994) . 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).

The study was approved by the Ethics Commitee of SJDH prior to the beginning of activities (ref.

PIC-240-20). Use of samples collected from participants in the "Kids Corona Study of SARS-CoV-2 transmission at Football Club Barcelona Academy "La Masia" for the present and future studies was covered in the informed consent process and approval of that study (ref. .

A non-significantly higher median SARS-CoV-2 Ct value was obtained in saliva using GeneFinder (Table 2 ) and in a range of -5.57 to 4.28 between diferent runs (Table 3) .

Minor differences were found between Ct values using GeneFinder SARS-CoV-2 gene targets for samples stored at room temperature 24 hours compared to preserved in refrigerator 24 hours (range, -1.11 to 0.78) or frozen at -80ºC (range -0.41 to 0.76). Ct value differences using the TaqPath kit were more noticeable (24-hour room temperature vs. 24-hour refrigerator preservation, range -2.59 to 3.90; 24-hour room temperature vs.freezing at -80ºC, range -2.37 to 1.06) ( Table 4 ).

A total of 183 out of 230 participants in the "Kids Corona Study of SARS-CoV-2 transmission at Barça" (185 teens and young adults, 45 older adults) met inclusion criteria and were followed up from August to October 2020. 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. Seven NP samples had inconclusive results by standard RT-qPCR within the follow-up period, including 6 negatives and one positive in paired saliva, and were excluded from analysis together with their saliva pairs. A total of 100 paired serial saliva-NP samples were found negative during follow up whereas a positive 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 specimens of three participants one week earlier than being detected for the first time in NP specimens (Fig 1) . Sensitivity and specificity values (1.0%). NP swabs were collected from participants with positive or inconclusive saliva results and tested by standard RT-PCR. All 24 (100.0%) participants with saliva-positive results were also found positive by standard RT-qPCR in NP swab. Four (14.8%) out of 27 participants with inconclusive saliva results were positive by RT-qPCR in NP swab and 23 were negative (Fig 2) .

There is a lack of evidence on feasibility and usefulness of saliva-based RT-qPCR protocols for early SARS-CoV-2 infection. This study reports the results of validation and subsequent implementation of a direct RT-qPCR method based on end-user self-collection of raw saliva.

Despite by-passing use of VITM and RNA extraction reagents, this method achieved high accuracy for screening asymptomatic individuals. Sensitivity (95.7%) and specificity values (100.0%) validated in a diverse cohort of teenagers and young and older adults without symptoms were comparable to those of standard RT-qPCR protocols that use NP samples for clinical diagnosis. Of note, the only saliva result discrepant from a positive result in NP sample was inconclusive. Thus direct RT-qPCR in saliva flagged the need of confirmatory testing for the individual with this inconclusive saliva result and fulfilled its screening purpose. Interestingly, we identified three subjects in the validation cohort that were positive in saliva one week before giving a positive result in NP sample. Since subjects were screened in saliva weekly and in nasopharynx every second week, this finding suggests that serial screening for SARS-CoV-2 should not consider frequencies longer than one week between successive tests to be effective.

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 productivity system allowed fast analytical workflow for close surveillance and timely control of potential SARS-CoV-2 nosocomial infection in the setting. 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 ( 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. Some limitations for generalisation of results need to be noted. First, the number of samples tested for analytical accuracy was limited. Second, 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. Differences in Ct values between kits may suggest limited usefulness of SARS-CoV-2 RNA load as a potential marker of active and transmissible infection, since the definition of a cut-off value with adequate discriminatory power appears to be highly dependable on the specific reagent used for viral detection. Third, optimal sensitivity (100%) of direct RT-qPCR in the pilot screening was determined upon a relatively low number of direct-saliva positive samples (n=24). Four, adequate performance of direct RT-PCR on saliva was achieved by engaging participants to collect their samples in the first morning hours or after a fasting period of 2 hours. We cannot assume that similar results would be obtained under other sampling conditions. Fasting before selfsampling certainly generates minor inconveniences but, in our view, such inconveniences are clearly outweighted by improved user experience, as shown during the pilot implementation.

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 self-collected, saliva-based direct RT-qPCR for enhanced community-wide screening of SARS-CoV-2. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CMA reports past grants to her organization from BioMérieux, Roche Diagnostics, Qiagen, BioFire Diagnostics, Alere, and Genomica, outside the submitted work and personal fees from BioMérieux, Roche Diagnostics, and Qiagen for presentations in satellite symposiums outside the submitted work. PB reports personal fees from Roche Diagnostics for a presentation in a satellite symposium outside the submitted work. The rest of authors declare no conflicts of interest. The three replicates yielded negative results for the negative controls The three replicates yielded negative results for the negative control 

The Microbiology Technical Support Team, et al. Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples

Diagnostic tests. 1: Sensitivity and specificity

Presumed asymptomatic carrier transmission of COVID-19

Direct RTqPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

Occurrence and transmission potential of asymptomatic and presymptomatic SARS-CoV-2 infections: A living systematic review and meta-analysis

Overview of testing for SARS-CoV-2 (COVID-19)

European Centre for Disease Prevention and Control. An overview of the rapid test situation for COVID-19 diagnosis in the EU/EEA

Assessment of the use and quick preparation of saliva for rapid microbiological diagnosis of COVID-19

Evidence supporting transmission of severe acute respiratory syndrome coronavirus 2 while presymptomatic or asymptomatic

Early-morning vs spot posterior oropharyngeal saliva for diagnosis of SARS-CoV-2 infection: implication of timing of specimen collection for community-wide screening

Self-collected oral fluidand nasal swabs demonstrate comparable sensitivity to cliniciancollected nasopharyngeal swabs for Covid-19 detection

Variation in false-negative rate of reverse transcriptase polymerase chain reaction-based SARS-CoV-2 tests by time since exposure

Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV-2)

Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices

Saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease 2019: a cross-sectional study

A direct comparison of enhanced saliva to nasopharyngeal swab for the detection of SARS-CoV-2 in symptomatic patients

Critical supply shortages -The need for ventilators and personal protective equipment during the Covid-19 pandemic

Saliva-based molecular testing for SARS-CoV-2 that bypasses RNA extraction

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

Laboratory diagnosis of COVID-19: current issues and challenges

Evidence for transmission of COVID-19 prior to symptom onset

Consistent detection of 2019 novel coronavirus in saliva

Accelerated emergency use authorization (EUA) summary: SARS-CoV-2 assay

SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

Saliva as a noninvasive specimen for detection of SARS-CoV-2

World Health Organization. Laboratory testing for 2019 novel coronavirus disease (2019-nCOVID) in suspected human cases

Saliva or nasopharyngeal swab specimens for detection of SARS-CoV-2

We are indebted to SJDH's Biobank The Neetherlands for sharing their experience on RT-PCR in saliva.