INTRODUCTION:

Saliva is considered to be one of the essential fluids present in our body. It comprises of various compounds (organic and inorganic) that are useful for most of the body functions such as lubrication, antimicrobial actions, cleansing, maintenance of mucosal membrane, breaking down food for swallowing, etc. The transfer of these compounds into the salivary fluid is passively from the blood^1-3. It is a hypotonic secretion from parotid, sublingual and submandibular regions which are>95% water^4-6. Submandibular glands contribute to the highest amount of secretion of saliva which is about 70-75% of the daily output of 1.0-1.5L in a healthy individual^7,8.

In the recent decade, about the advances made in the technology, saliva has been proved to be a useful and reliable tool for diagnosis of various diseases. Saliva as a diagnostic tool is an early, inexpensive and non-invasive method of detection of a disease and thus provides a higher chance of treatment being provided in time. It is also used for keeping a check on the progress in the health of patient^7,9.

The most advantageous part of using saliva as a diagnostic tool, concerning both, medical practitioner and the patient, is the accessible sample collection which is painless and quick. Also, it requires less care during transportation⁷. Salivary excretion can be used for diagnosis of diseases and disorders like auto-immune disorders, cardiovascular diseases, Endocrinology, Infectious diseases such as viral and bacterial infections, nephrology, Cancer treatment, Bio-molecular changes, Pharmaceutical etc. This review article is more focussed on the usage of saliva as a diagnostic aid for viruses.

Viruses enter the human body through various routes such as oral, infected food, contaminated blood, sexual contact, salivary exchange or via particulate matter from sneezes or a cough. Some common viruses that can be transmitted to humans are Herpes Simplex Virus (HSV), Epstein Barr Virus (EBV), Hepatitis, Human Immunodeficiency Virus (HIV), Rotavirus, Human Papillomavirus (HPV) etc.¹⁰.

Diagnosis kits based on antigen-antibody interactions are commercially available for the detection of a multitude of viruses such as Rubella, Rubeola and Parotitis virus¹¹. Polymerase Chain Reaction (PCR) is typically used for early detection of the viral genome from the saliva¹². Presence of antibodies for HIV is the basis for analysis of congenital infections, acute infections and recurring of infections¹³.

HEPATITIS VIRUS:

Hepatitis B (HBV) and Hepatitis C (HCV) virus infect more than 400 million people around the world. These infections are the primary cause of chronic liver disease and death worldwide. With the availability of new technology and advanced treatment, we are now able to eradicate the viruses in 60% of the cases and reduce the chances of progression in the remaining cases¹⁴. Unfortunately, because of the inadequate and late detection, many death cases are still reported. Therefore, there is an urgent need to shift from the traditional methods to fast and efficient methods like salivary diagnostics. Research has shown saliva to be an effective alternative method to serum for the diagnosis of hepatitis virus¹⁵.

Presence of IgM antibody in the saliva can be used to diagnose acute infections of Hepatitis A virus (HAV) and HBV. As compared to serum levels, antibodies of HAV in saliva showed a remarkable amount of sensitivity and specificity of nearly 99%¹⁶. Similarly, salivary analysis has proven to be extremely specific in addition to the sensitive method for the identification of other hepatitis such as B as well as C¹¹. Oral fluid samples collected using Orasure ®, when analysed proved to be an effective means for the detection of these viruses. In comparison to serum antibodies, oral fluid samples have reported 100% specificity and sensitivity for the presence of antibodies for viral Hepatitis B and C¹⁷. Hepatitis B surface antigen (HBsAg) has also been screened using saliva in some epidemiological studies. Serological kits used for recognition of HBsAg antibody in saliva have reported specificity of 86.8% and sensitivity of 92%¹⁸. Hence, it can be concluded that saliva is proved to be an effective and convenient substitute to serum for the diagnosis of hepatitis virus infection.

HUMAN PAPILLOMAVIRUS:

Oral cancer is the highly prevalent cancer being the chance of death in the developing countries. It is also the sixth most common malignancy all over the globe¹⁹. Leading causes of oral cancer include infection with human papillomavirus (HPV), bad oral hygiene, dietary habits and nutritional deficiency. Of all HPV types, HPV16 and HPV18 are considered to be the high-risk and most identified strains in the oral biopsies²⁰. Due to the underlying connection amid HPV and malignancy, there is an immediate need for quick and easy detection and quantification of specific HPV types whenever a lesion is suspected to cause an HPV infection. This would help to detect the precancerous abrasion and will influence the treatment procedure before it becomes more severe²¹.

Salivary diagnostic tests showing high specificity and sensitivity of 90% and 89% respectively are available for HPV, but mostly these tests involve the use of PCR. Hence, POC test is yet not available. Use of oral swabs, expectorated oral rinse or expectorated saliva has been a common method for the collection of oral samples for HPV detection. Among these, the expectorated oral rinse with mouthwash samples the entire oral cavity and dislodges the mucosal cells hence showing the highest sensitivity. OraRisk HPV test is the commercially available salivary diagnostic test for HPV infection in the United States²². This test provides a quick method for the diagnosis of HPV infection which critical for the survival of the patients with OSCC²³. Hence, the use of salivary diagnostic kits for HPV infection is likely to increase with the course of time.

ZIKA VIRUS:

The Zika virus is the member of Flaviviridae family of flavivirus genus²⁴. Zika fever has a non-specific clinical presentation. The symptoms include fever, rashes and asthenia²⁵. Zika fever has no sudden clinical onset, and due to this, accurate dating of the commencement of the infection is not possible²⁶. Symptoms increase after transmission of the virus by a Zika-infected mosquito vector, with an incubation period of 2–14 days²⁷.

Diagnosis of Zika fever in the laboratory is a challenge as no standard diagnosis test or tool exists for this purpose. Use of serology for diagnosis is limited; the culture of viruses is not conducted regularly, and antigenic detection tests are not available in the market due to cross-reactivity between antibodies due to different Flaviviruses²⁸ [28]. Hence, diagnosis in the acute phase employs molecular techniques such as PCR to amplify the viral nucleic acid material and detect it. For instance, a protocol using qRT-PCR (ZIKV RT-PCR) was reported by Lanciotti et al. while investigating the ZIKV epidemic on Yap Island²⁹.

According to a study, ZIKV RNA in blood is lower than in saliva. Nevertheless, ZIKV nucleic acid has been found in blood samples but not in saliva. Hence, they concluded that saliva could not be an alternate for ZIKV diagnosis³⁰.

The recent outbreaks of Zika virus have led to the need for active surveillance and diagnostic tool. Recently, a microfluidic assay to detect ZIKV RNA which uses reverse transcription LAMP coupled with reverse dot blot analysis in saliva has been developed. This test provides much quicker results compared to conventional methods and can be developed into for POC testing³¹.

HUMAN IMMUNODEFICIENCY VIRUS:

Human Immunodeficiency Virus (HIV) is the cause of Acquired Immunodeficiency Syndrome (AIDS). Characteristics of this disease include susceptibility to infection by opportunistic pathogens and more hostile form of B-cell lymphoma. CD4 T cells are also reported to decrease³².

In the laboratory, HIV infections can be acknowledged by the recognition of antibodies in the plasma and serum, which is confirmed by a Western blot or a polymerase chain reaction (PCR), or by detecting the presence of viral nucleic acid through PCR or by p24 antigen testing³³. Testing for antibodies is usually used to detect HIV. When antibody testing is not sufficient to conclude if a patient has been infected or not, DNA PCR is performed¹⁵.

Testing for antibodies in saliva for the diagnosis of HIV offers several unique advantages over the serum. Saliva collection is non-invasive and also eliminates the risk of infection for whoever collects the sample as transmission of the virus through saliva is not likely³⁴. Collection of saliva is easy for people with compromised venous access, haemophilia patients, and children³⁵.

Presently, varied tests that use saliva exist for the diagnosis of HIV which detects antibodies to HIV-1, HIV-2 or both. Typically, the tests contain nitrocellulose flow strips with capture areas for the detection of all antibodies in the sample and exclusively for HIV-1 or, ideally, with HIV-1 as well as HIV-2³⁵. A positive result is required to be confirmed by a second test such as a Western blot that detects antibodies to numerous HIV antigens, or a blood-based PCR test that detects HIV RNA.

Several test kits that use oral fluids to diagnose HIV are available in the market. Out of these only OraQuick In-Home HIV Test shows a response within a half hour, and has a specificity of 99.98 % and a sensitivity of 92 %, is approved by the FDA³⁶. Results from numerous studies have shown that the sensitivity and specificity these oral tests possess are comparable to those of antibody detection tests involving the use of blood serum or plasma^37,38. Developments are being made to make these antibody tests for HIV detection more accurate and enable early detection.

Due to differences in stability and concentration, targets such as antigen and nucleic acid are may not be detectable in saliva. For instance, immune-assays that detect p24 antigen and antibodies against HIV for early HIV detection from blood-based samples³⁹ have not yet been shown to work with oral fluid samples. Likewise, detection of viral RNA is harder in saliva than in blood due to decreased viral load⁴⁰. Current technologies make HIV RNA detection in saliva possible by employing concentration and purification to attain the required sensitivity.

Recently, an assay based on Antibody Detection by Agglutination–PCR (ADAP) has been developed. In this, the agglutination of antigen–DNA conjugates is driven by multivalent binding of antibodies. The resulting proximity allows ligation of DNA fragments to give a full-length DNA amplicon, which can be quantified using qPCR⁴¹.

INFLUENZA VIRUS:

Influenza is a genetically and antigenically diverse set of viruses that belong to the Orthomyxoviridae family and contain a negative-sense, segmented ssRNA genome⁴². Influenza virus leads to acute febrile respiratory infection and sometimes causes severe deadly complications^43,44. Hence, rapid diagnosis is crucial for treatment and isolation of the diseased.

In clinical laboratories, several methods such as isolation of virus in cell culture, immunofluorescence assays, serological assays, nucleic acid amplification, immunochromatography-based rapid diagnostic tests are employed to detect influenza virus⁴⁵. Detection of viral genomic RNA by PCR is considered the optimum method of identification and classification of the virus^46,47.

Nasopharyngeal swabs (NPS) are used to detect it. However, sampling saliva is more accessible compared to nasopharyngeal swabs. Also, this non-invasive test would provide to be potentially valuable, particularly for children. Subjecting the saliva sample to Reverse Transcriptase PCR is an essential technique for the same⁴⁸. As the viral load is lesser in saliva, PCR would amplify the nucleic acid, making it an essential tool for influenza virus detection in saliva. Use of rapid influenza diagnostic tests (RIDTs) to detect influenza virus in saliva has also been tested, but these showed that diagnostic sensitivity in NPS was higher than in saliva and combining saliva and NPS could improve the sensitivity of RIDTs⁴⁹.

Hence, even though NPS have elevated viral loads and is preferred by patients, additional molecular testing of salivary samples result in the improved treatment received by them⁵⁰.

CONCLUSION:

Saliva is easy to obtain from patients in a non-invasive manner, whereas the collection of serum requires professional guidance and some patients might face difficulties while drawing blood. Therefore, salivary diagnostic tests in comparison to serum offer cost-effective, easy and quick approach for the diagnosis of few major viral infections.

Saliva contains both serum-derived as well as locally produced markers that are essential for the detection of these viral infections. Hence, saliva has facilitated the availability of POC test kits for viral infections. This review shows that use of saliva for the detection of few major viral infections hold considerable promise. Monitoring of viral infections, including hepatitis, HIV, Influenza, Zika and HPV using salivary diagnostic test has proved valuable in early detection and hence proper treatment of infected individuals. Some of these kits are available in the market and are extensively used in clinics, by patients and by researchers. However, due to the unreliable reflection of the levels of specific markers in saliva, it is specifically used for qualitative diagnosis instead of a quantitative diagnosis.

ACKNOWLEDGEMENT:

We would like to express our special thanks to Dr.I. Manjubala for her constant support for this project as a part of our J-Component in Medical Diagnostics course. We would also like to thank Dean SBST for giving us the desired infrastructure to complete this project.

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