by Dr T. Truong, Dr J. Dien Bard and Dr R. Yee

Nasopharyngeal swabs are recommended as the preferred sample type for detecting infection with SARS-CoV-2. However, there has been considerable interest in other collection methods, especially if they can circumvent supply shortages and the requirement for trained healthcare workers to administer them. Here we touch on alternative specimen types that may be pursued with a focus on saliva.

Testing for SARS-CoV-2

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has presented an extraordinary challenge to human health, the global economy and society at large. Diagnostic testing to confirm infection with SARS-CoV-2 has been an important tool for patient management, surveillance efforts and contact tracing. The Infectious Diseases Society of America (IDSA), Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) all strongly recommend using nucleic acid amplification approaches, which is typically a reverse-transcriptase polymerase chain reaction (RT-PCR) to target different regions of the SARS-CoV-2 viral genome, with nasopharyngeal swabs (NPS) being considered the gold standard specimen collection method.

Unfortunately, large-scale testing with NPS has been hindered by several challenges. As NPS must reach the nasopharynx, samples can often be painful or uncomfortable to collect and impossible in some patients with physical obstruction [1]. Collection of NPS requires skilled healthcare workers and puts them at risk through the potential of aerosol generation [2]. In addition, supply chain shortages have limited the availability of NPS, especially in the first few months of the pandemic [3]. Overall, these issues can potentially constrain testing capacity and impose a large burden on the healthcare workforce.

There has been considerable interest in the use of alternative specimen types to address the potential pitfalls of NPS collection. Currently, the IDSA/CDC have approved other specimen types in addition to NPS, such as oropharyngeal swabs (OPS), mid-turbinate swabs (MTS), anterior nares swabs (ANS), sputum, bronchoalveolar lavages (BAL), tracheal washes, and saliva. Several studies have explored the detection of SARS-CoV-2 from other non-respiratory specimens types including blood, stool and urine with poor outcomes [4]. Respiratory tract fluids are considered more physiologically relevant as they express high levels of the angiotensinconverting enzyme II (ACE-2) receptor required for cellular entry of SARS-CoV-2 [5].

To address the shortages of transport media, multiple transport systems are considered acceptable by the U.S. Food & Drug Administration (FDA). These include viral transport media (VTM), universal transport media (UTM), liquid amies, phosphatebuffered saline, normal saline and even dry swab transport with a rehydration procedure.

Important considerations when selecting specimen type

Although the CDC and IDSA have listed several specimen types alongside collection instructions, it must be noted that there are no specific recommendations on what specimen type to use and when. It is up to each institution to determine the optimal approach based on available testing platforms, validated specimen types, and communication with the providers and clinical microbiologists [6]. Specimen types such as ANS and MTS are a less invasive option for self-collection. However, self-collection would likely be precluded in patients who are extremely ill, cognitively impaired or incapable (e.g. younger children) of obtaining an appropriate specimen by themselves [2]. The condition of the patient is also important; as demonstrated by intubated patients, tracheal aspirates rather than BAL are easier to obtain compared to NPS. Similarly, understanding the patient’s severity of illness may also dictate which specimen types are more appropriate (e.g. lower respiratory tract sample from patients with pneumonia). Combination testing from different sources (i.e. OPS+ANS) may have increased sensitivity although this is not a practical solution owing to an increased demand on supplies [7].

Given the numerous specimen types and collection devices, it is critically important to measure their performance characteristics before considering them suitable for clinical use. Consequences of false positives may induce stress for patients and families, impact daily life (e.g. job/schooling) and initiate unnecessary isolation precautions. Conversely, a false-negative result may lead to increased transmission as the suspected patient fails to isolate or quarantine [2]. These characteristics include analytical sensitivity, which is the percentage of true positives a test accurately captures. Analytical specificity is the percentage of true negatives a test accurately captures. Ideally, a test should be both highly sensitive and specific to minimize false positives and false negatives. The performance should be measured against a gold standard test or procedure, which at this point in time continues to be NPS.

Performance characteristics of saliva as an alternative specimen for SARS-CoV-2 detection

Despite the approval of different alternative specimen types in addition to NPS, many still rely on collection swabs, UTM and personal protective equipment (PPE) to protect healthcare workers. Saliva as a specimen type has garnered huge interest because to its simplicity of collection as it can be self-collected without the need for swabs and transport media and reduces the need for PPE. Additionally, high ACE-2 receptor expression has been demonstrated in salivary glands making saliva an appropriate specimen also from a pathophysiological perspective.

Meta-analyses of greater than 46 studies compared the performance characteristics of saliva specimens to other specimen types and reported that detection of SARS-CoV-2 in saliva is comparable to NPS [5, 8, 9]. The sensitivities were 88% (95% CI: 81–93%), and 94% (95% CI: 90–98%), respectively. Interestingly, the performance of saliva was superior to ANS (82%, 95% CI: 73–90%) and OPS (84%, 95% CI: 57–100%) [7].

The presence or absence of symptoms at the time of testing had minimal effect on test performance. In asymptomatic and symptomatic patients, the sensitivities of saliva were 87% (95% CI: 70–98%) and 88% (95% CI: 79–95%), respectively. Similar to NPS, the ability to detect SARS-CoV-2 in saliva decreased at 7 days post-symptoms onset [>7 days: 74% (95% CI: 62–85%) versus ≤7 days 89% (95% CI: 73–99%)]. Importantly, many studies have reported SARS-CoV-2 detection from saliva even when other specimen types are negative, further supporting its use as a reliable specimen type. Specificity of both saliva and NPS are high at 99% (95% CI: 98–99%) and 98% (95% CI: 97–99%), respectively [8]. Given the increased costs associated with collection systems that include swabs and transport media, collection of saliva, particularly raw saliva samples, has been projected to have economic benefits if more widely used. One study estimates that at least US$600¦000 can be saved per 100¦000 people if saliva were used in lieu of NPS [9]. Additionally, pooling of saliva samples, particularly when used for surveillance testing in asymptomatic populations, may further reduce cost with minimal effects of performance [10].

Admittedly, the majority of studies have focused on the adult populations and only a few were dedicated to pediatric populations, often with small sample sizes. One study with a larger sample size (n=43 children) demonstrated comparable performance of saliva compared to NPS [11]. The overall concordances of saliva and NPS were 91.0% (273/300) and 94.7% (284/300), respectively. Performance of saliva (82.4%) and NPS (85.3%) was comparable when only first-time-positive pediatric patients were analysed regardless of clinical presentation (presence or absence of symptoms) and no significant differences in viral load were observed [11].

There are numerous approaches to saliva collection which may affect overall detection ability. Instructing patients to cough or clear their throats before saliva collection may be more effective than passive drooling or spitting as the former allows for representation of lower respiratory sample alongside saliva; however, the introduction of coughing may increase risk to healthcare workers [2]. Meanwhile, patients who avoided eating, drinking, or brushing teeth before saliva collection had a higher detection yield [91% (95% CI: 86–95%) vs 86% (95% CI: 79–92%)]. Additionally, sensitivity was found to be higher when saliva collection was supervised [86% (95% CI: 72–93%) vs 66% (95% CI: 50–80%), respectively] [12].

Testing platforms available for SARS-CoV-2 testing on saliva specimens

Currently, many academic testing institutions and diagnostic companies have obtained Emergency Use Authorization (EUA) from the FDA for inclusion of saliva as a specimen type for SARS-CoV-2 testing. While most have validated the saliva specimen using a traditional RT-PCR workflow which consists of RNA extraction followed by RT-PCR, other extraction-free protocols have been recently granted EUA. However, one pitfall is that sensitivity has been found to be lower for extractionfree protocols [60% (95% CI: 49–70%) vs 89% (95% CI: 83–94%)] [5]. One example of an assay that bypasses manual extraction is the SalivaDirect test (Yale School of Public Health, New Haven, CT, USA), which allows for testing of low volume specimens. Saliva is first treated with proteinase K, followed by heat-inactivation and testing on dual plex RT-PCR assay. Another example is the Simplexa™ COVID-19 Direct Molecular Test (DiaSorin Molecular, Cypress, CA, USA), one of the few sample-to-answer platforms that have modified their EUA to include saliva as a direct specimen with comparable performance to other specimen types [13].

Saliva as a specimen type in other respiratory infections

The burden associated with collection of swab samples for SARS-CoV-2 testing throughout the COVID-19 pandemic has elevated the potential utility of saliva as an optimal clinical specimen. Interestingly, saliva was previously explored as a sample type for the diagnosis of other respiratory infections, including influenza A/B, respiratory syncytial virus and adenovirus [14]. A prospective study of 236 adults comparing paired NPS and saliva samples reported increased sensitivity of saliva for detection of adenovirus, and identical sensitivity for detection of non-SARS-CoV, enterovirus and parainfluenza virus [15].

Summary and future directions

Considerable diagnostic testing efforts are essential in combatting any pandemic including the current COVID-19 pandemic. Saliva has been implemented globally, alongside other sample types, as a primary sample type for the detection of SARS-CoV-2. Although there is no specific guidance available in selecting the more appropriate specimen type, it is important to weigh up the feasibility of collection, test performance, staffing and supply availability, patient age (e.g. young children will not be able to provide saliva) and patient population (i.e. symptomatic testing or asymptomatic surveillance). Additional studies comparing the head-to-head performance of different testing platforms and protocols, specimen collection and patient populations (age, symptomology, disease severity) are needed to better understand the clinical performance and significance. Moreover, it is highly recommended that studies and testing protocols specifically describe the collection and testing procedures used to minimize inter-protocol variations. Nonetheless, the data currently available indicate that saliva is a suitable specimen type for the detection of SARS-CoV-2.

The authors
Thao Truong1 PhD; Jennifer Dien Bard*1,2 PhD, D(ABMM); Rebecca Yee1 PhD

1. Department of Pathology and Laboratory Medicine, Children’s Hospital
   Los Angeles, Los Angeles, CA, USA
2. Keck School of Medicine, University of Southern California, Los Angeles,
   CA, USA

*Corresponding author
E-mail: jdienbard@chla.usc.edu

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