key: cord-0921311-nr4lre47
authors: Rubio, Camila P.; Franco-Martínez, Lorena; Resalt, Cristina Sánchez; Torres-Cantero, Alberto; Morata-Martínez, Irene; Bernal, Enrique; Alcaraz, María J.; Vicente-Romero, María R.; Martínez-Subiela, Silvia; Tvarijonaviciute, Asta; Cerón, José J.
title: Evaluation of different sample treatments options in a simple and safe procedure for the detection of SARS-COV-2 in saliva
date: 2021-05-24
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.05.053
sha: 4531b98d91d3a24293b55271dce0d9078332a61b
doc_id: 921311
cord_uid: nr4lre47

Objectives To evaluate four sample treatments in a safe and simple procedure for SARS-CoV-2 detection in saliva. Methods Four sample treatments in three-step procedure for the detection of SARS-CoV-2 in saliva, consisting in 1) heating at 95 °C during 5 minutes for sample inactivation, 2) sample treatment, and 3) analysis by RT-LAMP were evaluated using saliva samples with known amounts of added viral particles and saliva from infected individuals. Results Three treatments had a limit of detection of 500.000 viral particles per ml of saliva and could have a practical use for detecting those individuals that potentially could transmit the disease. The treatment consisting of a combination of phosphate buffer, dithiothreitol, ethylenediaminetetraacetic acid and proteinase K, and an additional 95 °C heating yielded the lower LOD 95 and the sensitivity ranged from 100% in patients with RT-PCRs NPS of Ct<20 to 47.8% in patients with Cts>30. Conclusions This report highlights the importance for an adequate sample treatment in saliva for the detection of SARS-CoV-2 and describes a cheap and flexible procedure that can be adapted to-point-of-care and, although its sensitivity is lower than RT-PCRs, can contribute to the Covid-19 control by the detection of individuals able to transmit the disease.

experienced laboratory technicians to perform the assays, preventing its use as a point of care technique and limiting its application in low-income areas [1] .

Loop-mediated isothermal amplification (LAMP) has been developed as a novel molecular method that amplifies genetic material with high specificity using strand-displacement activity of a DNA polymerase and unique primer design to enable amplification at a single temperature [2] . Its isothermal nature has various advantages such as the compatibility with simple instruments such as a heat block or water bath and the possibility of reading results directly with minimum equipment [2] . In particular, loop-mediated isothermal amplification with simultaneous reverse-transcription (RT-LAMP) allows the detection of nucleic acids in a fast and easy way [3] .

Naso-and oropharyngeal swabs are the two main specimens for SARS-CoV-2 diagnosis. However, obtaining these swabs requires a trained healthcare worker, has a potential risk for nosocomial transmission and produces discomfort in the person to be sampled. Therefore, their use is not practical, especially in serial monitoring or mass screening programs. These drawbacks may be reduced by using saliva samples that are non-painful and non-stressful to obtain, and can be collected by the patient, even at home. The use of saliva for the diagnosis of the infection was suggested early in the pandemic, and it is now being considered a suitable alternative with various tests approved by the US Food and Drug Administration [4] .

Several procedures, with different sample processing and treatment conditions, have been developed for the diagnosis of COVID-19 in saliva without the need for an extraction step [5] [6] [7] . Most of these procedures require a direct manipulation of saliva that has to be diluted and/or treated with chemicals before its inactivation.

This requirement of direct manipulation may limit the widespread use of these methods for routine surveillance or testing outside the clinical laboratory due to concerns about the risk of infection during the analytical process. Methods in which the manipulation of saliva occurs after inactivation would be safer and more practical, and therefore, information about their efficacy and possible use might be beneficial to efforts aimed at expanding testing.

In this report, we evaluated four different sample treatments in a procedure developed in our laboratory to detect SARS-Cov-2 in saliva. We called this procedure "SAFE-SAL" and consisted of three steps: (1) heat inactivation, (2) sample treatment involving the addition of a chemical solution, and in some cases heating, and (3) a RT-LAMP assay. These four different sample treatments ranged from a simple phosphate buffer solution to a more complex treatment involving a mixture of chemicals including dithiothreitol (DTT), Proteinase K (PK) and EDTA, and an additional heating. This procedure has three main characteristics: (a) low risk of disease transmission since its first step is the sample inactivation; (b) use a simple and flexible way of sample processing, requiring only a heat source and a treatment solution; and (c) the use of an easy to perform and economical assay for the viral J o u r n a l P r e -p r o o f detection such as RT-LAMP. The procedure also flexible at this point since other detection assays could be potentially used.

In all cases, saliva samples were collected by the individuals themselves in a sterile sputum container. In samples from clinical cases, saliva was frozen at -80ºC until analysis. Informed consent was obtained for all samples. This study was approved by the Ethical Committee of the University of Murcia and the Ethical Committee of the IMIB-Arrixaca.

For initial testing to set-up the sample treatments and the calculation of the limit of detection, saliva confirmed to be SARS-CoV-2 negative by RT-PCR was spiked with known amounts of heat-inactivated SARS-CoV-2 virions (VR-1986HK, ATCC, Barcelona, Spain) at different concentrations. Different spiked samples were prepared and used in the tests, from 1x10 6 , to 0.5x10 6 , 0.2x10 6 , 0.1x10 6 and 0.05x10 6 /mL.

The procedure for analysis has three steps ( Figure 1 Step 1. Heat inactivation. Saliva samples were inactivated at 95ºC for 5 minutes in a heat block. After the heating, saliva samples were kept at ambient temperature for two minutes to avoid droplet production and then kept in ice during all the analytical process.

Step 2. Sample treatment. Addition of the treatment solution to the sample in 1:1 volume and, in some cases, additional heating.

Overall, the treatments tested for the calculation of the limits of detection were A final volume of 3 microliters of any of these mixes was used for the RT-LAMP reaction.

Step 3. Assay A RT-LAMP assay was used in this step. The total volume of the reaction was 20 µL including 3 µL of treated sample, 2 µL of DEPC-treated water, 2.5 µL of 10X primer mix N2 [24] , and 12.5 µL of WarmStart Colorimetric LAMP 2X Master Mix (New England Biolabs, M1800 L). In table 1 is the description of the primer mix. In all the experiments, we added a negative control (buffer) and a positive control (saliva with 1x10 6 /mL particles added).

To determine the limit of the detection of the procedure with the different treatments at step 2, saliva spiked with different concentrations of virus particles were analyzed in 10 replicates. The dilutions were made by spiking known amounts of heat-inactivated SARS-CoV-2 virions into fresh human saliva that was confirmed to be SARSCoV-2 negative by RT-PCR.

Initially, we tested 1x10 6 particles per mL, and then dilutions of this limit representing different numbers of particles: 0.5x10 6 , 0.2x10 6 , 0.1x10 6, and 0.05x10 6 /mL were analyzed.

The procedure using at step 2 the treatment which gave the best results of limit From these saliva samples, those that have enough volume available (samples from 48 positive patients and 33 negative in NPS) were also analysed by a commercially available RT-PCR assay (AllPlex 2019-noCoV assay, Seegene Inc). In a previous study, this assay had the best sensitivity for saliva specimens when compared to other commercially available assays [8] . The treatment of DTT, EDTA, PK and heating at 95ºC during 5 minutes, was used for the analysis of these samples.

The results of our procedure were interpreted by the naked eye by two independent persons who were unaware of SARS-CoV-2 RT-PCR results. There were no discrepancies between the two readers.

J o u r n a l P r e -p r o o f

The LOD was defined as the last sample target concentration at which all ten replicates were tested positive for the respective target. In addition, the amount of viral particles per mL of saliva that could be detected by each treatment with a probability of 0.95 (LOD95) was calculated by using a probit regression model. In the pilot study to evaluate the procedure, sensitivity was calculated with Excel and SPSS 24.0.

The results obtained in the test of the limit of detection, with the probability of the detection at the different concentrations of particles used in saliva of the different sample treatments appear in Figure 2 . As a representative example, the results of the treatment consisting in DTT, EDTA and PK and heating at 95ºC for 5 minutes appear in Figure 3 . EDTA and PK with heating at 95ºC during 5 minutes. In all 29 samples that were negative to RT-PCR, the results of our procedure were also negative. 

In this report, the use of different sample treatments is evaluated in a procedure for SARS-CoV-2 detection in saliva developed in our laboratory. This procedure consists of three main steps: initial heating for sample inactivation, sample treatment based on the addition of different solutions and, in some cases, J o u r n a l P r e -p r o o f additional heating, and, finally, a RT-LAMP assay. One of the main advantages of this procedure is that saliva inactivation is performed in the first step and, therefore, the rest of steps can be made with a significantly reduced concern about the risk of transmission. This is a very important issue since biosafety is of paramount importance in dealing with the analysis of Covid-19 in biological samples, and in particular with saliva that is highly contagious [9] . Heating the sample at 95 ºC, besides inactivating the virus, allows for the detection of RNA in the biological samples without the need for extraction procedures, and potentially inactivates components that can inhibit PCR or LAMP reactions [10, 11] .

The first treatment evaluated was just a dilution with phosphate buffer. An important issue is the pH of the buffer since the RT-LAMP used in this procedure is based on the decrease in the pH, which is made by the amplification of a target, and when this pH is lower than 7, the pink colour of the mix is transformed to yellow. In our conditions, the mix of the sample with the buffer phosphate a pH 7.7 allowed to (1) increase the saliva pH in cases of individuals that could have an acidic pH, since saliva pH can range from 6.2-7.6 [12] and therefore avoid false positives, (2) not to increase the pH over 8 that could inhibit the reaction. This could be the reason why in the process of setting up the procedure, when we tested phosphate buffer at pH 8.4 and also borate buffer at pH 8.2 we found false negatives. We also found that the addition of Triton X-100 and Np-40 interfered with the colorimetric lamp even at low concentrations (0.5%). Phosphate buffer as a sample treatment can also be used with PCR assays for Covid-19 diagnosis, with J o u r n a l P r e -p r o o f the caution that high concentrations can inhibit the assay [10] . Despite being a very cheap treatment, in our conditions, phosphate buffer gave false negatives with all the viral concentrations analysed, therefore the risk of false negatives should be considered when used. Other conditions or treatments such as additional heating at step 2 with phosphate buffer could be tested in order to determine if they could increase its sensitivity.

The other three treatments tested involved the use of DTT and EDTA. DTT is a sulphydryl reagent that specifically reduces mucoprotein disulfide bonds, being a mucolytic agent widely used for sample homogenization in sputum or other viscous fluids [6, 13] . The presence of mucus or viscosity in saliva can result in the trap of virus-containing cell components in mucus, leading to low yield RNA. It can also cause pipetting errors, clot formation, and failed amplification; therefore it should be avoided for the analysis of SARS-Cov-2 [14] . The chelating agent EDTA was added to DTT to increase its stability since DTT oxidation is catalyzed by free metals [15] , and sequester cations necessary for RNAse activity preserving the viral RNA [6] . At pH 7.5 and 20ºC, the half-life of DTT is 10 hours, and its stability can be increased with the addition of chelating agents. For example, DTT had less than 15% of oxidation in 1 week at 4% in the presence of Ethylene glycol tetraacetic acid [16] . Therefore, in the conditions of our procedure, we could postulate that DTT could be stable at least for one day. Ideally, storage at -20ºC in aliquots and daily use of fresh solution would be recommended until specific data on the stability of J o u r n a l P r e -p r o o f the solution used in this procedure will be generated. TCEP has been proposed as an alternative to DTT as reducing agent in saliva since it is described as a more stable solution [6] . However, in our study, we prefer to use DTT since it was cheaper and because TECP stability can decrease with phosphate buffers and also chelating agents [16] .

The three different treatments tested in step 2 using DTT combined with EDTA and heating for 5 minutes at 95ºC gave a detection limit of 500.000 viral particles/mL and a LD95 lower than this value. No evident improvements in the DTT treatment with a previous incubation for 30 minutes at 37ºC were observed in our conditions, so just one heating at 95ºC could be performed when DTT is used at step 2. Further studies should be done to elucidate the mechanisms involved in the positive effect of the additional heating at 95ºC on step 2 of this procedure. The addition of PK to the treatment solution produced the lowest LD95. PK is a serine protease that degrades RNAases in samples resulting in prevention of RNA degradation, and also homogenizes sputum samples [17] . Possibly the combination of DTT and PK allows for a larger decrease in viscosity of the sample and the removal of substances that inhibit amplification, therefore improving the detection of virus RNA.

It has been described that samples collected from patients displaying less than 1.000.000 viral copies per mL contain minimal or non-measurable infectious virus, that cannot be cultured and, consequently, can have a potentially lower risk of infection [18, 19] . Based on this, our procedure, with any of the 3 treatments tested at step 2 J o u r n a l P r e -p r o o f that had limit of detection of 500.000 viral particles, could have a practical use for detecting those individuals that could transmit the disease [20] . However, it should be pointed out that it would be less sensitive than RT-PCRs, (for example the RP-PCR used in our study for comparative purposes has a limit of detect 250 copies per mL in saliva [8] and, therefore, can have limited sensitivity at the very early stages of infection. In this line, the use of a straw or any collection device that avoids the creation and expansion of drops should be encouraged [4] . Finally, it is important to point out that the data presented in this paper is specific for the procedure and assay used, and the treatments evaluated could have different effects if tested in other different procedures of assays. Also, results could vary if other sample types are used such as sputum that can have a higher viral load that saliva.

This procedure can be subject to improvements. In addition to further trials to test possible new sample treatments at step 2 that could increase the sensitivity of J o u r n a l P r e -p r o o f the procedure, the step 3 could be adapted to microtiter plates, increasing the throughput of the assays. Besides, though the set of primers used in our study was one of the most sensitive according to a previous report, the use of other primers or primer combinations could even enhance the sensitivity of the assay [25] and other types of LAMP or different types of assays could be used in the step 3. An important consideration for molecular diagnostic assays and particularly LAMP is the sensitivity to carryover contamination [26]; therefore, the use of separate spaces and equipment for the different phases of LAMP preparation is recommended where possible and also the inclusion of dUTP and uracil-DNA glycosylase in the reaction can help to prevent carry-over contamination.

In this report, we described an optimized sample treatment for a procedure for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva. This procedure is safe and flexible since it can work with modifications in various steps. It's main limitation is its lower sensitivity compared to RT-PCRs;

however, it can contribute to the Covid-19 control by the detection of individuals able to transmit the disease. Also, it is cheap, and it could be applied in contexts of reduced economic resources and limited access to high-cost equipment, personnel, or reagents. In addition, it has the advantage of using saliva as a sample and the potential of being adapted to point of care. It is expected that the analytical

Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On

Site Screening of COVID-19

Loop-Mediated Isothermal Amplification of DNA

Visual Detection of Isothermal Nucleic Acid Amplification Using PH-Sensitive Dyes

Use of Saliva for Diagnosis and Monitoring the SARS-CoV-2: A General Perspective

Rapid and J o u r n a l P r e -p r o o f Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification

SARS-CoV-2 Detection Using Isothermal Amplification and a Rapid, Inexpensive Protocol for Sample Inactivation and Purification

SalivaDirect: A Simplified and Flexible Platform to Enhance SARS-CoV-2 Testing Capacity

The Use of Saliva as a Diagnostic Specimen for SARS CoV-2 Molecular Diagnostic Testing for Pediatric Patients

Risks of Transmission, and Preventing COVID-19: A Comprehensive Evidence-Based Review

Massive and Rapid COVID-19 Testing Is Feasible by Extraction-Free SARS-CoV-2 RT-PCR

Stability of SARS-CoV-2 in Different Environmental Conditions

Comparative Evaluation of Three Preprocessing Methods for Extraction and Detection of Influenza A Virus Nucleic Acids from Sputum

Direct Clinical Evidence Recommending the Use of Proteinase K or Dithiothreitol to Pretreat Sputum for Detection of SARS-CoV-2

The Stabilities of Various Thiol Compounds Used in Protein Purifications

A Comparison between the Sulfhydryl Reductants Tris(2-Carboxyethyl)Phosphine and Dithiothreitol for Use in Protein Biochemistry

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids from Sputum Samples for Real-Time Reverse Transcription PCR

Test Sensitivity Is Secondary to Frequency and Turnaround Time for COVID-19 Screening

Virological Assessment of Hospitalized Patients with COVID-2019

Rethinking Covid-19 Test Sensitivity -A Strategy for Containment

Rapid Point-of-Care Detection of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)

Mass Screening of Asymptomatic Persons for SARS-CoV-2 Using Saliva

The Role of population-wide saliva sampling for COVID-19

SARS-CoV-2 Detection by Fluorescence Loop-Mediated Isothermal Amplification with and without RNA Extraction

Enhancing Colorimetric Loop-Mediated Isothermal Amplification Speed and Sensitivity with Guanidine Chloride