Saliva as a sample type for SARS-CoV-2 detection: implementation successes and opportunities around the globe

ABSTRACT

Introduction

Symptomatic testing and asymptomatic screening for SARS-CoV-2 continue to be essential tools for mitigating virus transmission. Though COVID-19 diagnostics initially defaulted to oropharyngeal or nasopharyngeal sampling, the worldwide urgency to expand testing efforts spurred innovative approaches and increased diversity of detection methods. Strengthening innovation and facilitating widespread testing remains critical for global health, especially as additional variants emerge and other mitigation strategies are recalibrated.

Areas covered

A growing body of evidence reflects the need to expand testing efforts and further investigate the efficiency, sensitivity, and acceptability of saliva samples for SARS-CoV-2 detection. Countries have made pandemic response decisions based on resources, costs, procedures, and regional acceptability – the adoption and integration of saliva-based testing among them. Saliva has demonstrated high sensitivity and specificity while being less invasive relative to nasopharyngeal swabs, securing saliva’s position as a more acceptable sample type.

Expert opinion

Despite the accessibility and utility of saliva sampling, global implementation remains low compared to swab-based approaches. In some cases, countries have validated saliva-based methods but face challenges with testing implementation or expansion. Here, we review the localities that have demonstrated success with saliva-based SARS-CoV-2 testing approaches and can serve as models for transforming concepts into globally-implemented best practices.

KEYWORDS:

• COVID-19
• diagnostic testing
• PCR
• saliva
• SARS-CoV-2
• screening
• surveillance

1. Introduction

The ‘gold standard’ sample types for SARS-CoV-2 detection defaulted early in the pandemic to nasopharyngeal or oropharyngeal swabs, as these are established diagnostic practices used in identifying other respiratory viruses [1,2]. However, swab-based sample collection can be costly, invasive, and put health personnel at risk by necessitating close contact during sample collection. Furthermore, the rapid evolution of the pandemic placed pressure on supply chains, increasing the need for alternative diagnostic tools to enable testing expansion (an overview of common approaches are shown in Figure 1). This led to the consideration of additional specimen types with saliva emerging as a quality alternative [3], a strategy supported by the continued growing body of evidence demonstrating optimization of saliva-based methods.

Figure 1. Overview of the most common sample types and testing methods for SARS-CoV-2 detection. The table in blue summarizes characteristics for the different sample types depicted, beneath. The table in grey summarizes typical characteristics of the testing methods depicted above.‘Nasal swabs’ include both mid-turbinate and anterior nasal swabs. Rapid antigen tests may also be known as lateral flow assays. POC = point of care. H = hour. Created with BioRender.com.

With appropriate treatment, saliva can be more sensitive than swabs for SARS-CoV-2 detection [4], particularly in early infections where saliva can be twelve times more sensitive compared to mid-turbinate swabs [5–7]. Additionally, less-invasive forms of testing are often preferred by patients over other specimen-collection options [8]. Public preference and accessibility of desired options directly influence testing participation rates [9]. Adding options like saliva-based testing that have clear diagnostic benefits and broad public acceptance serve to both remove barriers and strengthen public health response strategies. As a result, saliva-based testing continues to increase worldwide health security.

Many countries have demonstrated the successful introduction of saliva-based diagnostic testing as part of their public health responses. At the same time, misinformation and skepticism generated by the debate surrounding saliva as a reliable sample type have impeded the implementation and expansion of saliva testing programs. To demonstrate the current state of saliva-based SARS-CoV-2 testing internationally, this review highlights the best practices employed by successful testing operations, including effective methodologies and the use of low-cost materials, while addressing barriers and challenges that should be considered when developing or implementing salivary diagnostic programs. With this information, national leaders, health agencies, and research institutes hesitant about implementation can better consider saliva-based testing as a viable addition to the robust testing programs seeking to reduce preventable loss of life. As local context and populations requiring testing (e.g. asymptomatic adults vs. symptomatic children) vary, optimal methods for successful program implementation may differ. A protocol that is most suitable for one country’s population may not be the most appropriate for another. With a sound knowledge base, diverse populations can tailor methodologies and practices from the plethora of successful validation studies that have emerged globally throughout this pandemic and build a program that reflects their needs.

We note that this review is not systematic in nature, nor is this review a meta-analysis. Rather, the purpose of this review of the available literature is to highlight the regional acceptability, and the successes of various saliva-based methodologies for the detection of SARS-CoV-2 across the globe. It is intended to showcase examples as well as generate leads that researchers, public health decision-makers, and others can take from the trials experienced during the COVID-19 pandemic and apply to ongoing or future response efforts. Both systematic review and meta-analysis methodologies would have been too narrow to gather the insights this review has generated, as our primary concern was not just published research, but how research on saliva-based testing has been received by governments and regulatory bodies as well. Furthermore, meta-analyses conducted on this topic to date, often mention the differences in sample collection and processing that exist, but do not take these factors into consideration in the analysis, leading to confounded results and reporting. Our search strategy thus included reviewing each major geographic region, using a variety of search engines, to find published research, government documents, press releases, and other materials to understand how saliva as a sample type has been explored or implemented for the detection of SARS-CoV-2. The examples reported on were studies and/or technologies that showed the high performance of saliva as compared to swab-based approaches. We hope that this review encourages additional international collaboration to share and employ best practices for COVID-19 diagnostics, screening, and surveillance, and guide future research efforts.

2. Geographic regions

2.1. North America

In Canada, saliva was first suggested to be a viable diagnostic sample type in February 2021 [10]. As of March 2022 [11], the Canadian Government acknowledges saliva as a sample type for general COVID-19 diagnostics, but has yet to approve any of the saliva tests that have been submitted for consideration to their health agency (Health Canada). Despite this, the British Columbia Center for Disease Control has approved a gargle-and-spit testing method, involving the gargling of a saline solution then expelling saliva into a tube [12].

Saliva testing in the United States of America (USA) has been expansive: the Food and Drug Administration (FDA) has awarded Emergency Use Authorization (EUA) to 33 saliva-based molecular assays for SARS-CoV-2 detection [13]. Saliva as a sensitive sample type was first formally recognized when the FDA awarded an EUA to a PCR test developed by Rutgers University [14] in early April 2020 [15]. Other universities across the USA have also been integral to the innovation of saliva methods, working to address ease, affordability, and the ability to circumvent collapsing swab supply chains, including the Yale School of Public Health’s open-source SalivaDirect™ protocol [16], and the University of Illinois Urbana-Champaign’s covidSHIELD program for asymptomatic screening [17]. Many other universities in the USA have implemented high-volume, saliva-based testing programs specifically for their campuses and communities [18–24]. Saliva-based assays have also permitted safer returns to community centers, refugee resettlement projects, workplaces, and summer camps across the nation [25,26]. With lower vaccine coverage among children relative to adults [27], the urgency of testing vulnerable school populations was accommodated by Mirimus Labs (Brooklyn, NY), who were early innovators that offered pooled saliva testing affordably to schools so they could retain in-person learning for hundreds of thousands of students [28].

Commercialized saliva-based testing is also prevalent across the USA, servicing multiple private sector industries. Biotechnology companies have developed convenient saliva-based tests that can offer safer at-home sample collection by reducing viral exposure compared to testing centers [29], avert the more costly RNA extraction step [30,31], process results with portable PCR machines [32,33] or point-of-care instruments [34] to increase accessibility, streamline test processing through automated laboratory equipment [32,33] and enable sample pooling [35], or the detection of other respiratory illnesses via multiplexing [36,37].

Biotechnology companies have also partnered with state health departments to deliver saliva testing services to the public sector [38–42]. For example, the Howard County Health Department in Maryland contracted a company to provide free PCR saliva testing to the general public for both on-site or at-home testing [38], while the Minnesota Department of Health has offered on-site saliva testing for all Minnesota residents at no cost [40]. Biotechnology companies have also made partnerships with the aviation sector to provide saliva-based testing for inbound and outbound airport travelers [43–45].

An alternative saliva testing methodology, loop-mediated isothermal amplified testing (RT-LAMP), has been adapted in the USA as a simplified COVID-19 diagnostic option relative to PCR. The performance of some RT-LAMP assays is comparable to RT-PCR [46], demonstrating concordance as high as 98.8% [47], with options for extraction-free approaches [48], multiplexing for SARS-CoV-2 and influenza [49], or optimized for point-of-care devices [50,51].

More recently, American researchers have investigated the use of less common alternative diagnostic technologies, developing a silver nanotriangle array-based localized surface plasmon resonance (LSPR) sensor for use on saliva samples (February 2022) [52] and chip technology for detection of SARS-CoV-2 in saline gargle samples via smartphone (March 2022) [53]; both with the goal of providing quicker, easier testing. Overall, the abundance of robust saliva methods that have emerged in the USA validate saliva as a sustainable, reliable sample type to facilitate mass-testing programs and allow communities to more safely re-open.

In Mexico, saliva has reliably detected SARS-CoV-2 in healthcare workers [54], patients [55,56], and children [55,56]. The inaccessibility of RNA extraction kits forced health authorities to validate RNA-extraction-free protocols at a local level [57]. Saliva was successfully validated and implemented for primary school surveillance testing [57] as well as sample pooling for high-throughput testing [58]. As a result of nasopharyngeal swab shortages, saliva was also identified as a suitable sample type for testing asymptomatic healthcare workers (April 2021) [59]. Thus, saliva permitted more affordable and simpler diagnostic procedures appropriate for scalable SARS-CoV-2 detection [58].

2.2. Central America and the Caribbean

Countries throughout Central America and the Caribbean have been operating against intense resource and staffing constraints, challenging the region’s ability to effectively respond to the pandemic [60]. Hospitals and community health centers have had to prioritize the acquisition of other essential healthcare resources, such as ventilators and personal protective equipment, limiting the efficiency and availability of testing [61]. The lack of PCR testing options has imposed further stress on many of the region’s fragile healthcare systems by impeding the identification and isolation of infected healthcare professionals and other populations vulnerable to SARS-CoV-2 infection. Most of the region follows testing recommendations set forth by the Pan American Health Organization, which has called for an expansion of testing networks with a focus on rapid antigen tests (January 2022) [62,63].

Much of the Central America and Caribbean region abides by the current global standards of testing for incoming and departing international travelers, making the availability of PCR tests especially crucial in a time of high regional tourism. To meet testing demands related to international travel, some countries, including Costa Rica, have utilized saliva-based assays [64] or will accept saliva-based tests for border entry [65]. Island nations with high tourism rates, such as Aruba, Bonaire, and the Dominican Republic, boasted convenient saliva PCR testing locations and concierge services for outbound travelers in collaboration with Delta Airlines [66–68].

Earlier in the pandemic, a Nicaragua-based trial among healthcare workers (July 2020) found saliva-based LAMP to be an attractive testing option in the face of scarcity and inefficient government testing [69]. For testing of the general public, the SalivaDirect™ protocol was implemented in the U.S. Virgin Islands [70] and The Bahamas (November 2020) to expand testing for asymptomatic individuals [71], and a saliva-based PCR test developed in Puerto Rico (March 2021) is now used in major laboratories across the territory [72,73]. Although saliva-based PCR testing has been considered, most Caribbean nations solely rely on nasopharyngeal or oropharyngeal swab PCR testing for incoming travelers [74,75]. Thus, the region still faces a slow uptake of saliva-based PCR testing for COVID-19, despite demonstrations of its feasibility in implementation [76].

2.3. South America

The rapid and dramatic spread of COVID-19 throughout many South American health care systems drove the need for expansive testing efforts. Throughout the region, saliva collection has been praised as a non-invasive approach [77] that poses lower risk to healthcare providers [77,78] and is well-suited for low-resource settings [79]. Studies conducted in Argentina (September 2020)[80], French Guiana (October 2020) [81], Chile (February 2021) [77], and Colombia (October 2021) [79] all found saliva to have high sensitivity (91–100%) and to serve as an effective alternative sample type to nasopharyngeal swabs. In Peru (March 2021), a study found high household incidence of COVID-19 through testing saliva specimens [82]. In Ecuador, National Reference Laboratories have been recommended to use saliva samples alongside nasal and nasopharyngeal swabs when validating diagnostics for SARS-CoV-2 [83]. In Brazil, RT-PCR screening of saliva samples was found to be a comparable method for the detection of certain SARS-CoV-2 variants, as a time-, cost-, and contact-saving alternative to genomic surveillance [84].

Government authorization and commercial implementation currently varies considerably by country in South America. In Brazil, self-collected saliva specimens for PCR testing [85], rapid antigen testing [86,87], and electrochemical RNA detection [86,88] are available for at-home testing and at drive-through community testing sites. Chile’s Health Minister has endorsed saliva testing in primary care settings [89] and saliva testing results are accepted by Chilean airports [90]. A novel, saliva CRISPR-Cas-based COVID-19 test was developed in Peru [91] and two new isothermal amplification-based tests (iAMP and LAMP) using saliva have been validated in El Salvador and Paraguay [92]. Although strong uptake by the private sector has yet to occur, South American research demonstrates continental investment in saliva for the detection of SARS-CoV-2.

2.4. Oceania

In Oceania, the COVID-19 response has varied among nations and their internal geographies. Some island nations have reported limited COVID-19 case data, such as Micronesia which reported no cases within their borders through 2021 [93]. However other countries, such as Fiji and Papua New Guinea, reported zero or very few COVID-19 cases in 2020, but saw increases in case numbers during 2021, prompting a need for heightened testing and prevention measures [94].

Saliva test findings in this region have mainly emerged from Australia and New Zealand. Although saliva was recognized early in the pandemic (April 2020) in Australia as a viable diagnostic sample type [95], uptake was slow due to initial reports of reduced sensitivity relative to nasopharyngeal swabs. While this earlier data was potentially skewed by historic cases or methods not yet optimized for sensitive detection in this sample type, the authors later reported saliva to be effective for the detection of SARS-CoV-2 (December 2020) [96] and an observational study (October 2020) found higher positive predictive agreement (PPA) for saliva (72%) than for combined oropharyngeal-nasal swabs (63%) [97]. Furthermore, the study ran an optional preferences survey for participants finding that the saliva collection method was preferred by 79% of the respondents, including 92% of children under 10 years old [97].

In early 2021, the Australian government called for heightened use of saliva testing for vulnerable populations and essential workers that require regular testing [98]. Since November 2021, saliva testing has been utilized for essential workers within the New South Wales Ministry of Health’s COVID-19 Saliva Surveillance Program [98]. There are currently three COVID-19 saliva tests approved by the Australian government, meaning that most Australian laboratories are required to perform their own validation of assays for saliva testing [99]. A saliva rapid antigen test for at-home use has also been approved [100]. A proof-of-concept screening method using infrared light technology is in development that measures the SARS-CoV-2 RNA signature (sensitivity = 93%) in the infrared spectra of saliva in symptomatic hospital patients [101]. The portable test displays results in ~5 minutes greatly supporting reduced response times, a significant factor for interrupting transmission chains. The Australian Department of Health acknowledges the literature on saliva testing includes numerous methods that have demonstrated high sensitivity, but states that further studies are required to evaluate the best collection and processing methods for saliva [98]. The current Australian Testing Framework (updated 7 March 2022) includes reference to many Australian laboratories that have validated saliva for RT-PCR testing to facilitate expanded surveillance. It also posits the use of saliva as an alternative specimen to nasopharyngeal swabs as a way to increase testing uptake and frequency as well as sample pooling and extraction-free PCR methods [98].

In New Zealand, USA-developed RNA extraction-free, saliva-based PCR tests have been validated locally and offered commercially [102,103]. In May 2021, routine saliva testing was mandated for individuals at high risk of exposure [104]. The initial legislative act on saliva testing required confirmatory nasopharyngeal swab testing for positive saliva results. However, this order was amended in December 2021 as New Zealand’s Ministry of Health updated their position on the use of saliva for diagnostic COVID-19 testing, reducing the required frequency of testing and eliminating the need for confirmatory testing of nasopharyngeal swabs. The update also endorsed the use of saliva as a sample for diagnostic testing in the aviation, maritime, and education sector [105]; there are now designated locations for employees of these sectors to self-sample at home and then drop-off their samples in collection boxes [106]. In the private sector, saliva-based testing is used for elder care settings [103], certain workplaces, sports teams, and pre-departure travel certificates [107]. In January 2022, a New Zealand-based, inexpensive, and portable RT-qPCR device was added onto the SalivaDirect protocol’s FDA EUA [108] after demonstrating performance comparable to larger, immobile instruments [109]. In addition to its implementation in the maritime sector, this has also been implemented in an endoscopy clinic for saliva-based screening of patients and staff [110].

2.5. East and Central Asia

East Asian nations have spearheaded saliva-based COVID-19 testing, especially as some East Asian governments have been the first in the world to normalize it as the predominant sample type through legislation.

Hong Kong has been at the forefront of much of the saliva-based SARS-CoV-2 diagnostic research, innovation, and implementation. As early as February 2020, researchers in Hong Kong had identified that saliva samples contained SARS-CoV-2 in infected patients and therefore concluded that saliva showed promise as a noninvasive specimen for diagnosis, monitoring, and infection control in COVID-19 patients [2]. Importantly, differences in viral shedding dynamics were observed between sample types, with tests utilizing nasopharyngeal swabs taking longer to turn negative than those that utilized saliva [111]. This finding was particularly significant with respect to quarantine measures, as many countries have required a negative test to end isolation. A saliva-based RT-LAMP test was developed for mass on-site screening (July 2020) [112], and self-collected mouth gargle samples demonstrated similar sensitivity for COVID-19 detection compared to deepthroat saliva samples (December 2020) [113]. The Hong Kong government has a mixed-specimen approach to SARS-CoV-2 diagnostics, and recommends that oropharyngeal saliva specimens be used for asymptomatic individuals while symptomatic individuals receive both a nasal and a throat swab from a community testing center [114].

In Taiwan (October 2020), researchers argued that self-collected saliva should be included in the arsenal of COVID-19 diagnostics, particularly as it protects healthcare workers and the broader community from unnecessary exposure and is minimally invasive [115]. In South Korea (March 2020), the early identification of saliva as a useful specimen for diagnostic purposes led to the adoption of saliva-based methods by testing centers [116,117]. Since then, South Korean laboratory and health agencies have released guidelines on acceptable clinical diagnostics of SARS-CoV-2 (February 2022), recognizing saliva as a sample type that may benefit patients who require repetitive or less invasive sample collection [118]. In Japan, after a direct SARS-CoV-2 salivary detection kit (one that does not require RNA extraction or purification) was developed (June 2020) [119] and self-collected saliva demonstrated comparable sensitivity to nasopharyngeal swabs (June-July 2020) [120,121], saliva became the preferred sample-type for certain mass screening programs [122,123]. These findings drove innovation and enabled the implementation of saliva-based COVID-19 testing. As early as July 2020, Taiwan utilized PCR testing of oropharyngeal saliva samples for entry into the country [124], and had developed rapid detection approaches that use biosensors (electrical double layer (EDL)-gated field-effect transistor-based biosensor [BioFET]) for use in screening programs [125,126]. At least two different saliva-based chemiluminescent enzyme immunoassays have been approved by the Japanese Ministry of Health, Labor, and Welfare, and are currently used in airports [127]. For the purpose of contact tracing, most health care centers in Japan collect saliva samples, demonstrating its commonplace use as a screening tool for potentially-infected individuals. A self-collected PCR test developed in South Korea gained approval both domestically and internationally early in 2021 [128]. In November 2021, a saliva-based RT-PCR kit was authorized by the Taiwan Food and Drug Administration [129].

With early work from China demonstrating the utility of saliva for monitoring SARS-CoV-2 infection dynamics [130], China has embarked on many new saliva-based diagnostic endeavors in 2022. Among these are the development and evaluation of a MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-time of Flight Mass Spectrometry) assay coupled with isothermal gene amplification from saliva (February 2022) [131], the establishment of an enhanced substrate to be used saliva samples (February 2022) [132], the amplification of nanoparticles for rapid detection of SARS-CoV-2 in saliva (March 2022)[133], and the proposition of an electrochemical biosensor that can identify viral targets in samples containing only 10% saliva (April 2022) [134].

In Central Asia, clinical studies evaluating saliva-based tests are underway in Kazakhstan [135] and Mongolia [136] with the aim to increase testing accessibility, comfort, and turnaround time of SARS-CoV-2 diagnostics in the region.

2.6. Southeast Asia

In January 2021, the Philippine Department of Health approved saliva-based testing by the Philippine Red Cross (PRC)[137]. They validated the SalivaDirect™ protocol [138] to provide a more equitable and affordable option for the region’s testing, which is offered to the public at testing sites and through at-home collection kits [139]. Eventually, the Filipino government officially recognized the PRC’s saliva tests by including positive results in their national case counts [140]. In Thailand (April 2020), a cross-sectional study found saliva to be a suitable alternative to nasopharyngeal swabs especially in low resource areas [141]. As of February 2022, saliva appears to be a mainstream sample type as the Thailand government has authorized 63 at-home saliva-based rapid antigen tests [142], although there is little information available on saliva-based PCR tests. Prior to the emergence of the Omicron variant, quarantine-free entry into Thailand was permitted for specific cities when inbound travelers produced a negative swab or saliva-based RT-PCR test result [143].

In Singapore (August 2020), self-collected saliva specimens outperformed self-collected nasal swabs in sensitivity [144]. Initial studies proposed that nasopharyngeal swabs should be used for confirmatory testing following a positive saliva-based test result [145]. Furthermore, as the validity of saliva as a sample type became more widely accepted in diagnostics, Singapore implemented PCR saliva testing in airports for pre-departure testing [146] the collection kit used in airports is registered as a medical device with the Singaporean Health Sciences Authority (September 2020). Approval of a saliva-based antigen test to be used in airports is still underway; however, one is in development that uses a proprietary on-kit amplification method that reports sensitivity close to laboratory RT-PCR assays [147]. In Vietnam (April 2020), asymptomatic COVID-19 infections have been accurately detected by saliva-based PCR testing, determining that, despite higher yields of viral load being detected in nasal/throat swabs, saliva’s simplified collection process facilitates mass-screening initiatives [148]. Additionally, saliva was validated in Indonesia (August 2021) on nine commercial kits using RT-PCR, including RNA-extraction free methods, to demonstrate its versatility in use across multiple existing workflows [149].

2.7. South Asia

An investigation of the unique impact of the COVID-19 pandemic on low- and middle-income countries (June 2020) focused on Nepal and used saliva alongside other sample types for diagnostic testing [150]. In a large-scale study at the COVID-19 Screening Unit of Dhaka Hospital in Bangladesh (November 2020) [151,152], paired saliva samples and nasopharyngeal swabs were collected from all research participants and the sensitivity of saliva (80%) met the criteria defined by the WHO [151,152].

Studies in India (September 2020 and May 2021) showed the advantages of saliva testing for the detection of SARS-CoV-2 compared to nasopharyngeal swabs, including cost-effectiveness, acceptability, and ease of collection [153,154]. Researchers in India (November 2020) also developed an RNA extraction-free approach (Cas13 Assisted Saliva-based & Smartphone Integrated Testing; CASSPIT), which exhibited a 97% PPA with RNA extraction-based methods [155]. In response to the Delta outbreak in July 2021, the IndiaCOVID SOS volunteer task force developed recommendations for diagnostic approaches to tackle the crisis, including the expansion of rural surveillance using PCR testing of nasal and saliva samples [156]. In August 2021, a Singaporean start-up based in India sought to provide a new method in addition to the traditional nasal samples by utilizing a ‘lollipop-like device’ to collect saliva samples from patients [157]. This new device has enabled frequent testing in public spaces such as schools and airports [158].

2.8. Middle East and North Africa (MENA)

While the region has predominantly used nasopharyngeal swab PCR testing, some MENA nations have demonstrated success with saliva. Notably, the United Arab Emirates (UAE) has achieved one of the highest testing rates worldwide [159]. Studies in the UAE (July 2020) [160], Bahrain (October 2020) [161] and Kuwait (July 2020) [162] have validated the comparability of saliva samples (sensitivity 73–83%) to nasopharyngeal swabs for SARS-CoV-2 diagnostics, with additional research teams in Lebanon [163], Qatar [164], and a collaboration between Saudi Arabia and Pakistan [165] recognizing the reliability of saliva as a sample type. Researchers in Turkey are working on the development of an electrochemical immunoassay that detects the SARS-CoV-2 spike S1 protein (March 2022), adding another saliva-based diagnostic tool for point-of-care testing [166]. Saliva-based PCR testing programs have been implemented for screening programs at primary schools in Qatar (September 2020) [167], Abu Dhabi (December 2020) [159], and Israel (October 2021) [168], driving the authorization and strong uptake in some regions.

In the private sector, scientists in Israel [169] and Iran [170] have developed and brought to market saliva-based tests for the detection of SARS-CoV-2. In Turkey, a nanotechnology-based diagnostic system that detects SARS-CoV-2 from oral cavity swabs was developed in January 2021 [171], producing results in as little as 30 seconds [171,172]. It has since received approval from Turkish authorities (June 2021) and seen increased demand globally [172]. In Israel, a simple, inexpensive mouth-washing (10–20 second gargle) method, previously evaluated for influenza, was adapted for SARS-CoV-2 detection [173].

In North Africa, 30-minute saliva rapid diagnostic tests are commercially available and could help increase testing accessibility as they do not require mobile technology [174]. In August 2021, Morocco shifted from requiring medical settings for diagnostic testing to permitting the sale of rapid saliva tests at pharmacies across the nation [175].

2.9. Sub-Saharan Africa

In February 2021, the Africa Centres for Disease Control and Prevention (CDC) acknowledged saliva as a reliable sample type for COVID-19 testing. Even so, few African nations have officially authorized saliva-based SARS-CoV-2 tests as many do not have central regulatory agencies for the approval of diagnostic tools [176]. In Nigeria (December 2019), a cross-sectional survey of healthcare workers highlighted that, despite participants being aware of the merits of saliva as a sample type for disease detection, few countries applied saliva-based methods in clinical settings [177]. Wider use of saliva was most often impeded by methods with lower testing sensitivity, portability, speed, or automation. In addition, the fee-for-service model in many of these countries proved a barrier for those in need of testing [176].

However, commercial saliva testing is offered in certain African nations. Saliva-based PCR is offered in Burundi, Sudan, the Democratic Republic of the Congo, and Seychelles [178,179]. Scientists in Nigeria are pushing for the government to fund SalivaDirect^TM PCR testing at the national level based on its proven success and ability to cut costs and turnaround time [180]. Saliva is also used as a sample type for rapid tests as part of Uganda’s surveillance program [181] and for entry to Djibouti via national airports [182].

A multitude of scientific publications further support the potential for saliva-based COVID-19 tests in this region. In South Africa, a randomized-controlled trial began in May 2021 to develop regionally-appropriate protocols for saliva self-collection (in place of self-administered nasal swabs) for SARS-CoV-2 detection [183]. In Ghana (December 2020) [184] and Ethiopia (November 2021) [185], the high viral load and prolonged viral shedding of saliva was found to be more advantageous than nasopharyngeal swabs for diagnostics, and an ideal sample type for settings with limited diagnostic resources. Crucially, saliva was identified in South Africa (December 2021) to be the most reliable sample type for detection of the Omicron variant due to higher viral shedding in saliva than mid-turbinate swabs (PPA: saliva = 100%, mid-turbinate swabs = 86%) [186].

While saliva is often used as a sample type for more traditional and widely-accepted PCR, LAMP, or antigen testing technology, researchers in Nigeria (April 2021) have considered training African Giant Rats, native to Sub-Saharan Africa, to detect SARS-CoV-2 olfactorily in saliva samples [187].

2.10. Europe

In September 2021, the European Center for Disease Prevention and Control (ECDC) reiterated that saliva-based RT-PCR tests generate similar sensitivities to nasopharyngeal swabs for symptomatic cases and attributed differences to variation in populations tested, sampling techniques, and time of sample collection [188,189]. The ECDC initially recommended saliva as a sample option only if nasopharyngeal swabs are not accessible, as they desired further clinical validation data prior to suggesting saliva as a primary sample type [190]. However, in August 2020, the ECDC endorsed saliva as an option to increase testing acceptance in younger populations [191]. As of October 2021, saliva was not yet validated for rapid antigen tests by the ECDC [192]. Nevertheless, an abundance of findings on saliva as a versatile sample type, for detection using various platforms on numerous populations, have emerged rapidly across Europe.

2.11. Central/Eastern Europe

Following robust validations, saliva-based SARS-CoV-2 testing is available in laboratories and pharmacies across much of Central and Eastern Europe including Montenegro [193], Georgia [194], Slovakia [195], Slovenia [196], Latvia [197] and Ukraine [198]In Romania, school students are required to perform self-administered, at-home saliva tests under parental observation twice a week [199]. In Poland (January 2021), saliva-based LAMP has proved efficient and reliable [200]. Russia also sought to quickly utilize saliva as a diagnostic sample type. The Russian biotechnology company, Sistema-Biotech, federally registered a saliva-based rapid test in early 2020 [201,202].

Additional assays across Central and Eastern Europe have demonstrated success. The saliva-based Advanta Dx SARS-CoV-2 RT-PCR assay (Greece, December 2020) reported high sensitivity in both diagnostic (88.5%) and screening (98.1%) samples [203]. Good agreement was found between the saliva-based laboratory kit, Diagnolita (Lithuania. March 2021) and nasopharyngeal swabs (PPA = 98.28%, NPA = 98.11%) with a moderately strong correlation between the Ct values of the two samples (R = 0.53) [204]. In Slovakia, LAMP methods that include or bypass the RNA extraction step were evaluated to reduce cost and processing time [205,206]. In Slovenia (October 2021), saliva-based RT-qPCR testing demonstrated sensitivity of ≥ 95% in people with subclinical infection, outperforming RT-LAMP (≥ 70% sensitivity) [207]. Additionally, passive drool saliva demonstrated higher diagnostic sensitivity (95%) compared to oral cavity swab (87%) [207].

2.12. Western Europe

Across Western Europe, numerous developments and applications of saliva-based assays for SARS-CoV-2 detection have emerged. Studies conducted in Italy (April 2020) [208], Switzerland (January 2021) [209], Finland (March and August 2021) [210,211], Sweden (October 2021) [212], Belgium (December 2021) [213], and the United Kingdom (UK; January 2022) [214] have found saliva to perform with comparable or greater sensitivity than nasal-based swabs. Moreover, RNA-extraction free approaches have been validated in Sweden (May 2020) [215], Spain (October 2020) [216], Ireland (March 2021) [217], Finland (December 2021) [218], England (February 2022) [219], and the Netherlands (May 2022) [220]. Pooled saliva testing in France (October 2020) [221], Portugal (October 2021) [222] and Finland (2021) [218] has demonstrated high sensitivity while preserving resources and increasing mass testing capacity. When evaluated as a screening tool for nursing home staff in Belgium (December 2020), saliva-based RT-PCR testing led to decreases in COVID-19 prevalence (from 34.4% to 13.4%) and deaths among nursing home residents [223]. In Italy (April 2020), saliva swabs consistently tested positive throughout infection while nasopharyngeal swabs tested negative for some patients over the course of infection, indicating that saliva may reveal the clinical evolution of COVID-19 more reliably than nasal samples [208]. The Irish government has urged staff and students to participate in research to explore potential surveillance systems on college campuses, including saliva-based testing solutions [224]. Furthermore, a collaborative study (UniCOV) across four Irish universities aims to determine appropriate surveillance systems for COVID-19, evaluating both nasal antigen tests and saliva PCR tests [225]. In Spain (December 2020), saliva and oral swabs were the sample types of choice for a study on pandemic wave trends [226]. Researchers in the UK (January 2022) used saliva samples to validate SARS-CoV-2 CRISPR-Cas detection technology for point-of-care testing.

LAMP tests have also proven successful in Western Europe. In the United Kingdom (August 2021), following a large-scale study evaluating its potential [227], the National Health Service is offering saliva-based LAMP testing to healthcare workers in addition to rapid antigen tests [228]. However, other researchers from the UK evaluated the use of RT-LAMP to test saliva samples of asymptomatic individuals (March 2022) and while the specificity was 100%, the sensitivity (40.91%) was significantly lower than when testing the same saliva samples with RT-PCR [229]. Additionally, Trinity College in Dublin, Ireland implemented saliva-based LAMP testing for its students (November 2021) [230]. In France, a 40-minute rapid saliva-based LAMP test that can be performed entirely outside of a laboratory was designed to allow for mass testing [231].

An alternative approach – an electrochemical immunoassay – was developed in Italy (October 2020) to rapidly detect SARS-CoV-2 in saliva promising high potential for market entry, being portable, non-invasive, and highly sensitive [232].

Across Western Europe, there have been varying levels of commercial uptake of saliva-based COVID-19 testing. Saliva testing for the detection of SARS-CoV-2 was introduced in Sweden in June 2020 [233]. Labs located in cities such as Stockholm and Gothenburg monitor individuals using a saliva-based antigen test that meets the WHO’s recommended quality requirements [234]. Saliva-based PCR testing has been implemented as a screening tool for students and staff across Western Europe, at the University of Liège in Belgium[235], primary schools in Milan, Italy [236], and primary schools and universities in Monaco and Norway [237,238]. It is also being used extensively in Germany in homeless shelters, schools, and citywide [239–242]. The Netherlands created the Testing for Entry program to offer free testing for events, activities, or hospitality, which utilizes a combination of nasal swabs and throat swabs for antigen tests [243]. In December of 2021, the Federal Council in Switzerland announced they would cover the costs for certain pooled saliva testing for those requiring a COVID-19 certificate [244]. In the executive summary on the Conference Towards the Deployment of a Global and Collaborative Diagnostic Arsenal to Detect and Fight Against Pandemics that was hosted by Monaco in December 2021, saliva as a sample type is highlighted as a most useful strategy in detecting COVID-19 [245]. The UK has approved comparatively few saliva-based assays that have gained traction [246]. The lack of saliva testing across the UK may be in part due to the government’s prior emphasis on increasing access to rapid antigen tests [247]. In Norway, one expert noted that the country is currently hesitant to use saliva as a sample type for COVID-19 detection but that with additional data there is potential to reconsider the adoption of saliva [248].

3. Discussion

While nations differ in their public health capacity and implementation of saliva-based COVID-19 testing, every region, globally, has demonstrated success in using saliva as a sample type for SARS-CoV-2 detection. The extensive data that emerged from East Asian countries in conjunction with rapid action by their governments to deploy PCR saliva testing has led to the incorporation of saliva as a fundamental component in their pandemic response strategies. The USA’s saliva testing response has also been high-reaching, with interventions accessible through both the public and private sector (following federal authorization [13]) and for diverse populations, including asymptomatic and symptomatic children, adults, and healthcare workers. Similarly, uptake of saliva-based detection has spanned across Europe, including large-scale surveillance for schools, universities, and the general public, and involving PCR, LAMP, and rapid antigen methods [191]. In Oceania, testing has predominantly occurred in the high-income island nations of Australia and New Zealand, where the goal of safeguarding their borders has resulted in aviation, maritime, and quarantine hotel workers being the target groups for saliva surveillance. All regions have reported finding value in the non-invasive approach offered through saliva sampling for SARS-CoV-2 detection, whether in clinical diagnostic settings or for screening and surveillance testing.

The emergence of saliva as a sample type that is comparable in caliber and sensitivity to swab-based methods for SARS-CoV-2 detection offers a scalable and affordable solution to testing, warranting more widespread adoption. Though numerous studies cite concerns regarding saliva’s ability to match or outperform nasopharyngeal swabs, the efficacy of saliva-based COVID-19 testing has been demonstrated time and again. Poorer findings that resulted from less robust methods of this previously non-traditional sample type may account for some of the hesitancy by governments to broadly implement saliva-based diagnostics. As nasopharyngeal swabs were the default sample type at the start of the pandemic, unfamiliarity with saliva sampling may have led to apprehensions about switching collection methods at testing sites later on.

Saliva sampling can increase testing acceptability as it is less invasive than nasopharyngeal and oropharyngeal swabbing, and has been found to be the preferred method by patients. Almost two thirds of one sampled population reported that they would prefer a saliva-based test [81]. Furthermore, 76% would be willing to repeat a saliva test for confirmation of their COVID-19 status, whereas only 63% would repeat a nasopharyngeal or oropharyngeal swab test [81]. Saliva is also suitable for community settings comprising populations that may be more challenging to collect nasal samples from, including children and the elderly [151]. Thus, the minimally-invasive nature of saliva collection makes it ideal for mass testing, as more individuals are likely to participate in screening programs [249,250]. Additionally, saliva collection has a lower staffing requirement than swabbing, reducing transmission risk for healthcare workers while enabling scalable sampling even in resource-limited settings [251,252].

Greater focus on resource-limited settings (including low- and middle-income countries) is warranted, as many countries in Africa, Central America, the Caribbean, and the Middle East have yet to validate and implement saliva testing. Nations should seek to replicate aspects of the successful applications demonstrated across various populations globally, while remaining cognizant of barriers they may face. This is of utmost importance in narrowing testing gaps between high- and low-income nations. The cost of saliva sample collection can be a fraction of the cost of nasopharyngeal swabs, permitting a greater increase to testing accessibility [253]. Test costs can also be reduced with RNA-extraction-free approaches [254,255], requiring fewer materials and less labor while increasing efficiency. Moreover, saliva may prove a useful specimen type for occupied territories and conflict zones. Palestine, for example, has endured a restricted flow of necessary COVID-19 supplies to the region [256], and in May 2021, an Israeli airstrike damaged Gaza’s only COVID-19 diagnostic laboratory [257]. In Palestine, saliva-based antigen tests could increase diagnostic testing capacity, given the relatively low cost of production in the context of Palestine’s limited laboratory infrastructure and occupation-related resource constraints. Understanding the most useful tools for detecting SARS-CoV-2 in conflict zones continues to be relevant particularly as the conflict in Ukraine unfolds.

Additionally, the use of saliva for diagnostic testing has a history that precedes the COVID-19 pandemic. This includes salivary detection of norovirus in Peru [258]; tuberculosis in Ecuador [83]; Kaposi’s Sarcoma-Associated Herpesvirus and Epstein-Barr virus in Cameroon[259]; HIV in Uganda[260], Tanzania[261], Myanmar [262], and Jamaica [76]; HPV in Australia [263]; malaria in Cameroon [264,265] and the Philippines [266]; Leishmania DNA in Thailand [267]; and both Zika virus [268] and Chikungunya [269] in French Polynesia. The familiarity and versatility of saliva for the detection of numerous infectious diseases, especially in low-income nations, demonstrates its likely acceptability and feasibility for official government adoption by all regions for SARS-CoV-2 detection. Importantly, SARS-CoV-2 has been successfully sequenced from saliva by multiple groups around the world [270–272], further elevating its status as compared to nasal swabs. One method, SARSeq (Austria; May 2021) also allows for the concurrent detection of other respiratory RNA viruses, including influenza A and B and human rhinoviruses [271].

A plethora of global findings demonstrate the application of saliva as a sensitive, affordable, and sustainable sample type for SARS-CoV-2 detection. Expanding research around the world has led to government action, innovations in the private sectors, and implementation in a variety of populations. With great intra-regional disparities in the availability and uptake of COVID-19 testing, coupled with novel threats of new COVID-19 variants and low vaccine availability in some areas, the mass implementation of sustainable testing practices is imperative to enable the mitigation of COVID-19 burdens across all continents. In countries that may be under-resourced and unable to provide wide-scale oropharyngeal or nasopharyngeal swab testing, saliva testing is a strong candidate to augment existing efforts through the introduction of an easy, affordable, and comfortable testing option. Ultimately, international solidarity among research scientists, policymakers, and government agencies to standardize the most robust saliva testing methods and implementation practices will enable saliva to be a mainstream diagnostic sample type for the foreseeable future.

4. Expert opinion

Over two years into the pandemic, the term ‘gold-standard’ is still too often applied to the nasopharyngeal swab. This outdated terminology must be revised as it continues to fuel controversy within the scientific community and leads to skepticism of alternative sample types within the general public. It also does not reflect the utility of other sample types, such as saliva, which continues to demonstrate equivalent or greater efficacy to swabs. In line with this, saliva was even proposed as a potential new ‘gold-standard’ sample type after numerous international studies in 2020 already demonstrated strong performance and implementation success of saliva-based PCR testing [4]. The misconception surrounding the sensitivity of saliva is one of the biggest obstacles to mass implementation of saliva-based PCR diagnostics. This global review of saliva as a robust sample type for SARS-CoV-2 diagnostics is the first piece to summarize findings of multinational studies and implementation efforts, while elucidating innovative saliva-based tools that generated effective protection to communities. Together, these success stories from diverse global contexts of the SARS-CoV-2 pandemic exemplify the need for further evaluation of and support for saliva as a sample type for respiratory infections from the scientific community. This action is necessary to increase awareness of the validity of saliva and the opportunities this awareness can provide by expanding a range of accepted sample types for clinical testing.

Throughout the pandemic, we have witnessed disparities in access to testing all around the world. However, in many cases, saliva-based tests decrease cost and increase access. Moreover, we have also witnessed the consequences of delayed response and lack of access to testing. We need to be ready for future surges, new variants of concern, reduced vaccine effectiveness in the face of these variants, and waning population immunity. Following the trends of earlier variants [273], the further shift in SARS-CoV-2 infection profile reported for Omicron, resulted in dramatically poorer detection with nasal swabs and far more sensitive and earlier detection using saliva [186,274]. Here, the superiority of saliva for early and asymptomatic detection is essential to limiting virus transmission and ultimately, mitigating the potential for new variants to arise in the near future. Thus, we must remain flexible in our response and be open to the use of alternative sample types when the need arises and when they can better serve public health responses. Importantly, with many SARS-CoV-2 tests around the world only permitted for use under the current pandemic emergency response, formal approval of these diagnostic tools will be imperative to ensure they are available beyond this pandemic to provide reliable, easy testing for the long-term.

The demands of the pandemic spurred rapid development in diagnostic tools. Innovative saliva-based approaches are continuing to emerge with great potential for further creative applications that diagnostic test developers and public health decision-makers can pursue beyond COVID-19. This is particularly true for multiplexed approaches that enable non-invasive, routine screening for early warnings of multiple diseases in a single assay. Implementation science will be key to translating many of these diagnostic innovations into practice as on-the-ground barriers arise. These barriers will be especially prominent in resource-limited settings where frequent testing is still of great urgency yet great disparities exist in access to testing. Consideration should also be given to the optimal specimen type for point-of-care and rapid tests across a range of diseases for low- or middle- income countries that may not have adequate laboratory infrastructure to use PCR assays. To help accommodate affordability and convenience, methods such as pooled sample testing can be implemented, which has demonstrated success in settings such as workplaces and schools.

Countries all around the world are demonstrating how saliva-based sampling can increase the acceptance of clinical testing. With programs in place at schools, workplaces, older living facilities, prisons, healthcare centers, etc., there are numerous, robust examples that can serve as a framework for the quick implementation of scalable testing. These examples can act as a playbook for other communities, or even entire countries, to rapidly implement in response to new public health threats. The need for earlier detection and more accurate diagnostics becomes greater as COVID-19 treatments become increasingly available (as highlighted in the USA with The White House’s current Test to Treat initiative [275]). With many treatments most effective when administered early in the course of infection, test results must be returned in an actionable time frame for early treatment and better recovery.

Looking forward, these accurate and timely diagnoses have a larger role to play in protecting public health. As another major public health crisis looms with increasing antibiotic resistance, more accessible diagnostics can also promote antibiotic stewardship. While stewardship initiatives better equip clinicians to optimize the appropriate treatment of infections, this also reduces the risk of adverse events associated with antibiotic use. Thus, global solidarity is warranted to most effectively apply the learnings of saliva-based diagnostics as a way to expand diagnostic options, access, and efficiency. Through our review, we call upon these stakeholders to collaborate internationally and continue driving affordable, sustainable testing interventions. Actioning this compilation of implementation successes can subsequently enable saliva to transition nations into a safer tomorrow.

Article highlights

• The ‘gold standard’ sample types for SARS-CoV-2 detection defaulted early in the pandemic to nasopharyngeal or oropharyngeal swabs, but there is a growing body of evidence demonstrating saliva as a suitable sample type for SARS-CoV-2 detection

• Misinformation and skepticism generated by the debate surrounding saliva as a reliable sample type have impeded the implementation and expansion of saliva testing programs globally.

• This review highlights the best practices employed by successful testing operations, including effective methodologies and the use of low-cost materials, while addressing barriers and challenges that should be considered when developing or implementing salivary diagnostic programs.

• Global solidarity is warranted to most effectively apply the learnings of saliva-based diagnostics as a way to expand diagnostic options, access, and efficiency.

• Saliva testing is a strong candidate for countries that may be under-resourced and unable to provide wide-scale oropharyngeal or nasopharyngeal swab testing, to augment existing efforts through the introduction of an easy, affordable, and comfortable testing option.

Declaration of interest

AL Wyllie has received research support from the Rockefeller Foundation, Flambeau Dx, Tempus Labs, Pfizer and Merck. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

Salary support was provided from the Rockefeller Foundation (2021 COV 006 PC) and Flambeau Dx in the form of research funding to AL Wyllie.