Mass Screening Using Saliva Samples and Pooled Testing Strategy For SARS-CoV-2 RT-PCR Detection in Latvia

Background The number of COVID-19 cases is increasing globally and there is an urgency for a simple non-invasive method for the detection of SARS-CoV-2. Our study aimed to demonstrate that saliva can be used as a specimen for SARS-CoV-2 detection notably for the screening of extensive population groups via pooling. Methods We performed 36 serial measurements of 8 SARS-CoV-2 positive saliva samples to conrm the stability of the specimen and completed 37 pools of saliva samples by adding one positive specimen per pool. We collected paired nasopharyngeal/oropharyngeal swabs (NPS) and saliva specimens and processed them within 24 hours of collection. To demonstrate that saliva is an appropriate specimen for SARS-CoV-2 detection a eld study including 3,660 participants was performed between September 29 and October 1, 2020. A new solution for distribution and collection of self-sampled saliva was designed, produced and installed. All methods were performed in accordance with the relevant guidelines and regulations.


Introduction
Since the beginning of 2020 massive resources have been dedicated to control and mitigate the COVID-19 pandemic. Testing capacity and accessibility are crucial in monitoring COVID-19 outbreaks and allowing for the adjustment of measures put into place to reduce community transmission [1]. Viral nucleic acid detection using the real-time polymerase chain reaction (RT-PCR) assay remains the gold standard for the detection of SARS-CoV-2 and to diagnose COVID-19 [2]. Due to the increased demand for sampling and testing materials globally considerable constraints remain to conduct widespread screening for SARS-CoV-2.
Shortages of supplies and medical staff, logistical hurdles, as well the unpleasantness associated with obtaining a RT-PCR specimen making it di cult to practice the procedure on children and causing irritation for those who need to undergo repeated tests, all these factors have naturally accelerated research into alternative specimens and sampling methods for COVID-19 testing [3,4].
On September 21, 2020, Latvia had a 14-day cumulative number of COVID-19 cases of 5.1 per 100,000 people. By October 10 that number had risen to 66. During this period, laboratories experienced a signi cant overload [5]. Rapid and e cient solutions to increase testing capacity were needed.
The objective of this study was to con rm that saliva is a suitable specimen comparable to a nasopharyngeal/oropharyngeal swab for SARS-CoV-2 detection in speci c populations. We also evaluated the pooling approach to employ it in eld conditions for extensive screening of high-risk groups. Finally, a new solution was found for the distribution and collection of self-sampling kits for saliva testing. The new approach allows to increase sample collection capacity in a safe and effective way.

Patient sample collection
A single-center study was performed including all consented patients attending the laboratory for SARS-CoV-2 testing between May 12 and October 19, 2020. For the collection of the saliva samples, self-collection kits were distributed consisting of a specimen vial, registration form, alcohol pads for disinfection, and two safety plastic bags. Patients were requested to collect their saliva in an isolated location (keeping at least ve meters distance from other persons or objects), preferably outdoors, at home, or in their car. The saliva was collected in a container without any additive and delivered to the laboratory within 24 hours. Paired nasopharyngeal/oropharyngeal swabs (NPS) for RT-PCR were collected using the standard method used in our laboratory [6].
Sample preparation and analysis using RT-PCR Immediately upon arrival at the laboratory, the samples were pretreated by adding 1 ml of phosphate-buffered saline (PBS). The extraction of RNA and RT-PCR testing was identical for both swab and pre-treated saliva samples. Viral RNA was extracted with standard commercial extraction methods (QIAGEN, Germany and LifeRiver, China) used in our laboratory. RT-PCR was performed using our laboratory-developed and validated test method which detects S and N genes of the SARS-CoV-2 virus. Our validated and veri ed limit of detection (LOD 95 ) was 1 cp/rxn of the RT-PCR. Validated LOD of whole procedure, including extraction, was 50 cp/mL of unprocessed sample using data obtained from nasopharyngeal/oropharyngeal samples.

Stability of saliva specimens
To con rm the stability of saliva samples, we performed 36 serial tests on 8 primary SARS-CoV-2 positive saliva samples collected from patients 48 hours after their NPS tested positive. The data are summarized in Tables 1-3. Testing was randomized by different time intervals, the time of day, and the shifts of technicians. It includes repeated testing of two samples after 22h ( Table 2) and serial repeats of one sample (  The positivity rate in Latvia on September 23, 2020, was 0.5%. The approximate positivity rate at which the pooling of 5 samples becomes more e cient than the pooling of 10 samples is 3% (2-4% depending on test price, laboratory load, tests per sample).

Field study
To evaluate the convenience of saliva pool testing by RT-PCR in eld conditions, the town of Kuldiga (total population 10,352) with an ongoing outbreak of COVID-19 in a textile factory was selected. In collaboration with local authorities, 4,100 saliva self-sampling kits were distributed to inhabitants at a specially established distribution point. There were four persons dedicated to the distribution of the kits and four drivers to deliver the samples by car to the testing laboratory in Riga (150 km one way). During the study period, 3,660 saliva samples were collected (response rate 91.5%), delivered to the laboratory, and tested in pools of 10 samples. There were a total of 366 pools.
In the rst round of testing 43 pools were found to be positive and samples from these pools were re-tested individually. The saliva samples from 44 patients were con rmed as positive by RT-PCR. Mean Ct values of pooled samples were 13% higher than individually tested Ct (31.6 versus 27.6). The positivity rate of the tested population was 1.2%. In total 796 RT-PCR tests were performed. At the time of the sample collection, 68.2% (30/44) of patients did not report any symptoms on the questionnaire form.
Nasopharyngeal swab tests and saliva tests were compared with the number of new COVID-19 cases. The ndings illustrate that the second peak in new cases occurred 6-7 days after the initial peak. Such ndings correspond with the mean incubation period of the SARS-CoV-2. The results re ect a decrease in new cases in the following observation period (Figure 1 Comparison of con rmed new cases with testing in eld study).

Contactless self-service terminals and saliva testing on national level
In total, (between September 29, 2020 and April 10, 2021) 415 673 RT-PCR tests of saliva were conducted. Tests were used for screening purpose of local population in selected municipalities, healthcare workers and other essential teams, social care centers, schools, pre-school groups, participants of special meetings, sport competitions and award ceremonies. Contactless self-service terminals were accepted in hospitals, factories and municipalities as an additional way to submit self-collected saliva for testing. The order-sample-result ow for contactless solution is  Table 5. Percentage of saliva samples used for testing inceased continuously month after month reaching 67% of all SARS-CoV-2 tests performed in our laboratory in March 2021.

Discussion
The ndings of our study can be summarized as follows: (a) for the detection of SARS-CoV-2, saliva is a stable specimen with acceptable speci city and sensitivity at the early stages of infection; (b) saliva specimen is appropriate for pooling, with accurate diagnostic performance; (c) self-sampling of saliva was well accepted by the different groups of population and the new, automated, contactless terminal is the preferred option to increase the accessibility to testing.
Saliva has been identi ed as a reliable testing specimen for SARS-CoV-2 using the RT-PCR approach in several recent studies [7][8][9]. In the updated European Centre for Disease Prevention and Control (ECDC) recommendations, saliva is also mentioned as a convenient specimen for SARS-CoV-2 testing [10,11]. The FDA has approved methods for SARS-CoV-2 testing using saliva in several laboratories [12,13]. Nevertheless, saliva has been included in IFCC COVID-19 Guidelines on Molecular, Serological, and Biochemical / Haematological Testing as a promising sample type [14].
In addition, two recently published meta-analyses have concluded that self-administered saliva tests are on par with nose and throat swabs for detecting SARS-Cov-2 [15,16].
The high expression of ACE2 receptors in salivary gland cells is the main factor for virus a nity that could lead to active replication and transmission by saliva droplets expelled into the air during coughing or sneezing [17,18].
It is essential to establish standardized sample collection procedures, safe logistics, and reliable testing methods that meet performance requirements. Our stability testing con rmed that saliva samples stayed equally positive up to 24h.
In the analytical context, saliva can have the same or even better performance than NPS [19]. It has been previously con rmed to be highly sensitive and speci c at the early stage of infection 0-14 days after onset of symptoms and in asymptomatic cases [20]. Therefore, it is essential to take into consideration the relationship between the dynamics of viral load, Ct values, and the number of days after the onset of symptoms. As described in prior publications, the Ct value is important in determining the infectiousness of a patient sample. Ct values above 34 do not emit infectious virus particles, and values between 27-34 show low viability of the viral load. Samples with a Ct value of 13-17 show positive virus viability [21]. Ct values may vary depending on the different assays by up to 5 cycles for the same sample [22]. Variation also appears due to the quality of the specimen obtained and the different treatment methods used to prepare samples for testing [23]. As expected, we got some variation between the NPS and saliva sample types. In our pilot testing using 44 positive saliva samples we found slightly higher (13%) Ct values in pools, compared to individual tests. Similar ndings have been published by other researchers [24]. This observed slight difference still allows for the safe use of saliva pooling for surveillance purposes with su cient diagnostic accuracy. Nevertheless, pooling strategies are described in the latest update of COVID-19 testing strategies and objectives published by the ECDC [11]. There is evidence of bene ts from pooling in low prevalence countries with a low proportion of positive samples -up to 5%. Recent publications indicate that for populations with a prevalence of less than 1% the testing of saliva pools of 10 or 20 samples is more bene cial, and our independent calculations also bear this out. Comparing the correlation between different pool sizes (usefulness and e ciency) with test positivity rate, we concluded that a pool size of 10 is more e cient than a pool size of 5. The Additionally, improvements may reasonably be expected in terms of organizing the distribution of self-sampling kits to patients as well as the logistics of returning the kits to the laboratory that would further increase the usefulness of screening using a pool testing strategy. The contactless self-service terminals has shown a promising response to sample collection challenge, making preanalytical steps safer, more accessible and more convinient for selected groups of population. Self-testing kit distribution and collection points could also be further implemented for other dianostic purposes beside COVID-19 pandemia. The current capabilities allow further integration of urine and feces sample collection, based on the same process.
There were some limitations in our study: the size of the cohort of participants was limited, the selected laboratorydeveloped RT-PCR method for evaluation was used, also a lack of multicenter observation. Future studies extending the cohort are needed to con rm current ndings and provide the implications in clinical practice.
In conclusion our ndings con rm the stability of SARS-CoV-2 RNA in saliva, showing acceptable performance in terms of speci city and sensitivity at the early stages of infection, and the advantages of testing using a pooling strategy in a low prevalence population. Ct values and detected/missing gene information are favorable for the interpretation of the results from several aspects, e.g. epidemiological investigation, determination if the patient is infectious at the current stage, etc. Self-sampling makes the procedure faster, safer, and requires fewer resources.
The targeted distribution of test kits among a population with a known outbreak signi cantly increased the positivity rate. Saliva pool testing on a large scale provides an additional tool to take timely measures and contain outbreaks.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Committee of the Pauls Stradins Clinical University Hospital (record No 300720-18L).
Informed consent was obtained from all individual participants included in the study. Written informed consent was obtained from the parents of pediatric participants.

Consent for publication: Not applicable
Availability of data and material: The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: All authors declare that there are no con icts of interest.    Abbreviations: NPS -nasopharyngeal/ oropharyngeal swabs, RT-PCR -real-time polymerase chain reaction Table 5 Results of saliva testing by RT-PCR in E.Gulbja laboratory between September 29, 2020 and April 10, 2021 Figure 1 Comparison of con rmed new cases with testing in eld study