key: cord-0990547-glhgijvk authors: Michailidou, Evangelia; Poulopoulos, Athanasios; Tzimagiorgis, Georgios title: Salivary diagnostics of the novel coronavirus SARS‐CoV‐2 (COVID‐19) date: 2020-12-04 journal: Oral Dis DOI: 10.1111/odi.13729 sha: 11ee052c47f7417154190d759cecc247a44f0b2e doc_id: 990547 cord_uid: glhgijvk INTRODUCTION: Laboratory testing for the SARS‐CoV‐2 virus and the consequent respiratory coronavirus disease 2019 (COVID‐19) is categorized into methods that detect the viral presence and methods that detect antibodies produced in the host as a response to infection. Methods that detect viral presence into the host excretions measure current infection by SARS‐CoV‐2, whereas the detection of human antibodies exploited against SARS‐CoV‐2 evaluates the past exposure to the virus. OBJECTIVE: This review provides a comprehensive overview for the use of saliva as a specimen for the detection of SARS‐CoV‐2, the methods for the salivary diagnostics utilized till very recently, and the arisen considerations for the diagnosis of COVID‐19 disease. CONCLUSION: The major advantage of using saliva as a specimen for the detection of SARS‐CoV‐2 is that saliva collection is a non‐invasive method which produces no discomfort to the patient and permits the patients to utilize home self‐sampling techniques in order to protect health providers from the exposure to the pathogen. There is an urgent need to increase the active research for the detection of SARS‐CoV‐2 in the saliva because the non‐invasive salivary diagnostics may provide a reliable and cost‐effective method suitable for the fast and early detection of COVID‐19 infection. In December 2019, a new epidemic of pneumonia broke out in the province of Wuhan city, China. The disease was first reported by Zhou et al. (2020) to be caused by a novel coronavirus, probably originating from bats. The International Committee on Taxonomy of Viruses named this new single-stranded RNA virus as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2; Coronaviridae Study Group, 2020) . This new, zoonotic disease outbreak was in turn officially named as novel coronavirus disease 2019 . Τhe World Health Organization declared the supervening of SARS-CoV-2 a pandemic, a public health emergency of international concern with a very high global health risk assessment level. COVID-19 is causing deaths and restrictions all over the world. The most recent statistics render the scenario nightmarish. To date, more than 41,570,883 people worldwide have been affected, while 1,134,940 people have already died from the disease. Interestingly, Fan et al, in an article published back in March 2019, had foreseen this epidemic and had already warned us that: "Thus, it is highly likely that future SARS-or MERS-like coronavirus outbreaks will originate from bats, and there is an increased probability that this will occur in China" (Fan et al., 2019) . Laboratory testing for virus SARS-CoV-2 and the consequent respiratory coronavirus disease 2019 is divided into methods that detect the viral presence and methods that detect antibodies produced in the host as a response to infection . According to the report from the American Society for Microbiology in their COVID-19 International Summit, March 23, 2020: Methods that detect viral presence into the host excretions measure current infection by SARS-CoV-2 , while the detection of human antibodies deployed against SARS-CoV-2 measures the past exposure to the virus . Saliva can be used as a specimen in either of these methods aiding in the battle against this life-threatening disease. In order to identify a new pathogen such as a virus (as in the case of SARS-CoV-2), the virus must be isolated in cell cultures and its genome sequences must be fully analyzed and the viral nucleic acids are detected. At the moment, the "gold standard" for the diagnosis of COVID-19 disease is the real-time quantitative reverse transcription PCR (rRT-PCR) (Lippi et al., 2020) on specimens such as nasopharyngeal and oropharyngeal swabs or wash in ambulatory patients from the upper respiratory tract and sputum (if produced) (CDC, 2020) or BALF (bronchoalveolar lavage fluid) (WHO) from the lower respiratory tract (Gualanoa et al., 2020) but not saliva specimens. According to our research experience, real-time quantitative reverse transcription PCR (rRT-PCR) was successfully applied to saliva in different diseases (Michailidou et al., 2016) and viral detection protocols (Speicher et al., 2015) , despite the fact that this option is not yet mentioned by any health organization as a possible specimen in the detection of SARS-CoV-2. As soon as the viral sequence was discovered and released (Figure 1 ) , the first real-time reverse transcription polymerase chain reaction (rRT-PCR) test was developed in January 2020 by Charité Institute in Berlin and was adopted by the World Health Organization (WHO, protocols, 2020). Since then, different RT-PCR protocols have been established by different countries. Various accredited public health laboratories have developed their own techniques targeting different parts of the viral genome. The first PCR protocol was published in Berlin, Germany, on January 13 from the Charité-Universitätsmedizin with a modification on January 17, both protocols provided by WHO. The researchers downloaded all complete and partial (if >400 nucleotides) SARSrelated virus sequences available at GenBank by January 1, 2020. Artificial sequences and sequence duplicates were removed, resulting in a list of 375 sequences. The sequences were then aligned and used for assay design. They were later complemented by sequences released from the Wuhan cluster. The first protocol was complex and time-consuming, so on the 2nd version, the N gene assay was removed, single probe versions of RdRp assay added, and availability of controls updated. The RdRP_SARSr-P1 probe, the Pan Sarbeco-Probe, will detect 2019-nCoV, SARS-CoV, and bat SARS-related CoVs, while the RdRP_SARSr-P1 probe is specific for 2019-nCoV and will not detect SARS-CoV . China CDC targeted two loci in the viral genome: first the Open Reading Frame 1ab (ORF1ab) and second the N gene. Whereas Japan used nested RT-PCR and total RNA extraction by utilizing the QIAamp Viral RNA Mini Kit (Qiagen) following manufacturers' instructions detected two SARS-CoV-2 specific primers ORF1a and the S (spike) gene. Institut Pasteur, Paris, France, protocol, based on the first sequences of SARS-CoV-2 available on the GISAID database on January 11, 2020, describes procedures for the detection of SARS-CoV-2 for two RdRp gene targets (IP2 and IP4) spanning nt 12621-12727 and 14010-14116 (positions according to SARS-CoV, NC_004718) using the E gene assay from the Charité protocol as a confirmatory assay. The USA CDC using oligonucleotide primers and dual-labeled hydrolysis probes (TaqMan®) and control material target three loci in the N gene and RNase P. The National Institute of Health in Thailand suggested two monoplex assays reactive with all coronaviruses (target: ORF1b-nsp14) under the subgenus Sarbecovirus that includes 2019-nCoV, SARS-CoV, and bat SARS-like coronaviruses and none specific for SARS-CoV-2. The National Health Service in the United Kingdom adopted the RdRp assay of the Charité, Germany, protocol and uses these probes only. Canada carries out the E gene assay, and on indeterminate result, the RdRp gene also from the Charité, Germany, protocol. In Table 1 are summarized the methods (gene targets, primers, and probes) and their origin, according to WHO (WHO, protocols, 2020) . Interestingly, saliva, as emphasized above, is not yet mentioned in the instructions for specimen collection of any organization. However, when WHO mentions wash, does it mean an oral rinse? (WΗΟ, laboratory testing for COVID-19, 2020) and the CDC (Centers for Disease Control and Prevention), when it mentions the use of sputum it clarifies "have the patient rinse the mouth with water and then expectorate deep cough sputum." Either way, Hong Kong has already established a testing outpatient-program strategy where saliva is the protagonist specimen. Suspected patients (people over the age of 18 with fever, upper respiratory tract infection, and/ or pneumonia) stay home, while the emergency department supplies them with a specimen tube where they have to spit-first thing in the morning-and send it back and get a test result with a text message in a little while after. This way, people "who test negative will be kept out of the health-care system and away from patients who may actually be infected with the virus" (Beaubien, 2020) . At first, three articles, analyzing the potential of saliva as a specimen for the detection of SARS-CoV-2 and the diagnosis of COVID-19 disease, two of them originating from the same team of scientists in Hong Kong, came to the fore . In February 2020, To and collaborators investigated the detection of SARS-CoV-2 in the saliva of twelve patients, where they also conducted viral cultivation . Sabino-Silva et al. (2020) , in a letter to the editor, commented on the article of To et al., stressing the health risk during the practice of dental procedures in dental clinics and offices but also the potential use of saliva in detecting the virus using salivary diagnostic platforms (Sabino-Silva et al., 2020) . The scientific team of in March 2020 advanced their research one step beyond and reported on the temporal viral loads of SARS-CoV-2 in the saliva of 23 patients admitted in two hospitals in Hong Kong March 2020, where they also tested the serum antibody titers . They found that salivary viral load was highest during the first week after the onset of symptoms and subsequently declined with time (slope −0.15, 95% CI −0.19 to −0.11; R 2 = .71). This finding renders saliva as a very suitable medium for the early diagnosis of COVID-19 because SARS-CoV-2 exerts the highest viral load near the disease presentation while there is a report that nasopharyngeal swab specimens may run with a lag in SARS-CoV-2 viral detection (Lo et al., 2020) . In the article by , saliva was collected intraorally by a physician with the use of a pipette on the day of the patient's hospital admission. RNA was extracted from the saliva specimens using QIAmp Viral RNA Mini Kit (Qiagen) and primers targeting the 5′UTR region of SARS-CoV-2. SARS-CoV-2 was detected in all 25 patients with relatively high Ct values (range 18.12-32.23, mean value 27.16 ± 3.07)-all of the specimens presenting Ct lower than 33 . These first three research articles attempting to detect SARS-CoV-2 RNA in the saliva presented a very limited number of patients: 12 patients in the article of To and collaborators in February 2020, 23 patients by the same research team in March 2020 , and 25 patients in the research published by . Furthermore, by harvesting oral swabs and testing RNA among 15 patients, Zhang et al. found that half of them (50%) were 2019-nCoV RNA-positive . Meanwhile, much more research upon salivary COVID-19 diagnostics flew down the stream with more than 30 articles published on the topic, at the moment. A total number of 122 patients were recruited in the mentioned study, but three subjects were excluded from the analysis because their RST failed and was not repeated, consequently the total number of the patients was 119. There is still, however, a discordance in the exact type of saliva specimen used in these research papers. Some researchers use posterior oropharyngeal saliva Cheuk et al., 2020; Hung et al., 2020) or deep throat saliva , others drooling saliva Williams et al., 2020) , and many self-collected saliva (Iwasaki et al., 2020; Jamal et al., 2020; Nagura-Ikeda et al., 2020) . The contingency that this fact may pose a difference in the diagnostic results needs yet to be defined. Many research studies lack proper sampling technique and some don't even refer to it. Moreover, discrepancies in study design Zhu et al., 2020) , the use of control group samples, appropriate blinding, data analysis, and interpretation of the results, render the deduction of safe conclusions perilous (Sarode et al., 2020) . Besides this critical appreciation of the current research, the large number of studies conducted and published during the last months advocating the use of saliva in the diagnosis of COVID-19 disease manifests its importance as a possible diagnostic medium during this pandemic. Nevertheless, it is more than obvious that much more research is needed on the field, involving large cohorts of patients in order to validate saliva as a possible specimen and the experimental conditions for optimal results. Saliva can also be a suitable medium for the reaction: antigen to antibody recognition. While the "gold standard" for SARS-CoV-2 detection is qRT-PCR (real-time reverse transcription polymerase chain reaction), detec- Serology tests detect IgM, IgG, IgA, or total antibodies (usually in blood but saliva can be used also). Antibodies are usually checked in blood or serum, but saliva is sometimes an equally compatible medium for antibody detection. Antibody tests give us historical detail about infection. To make it plain, antibodies appraise immunity to severe acute respiratory syndrome coronavirus 2 (Petherick, 2020) . Still, in the case of SARS-CoV-2 whether this immunity is here to stay and will protect the patients from a future reinfection is under investigation at the moment. WHO warns that "In some people with COVID-19, disease confirmed by molecular testing, weak, late or even no antibody responses have been reported" (Gorse et al., 2020) . According to the American Society for Microbiology , early studies suggest that most patients present antibodies to SARS-CoV-2 between the 7th day and 11th day after exposure to the virus. Some patients may develop antibodies sooner, and some may develop after the 2nd week of infection (Okba et al., 2020) . SARS-CoV-2 is a new virus, and longitudinal studies concerning seroconversion, type, efficiency, and duration of antibodies and the strength of their prophylactic shield against reinfection are not known yet. Serology tests are most often performed using whole blood, plasma, or serum employing enzyme-linked immunosorbent assays (ELISA) in laboratory premises. In the case of SARS-CoV-2, they usually attempt to detect host (human) antibodies against the viral spike protein and the receptor-binding domain (RBD). Immunoglobulins are also present in the saliva. There is a reported similarity in IgG profiles between serum and saliva opening an opportunity for saliva-based antibody tests (Hettegger et al., 2019) . In viral diseases, antigens that their efficiency depends on "the time from onset of illness, the concentration of virus in the specimen, the quality of the specimen collected from a person and how it is processed, and the precise formulation of the reagents in the test kits" and that the sensitivity of these tests might be expected to vary from 34% to 80% (Bruning et al., 2017) . Saliva collection is a non-invasive method which produces no discomfort to the patient and allows for the patients to perform easily home low-cost self-sampling techniques in order to protect health providers from the close contact to the patient and exposure to the pathogen. Attention must be paid to the sample collection method which according to Bhattarai et al., (2018) "must be appropriately optimized to reduce error". Tong, in a letter to the editor (Tong, 2005) stresses the importance of minimizing the interaction between suspected patients and health providers highlighting a part of Chowell et al. (2004) article where the author analyzes that "the strong sensitivity of R 0 to the transmission rate β indicates that efforts in finding intervention strategies that manage to systematically lower the contact rate of persons of all age groups promise an effective means for lowering R 0 ". Furthermore, promoting saliva self-sampling "eases the burden on doctors, clinics and laboratories" (Tong, 2005) Saliva as a testing fluid has an easy and safe non-invasive collection bypassing venipuncture that a patient can even perform in-home alleviating the heavy workload during a pandemic from hospitals and the infection hazard from health professionals. Let's not forget that COVID-19 patients often present with a thrombocytopenia; therefore, nasopharyngeal or oropharyngeal swabbing may cause bleeding and distress and rendering instead saliva sampling more suitable. Last but not least, whole saliva by passive drooling provides a large sample. Consequently, this allows the sample to be tested for more than one biomarker. It also facilitates the researcher to freeze the left-over and use it at a later time (Bhattarai et al., 2018) . Saliva as a fluid easily accessed and collected, being at the entrance of the respiratory system, has also proved to incorporate 2019-nCoV nucleic acid . A study of Wyllie et al. not only detected more SARS-CoV-2 RNA copies in the saliva specimens than in the nasopharyngeal specimens, but also observed that a higher percentage of saliva samples than nasopharyngeal swab samples were positive up to 10 days after the COVID-19 diagnosis (Wyllie et al., 2020) . Taking into consideration the characteristics of non-invasiveness and less risk of exposure for the healthcare workers, saliva specimen collection for the diagnosis of coronavirus has the advantages of being more acceptable for patients, more safe for healthcare workers, and last but not least with significant lower economic cost. The provenance (origin) of SARS-CoV-2 RNA in the saliva must then be really elucidated. First of all, viral RNA may be derived in the saliva by the respiratory secretions frequently exchanged among the upper or lower respiratory tract and the oral cavity. Additionally, specific blood exudate often comes into the oral cavity through the crevicular fluid effluence and enriches saliva with blood derivatives such as circulating nucleic acids and blood antibodies. Moreover, specific antibodies are electively secreted in the saliva like sIgA (Gianchecchi et al., 2019) . In the case of COVID-19, however, some more mechanisms of SARS-CoV-2 viral presence and the subsequent viral load in the saliva must be mentioned and disambiguated. The specific receptor of SARS-CoV-2 on the cells is ACE-2 (angiotensin-converting enzyme 2) . COVID-19 is considered to be transmitted through respiratory droplets. Nonetheless, ACE2 is commonly reported to be highly expressed in other organs too, such as intestines and kidneys but also on the human epithelial cells of the oral cavity mucus membrane (Xu, Zhong, et al., 2020) . Therefore, different routes of transmission most probably exist. There is also a report, back in 2011, that "epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection (SARS) in the upper respiratory tracts of rhesus macaques" (Liu et al., 2011) . There is a possibility, which needs to be explored, that SARS-CoV-2 may affect the epithelial cell lining salivary gland ducts in human thus eventuating (resulting in) in the highly detectable salivary viral loads of SARS-CoV-2. Last but not least, a very recent research revealed a shell disorder in SARS-CoV-2 which transfuses greater resilience of SARS-CoV-2 (COVID-19) outside the body and in bodily fluids (Goh et al., 2020) . SARS-CoV-2 virus, may, this way, be more resistant to the RNAses and all the antiviral enzymes that naturally exist in the saliva. These data could explain the high detection rates and viral loads of SARS-CoV-2 in the saliva. Furthermore, SARS-CoV-2 virus has this way a greater chance to shed larger numbers of viral particles in body fluids, among them saliva, and can remain in an active stage for a longer period of time. Additional data that need to be investigated when dealing with the idea of using salivary diagnostics in COVID-19 are the viral load that it exhibits. Up today, there are still not enough research data upon viral kinetics and viral loads of SARS-CoV-2 in COVID-19. In particular, there are not any published articles that specifically compare viral loads and kinetics between nasal/throat swabs and saliva. Salivary viral loads are reported to be high in the first days of infection and then decline as the infection goes down affecting the lungs . There is one published research, referring to infection control of COVID-19 in Hong Kong, that uses various samples, among them saliva . It reports that saliva samples presented higher viral loads than pooled nasopharyngeal and throat swabs (pooled nasopharyngeal and throat swabs 3.3 × 10 6 copies/mL and saliva 5.9 × 10 6 copies/mL, respectively), but it does not report the exact viral kinetics or even the exact day of sampling. . The research in this aspect is essential and will furthermore shed light upon the role of saliva as a diagnostic specimen. The most frequent antibody in the human saliva is sIgA (secretory IgA) being on the front line of specific and non-specific immune defense against pathogens in the oral cavity. sIgA, as an antibody class, is found in various external secretions and differs in structure and function from other antibody classes. IgA presents as dimeric in the saliva and is also bound with the secretory component (SC) that furthermore aids in the stability of the molecule. Dimeric IgA is bound to the polymeric immunoglobulin receptor (pIgR) in the epithelium lining the lumen of the salivary glands. In this way, it is transported into the salivary lumen together with other compounds that constitute the salivary gland secretion (Brandztaekg, 2013) . The part of the immunoglobulin receptor (pIgR) that binds to the dimeric IgA splits and forms the molecule sIgA . Salivary IgG and IgM are, in their large proportion, as mentioned by Janket et al. (2010) , "an ultrafiltrate of serum IgG and IgM, which is modified by the host's general immune response and may not accurately reflect the strength of infection" (Brandztaekg, 2013; Janket et al., 2010) . Plasma contains approximately 12.5 mg/ml IgG and 2.2 mg/ ml IgA, while the concentrations for unstimulated whole saliva are estimated at approximately 0.014 and 0.19 mg/ml for IgG and IgA (Brandztaekg, 2013) . Salivary IgG is mainly exudated from blood circulation, while a minority (<20%) is produced by local plasma cells in gingival lesions or salivary glands. In contrast, more than 95% of salivary IgA is produced locally in the oral cavity by plasma cells in various salivary glands. In raged all over the world sweeping human lives, national health systems, and economies. The need for quick and effective restriction of the disease spread is urgent. Accurate and timely testing for the COVID-19 disease is a key ad hoc and is allocated into two directions. First direction is the detection of the viral presence into the host excretions and systems which measures the current infection with SARS-CoV-2 . Second direction is the detection of human antibodies deployed against SARS-CoV-2 something which measures the past exposure to the virus . Saliva, as a bodily fluid seems to meet the criteria for a suitable specimen toward these aims. Saliva is a complex, yet way informative, bodily fluid consisting of the secretion of a set of three major salivary glands and numerous minor salivary glands in the oral cavity. Gingival crevicular fluid (GCF), desquamated epithelial cells, and various microorganisms are also components of saliva and may be used in diagnostics. When a patient is ill or wounded, saliva may contain bronchial secretions, and serum and blood derivatives. Researchers advocating in favor of saliva as a testing fluid in the battle against SARS-CoV-2 propose the easy and safe non-invasive collection bypassing venipuncture that a patient can even perform in-home alleviating the heavy workload during a pandemic from hospitals and health professionals. The official pathogen detection is the confirmation of 2019-nCoV nucleic acid from throat swabs (Lippi et al., 2020) . Throat swabs are relatively invasive, induce coughing, and may cause bleeding, which is possible to increase the danger of clinical staff infection. From initial research data, it seems that the area and method of collection of the saliva specimens greatly influence the diagnostic effectiveness (Xu, Cui, et al., 2020; Zhang et al., 2020) . Saliva from deep throat, from oral cavity, and from salivary glands, respectively, documented a diagnostic tendency of decreased positive rate of 2019-nCoV RNA among COVID-19 patients (Xu, Cui, et al., 2020; Zhang et al., 2020) . Consequently, for the effective and reliable clinical application the saliva specimens from deep throat proved to have the highest positive rate of virus detection, which may be very helpful in the early diagnosis of COVID-19 (Xu, Cui, et al., 2020; Zhang et al., 2020) . Furthermore, very initial research data in a very small number of patients indicated that saliva specimens collected directly from saliva glands ducts are associated with severe COVID-19 and possibly could be a predictive and non-invasive method for critically ill patients in need of ventilator support (Xu, Cui, et al., 2020) . Despite the obvious advantages of diagnosis of COVID-19 by using saliva because of the ease of collection, the noninvasiveness, the less hazardous compared with throat swabs, the low cost, and the safety for the clinical personnel, comprehensive diagnosis should be combined by the acquisition and evaluation of detailed information of symptoms, epidemiological history, and analysis of multiple clinical examinations. Based on the data cited above, SARS-CoV-2 seems to affect the mucosal epithelial cells in the oral cavity (Xu, Zhong, et al., 2020) , is then probably released, and aggregating in the oral cavity displaying an extremely high shell resilience in the saliva (Goh65) supporting the idea of SARS-Co-V-2 transmission through oral droplets. Other issues to be addressed when dealing with SARS-CoV-2 salivary diagnostics are as follows: 1. Which viral RNA sequence we must target when trying to detect the SARS-CoV-2 virus in the saliva. For the moment in the three published articles concerning SARS-CoV-2 salivary detection, while two come from the same author yet, different RNA parts are amplified. To et al. in their first article targeted the S gene, while in the second, the RNA-dependent RNA polymerase helicase, whereas Azzi et al. used primers targeting the 5'UTR region of SARS-CoV-2 . Deciding the appropriate RNA sequence to be targeted in qRT-PCR is crucial because when refining the molecular targets many analytical pitfalls of PCR detection are well avoided (Lippi et al., 2020) . The ultimate goal is a viral RNA sequence easily targeted, captured, and amplified but specific enough for SARS-CoV-2 to avoid cross-reactions. The best approach toward salivary COVID-19 diagnostics is to apply the same protocols and assays published by WHO for respiratory samples. Coming to discuss the optimal protocol, therefore, the use of only a single probe is not credible enough. Moreover, gradual steps toward reaching a diagnosis are rather a safer yet more complex approximation. The Charité protocol, using the RdRP_SARSr-P1 (Lippi et al., 2020) ? The frequency of sampling must be also investigated. Moreover, salivary viral loads are reported to be high in the first days of infection and then decline as the infection goes down affecting the lungs there is a question whether saliva sampling will be informative enough many days after the disease onset. Additionally, when the cell-free part is employed the hypothesis that PCR might detect fragmented parts of the viral RNA for long periods of time not disclosing true viral viability and replication must be checked. 4. Last but not least, a great advantage in the use of saliva is the large sample size that is usually assembled (2-5 ml). The sample can be aliquoted, and if the first result is negative, an affirmative diagnostic test repetition is possible. Moreover, saliva sample is a suitable medium for both the necessary methods in COVID-19 disease: the ones that detect the viral presence (viral nucleic acids or antigen detection) but also the methods that detect antibodies produced in the host as a response to infection . In the opposite, different sample types-nasal swabs for molecular tests and blood samples for antibody tests-and probable collection in different timings must be employed. Transmission of SARS-CoV-2 through saliva droplets has to be elucidated, something that will change the perception about transmission routes and will furthermore alert dentists about the additional hazards while practising their profession. It is not therefore accidental that msn news publishes an article characterizing the new coronavirus tests as "game-changers." Research employing saliva as a suitable sample in order to detect viral RNA, antigens, and antibodies is not yet extensive for any of the 2. Defining the optimal RNA extraction protocol from the saliva and procedure of sample processing. 3. Detecting the levels of salivary immunoglobulins and the quality of anti-SARS-CoV-2 antibodies in the saliva. In conclusion, there is an urgent need to increase the active research for the detection of SARS-CoV-2 in the saliva because the non-invasive salivary diagnostics may provide a reliable and cost-effective method suitable for the fast and early detection of COVID-19 infection. None to declare. The peer review history for this article is available at https://publo ns.com/publo n/10.1111/odi.13729. Athanasios Poulopoulos https://orcid. org/0000-0002-6611-7506 Viral loads of SARS-CoV, MERS-CoV and SARS-CoV-2 in respiratory specimens: What have we learned? 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