Research Article

Correlation between Hepatitis C Virus Antibodies in Saliva and Serum: A Safe Method for Epidemiological Studies


  • Oya Özlem Eren Kutsoylu
  • Ziya Kuruüzüm
  • Ayşe Yüce

Received Date: 02.03.2020 Accepted Date: 22.06.2020 Viral Hepat J 2020;26(2):65-68


Hepatitis C virus (HCV) infection is an increasing public health problem in developing countries. A non-invasive method is required as blood sampling is an invasive method for detecting HCV antibodies. In this report, we examined the performance of a commercially available serological kit to detect HCV antibodies in saliva as a possible alternative to serum for epidemiological studies.

Materials and Methods:

A total of 150 paired oral fluid and blood samples were collected from 75 anti-HCV-positive and 75 anti-HCV-negative individuals. Homemade swabs were used for saliva sampling instead of commercial products.

Modified Ortho HCV 3.0 SAVe ELISA kit was used to detect HCV antibodies in saliva, and blood samples were analyzed for anti-HCV and HCV-RNA.


The sensitivity and specificity of this assay were 86.7% and 86.7% in saliva. Out of 38 participants who were positive for HCV-RNA in serum, 36 were also positive for HCV antibodies in saliva.


The implementation of a non-invasive method such as saliva collection is easy, economical, and can be done by unskilled personnel. According to our sensitivity and specificity results, the modified ELISA method for anti-HCV detection in saliva with the use of a different saliva collection system can be an alternative technique for epidemiological surveys.

Keywords: Hepatitis C virus, epidemiology, antibody, Saliva


Hepatitis C virus (HCV) infection is an important public health problem, with an estimated 170 million people infected worldwide (1). Although available data are limited, it is estimated prevalence of HCV is higher in developing than developed countries (2). Common risk factors for HCV infection are blood transfusion and intravenous drug use, and sexual and vertical transmission (3). HCV infection primarily affects liver and can cause cirrhosis and hepatocellular carcinoma later in life.

Although sensitive and specific serologic tests are available, difficulty in obtaining blood samples and risk of disease transmission to health personnel through needlestick injury are disadvantages that limit their acceptance outside a clinical setting. Therefore, a safer, non-invasive alternative to blood sampling is required. Collection of saliva is easy, non-invasive, painless and safe to carry out. In epidemiological studies, especially those involving screening large populations, saliva sampling is quicker than blood sampling and there is no need for specialized staff (4).

Several saliva collection methods have been developed, e.g. Omni-SAL (Saliva Diagnostic Systems, Inc., Vancouver, WA, USA), Orapette (Trinity Biotech, Dublin, Ireland), OraSure (Epitope Technologies, Inc., Bethlehem, PA, USA), and Salivette (Sarsted Ltd., Leicester UK). The study reports use of an in-house saliva collection device, which is easy to produce and offers a more affordable method for clinical and epidemiology studies conducted in developing countries.

Materials and Methods

Study group

The study group consisted anti-HCV seropositive and anti-HCV seronegative patients (n=75 for both groups). HCV-seropositive patients were those undergoing follow-up visits at the Infectious diseases and clinical microbiology outpatient clinic. HCV-seronegative participants were those who attended the same outpatient clinic for other reasons. The study was approved by the Institutional Ethical Comittee Dokuz Eylül University (approval number: 200895). Prior informed consent was obtained from all participating patients, who were >18 years of age.

Sample collection

Each participant donated two blood samples (16 mL) and one saliva sample (6 mL), collected at the same time. Serum from one sample was stored at -20 °C and the other at -80 °C until used. Saliva samples were collected using sterile foam swabs (3 cmX 1.5 cmX 5.5 cm). Patient was requested to hold the foam swab in mouth for two minutes, then swab was placed in one ml aliquot of Universal Transport Medium (Copan Italia S.p.a., Brescia, Italy), centrifuged at 3.000 g for 15 minutes and supernatant stored at -20 °C until used.

Laboratory assays

Serum samples were tested for anti-HCV antibodies using an Architect i2000 SR kit (Abbott Laboratories, Ltd. Saint-Laurent, Québec, Canada). Anti-HCV-positive serum samples were then tested for presence of HCV-RNA using Artus HCV-RG-RT-PCR assay kit (QIAGEN Gmbh, Hilden, Germany).

Saliva samples were tested for anti-HCV antibodies using Ortho HCV 3.0 SAVe ELISA kit (Ortho Diagnostics, Amersham, UK) with a modified protocol to increase sensitivity (5). In brief, 110 µl aliquot of saliva solutions and control samples were incubated for 16-20 hours at ambient temperature (15-30 °C) with shaking in a 96-well microtiter plate. Then plate was washed with buffer (supplied by the manufacturer), 200 µl aliquot of horseradish peroxidase-conjugated murine anti-human immunoglobulin G (IgG) monoclonal antibodies was added to each well, and plate was incubated for another three hours at ambient temperature, washed as described above before addition to each well of 200 µl of substrate and incubation for 30 minutes at room temperature in the dark. Reaction was terminated with 50 µl of 4 M sulfuric acid solution and A490 nm measured with a microplate spectrophotometer (Multiskan FC Microplate Photometer; Thermo Fisher Scientific Oy, Oulu, FINLAND). Final A490 nm values determined by a receiver operating characteristic (ROC) curve analysis.

Statistical Analysis

Detection of anti-HCV antibodies in serum samples was used as a gold standard for the assessment of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Ortho HCV 3.0 SAVe ELISA assay (Ortho Diagnostics) in saliva. Cut-off value was calculated according to ROC curve analysis as value with highest specificity and the sensitivity. Values ≥0.133 were considered positive and values <0.133 considered negative.


Mean age of the participants was 57±12.5 years in the test group and 58±17 years in control group, with 47% males. Nine participants in the test group were in a hemodialysis program.  All participants had negative Human Immunodeficiency Virus serology.

Difference between mean A490 nm value (1.32±1.023 and 0.17±0.24) of anti-HCV antibody ELISA in saliva samples of test and control group respectively is statistically sigificant (Mann-Whitney U test; mwu: 27, p-value <0.001), as was in serum samples (data not shown). Sensitivity, specificity, NPV and PPV value of saliva samples employing the modified Ortho HCV 3.0 SAVe ELISA (Ortho Diagnostics) was 87% for each parameter (Table 1).

In the HCV-seropositive group, 38 (51%) patients were HCV-RNA positive, of whom 36 (95%) were positive for anti-HCV antibodies in saliva. Of the 37 patients who were HCV-RNA negative, 29 (78%) had anti-HCV antibodies in saliva. Similar relationship was observed between serum HCV-RNA positivity and A490 nm values in saliva. Difference between mean A490 nm value (1.65±0.94 and 0.66±0.78) in saliva of test patients with positive and negative serum HCV-RNA respectively is also statiscally significant (Mann-Whitney U test; mwu: 277.5, p-value <0.001). Higher mean A490 nm value was observed in serum HCV-RNA positive than negative group.


Saliva sampling is an easier and less invasive method compared to serum sampling. Previous reports indicate that saliva is an appropriate specimen diagnosis of different infectious diseases, in particular viral infections that pose a hazardous risk to health personnel in the situation of needle prick accidents (5). In addition, there are various devices for collecting saliva as well as commercial tests geared for assaying saliva samples (Table 2).

In our study, an in-house saliva collecting device consisting of a sterile foam placed in a subject’s mouth was employed, a low-cost alternative to more expensive commercial saliva collecting devices. Using a saliva-dedicated commercial ELISA kit, there was good concordance between paired serum and saliva samples in detecting anti-HCV antibodies. Sensitivity and specificity of this assay for saliva samples were high (>85%).

In comparison with serum, Ig levels are 800-1000 folds lower in oral fluid, where IgA is predominant (6). Lee et al. (7) evaluated the use of an HCV antibody rapid test device with venous blood, fingerstick blood, serum, plasma, and oral fluid. They observed a slightly lower sensitivity (98.1%) with oral fluid, which they attributed to conditions of oral health, use of oral care products, and consumption of food and drink (7). Cha et al. (8) used the HCV antibody rapid test device with oral fluid and reported a clinical sensitivity was 97.8%. This may be responsible for low sensitivity of HCV antibody detection in saliva in the event of low anti-HCV antibody titers and negative HCV-RNA levels (9,10). It is worth noting a patient negative for saliva anti-HCV antibody was HCV-RNA positive and had previously been diagnosed as rheumatoid arthritis and treated with immunosuppressive agent.

Using saliva for antibody detection brings out new approaches which makes anti-HCV detection easier. Self-saliva collection is one these advantages. User friendly and easy to apply test is cost effective, convenient and time saving compared to complex laboratory test methods which requires highly skilled and experienced laboratory specialist and more accessible in underserved communities and isolated populations (11).

Cost-effectiveness also makes saliva sampling attractive. In a cost-effectiveness study rapid antibody saliva testing costs was determined much more lower when compared to testing via venipuncture (10).

Study Limitations

Discrepancies in sensitivity and specificity determined in other studies (Table 2) may be related to collection devices used, study populations and saliva ELISA method employed. Sensitivity and specificity of saliva anti-HCV antibody ELISA was lower in our study compared to literature data, likely related to use of a different method for saliva collection. A comparative study using the collection device of the present study and at least one of the commercially available collection devices should be able to provide an answer.


A combination of the new saliva collection method with a modified commercial ELISA assay yielded acceptable results. Sensitivity and specificity indicate that this method should be suitable for epidemiological surveys, obviating risk to health personnel using invasive procedure and more acceptable by infants and young children.


We thank Jacqueline Gutenkunst for refining the language of the article and Prof. Dr. Reyhan Ucku for statiscal analysis.


Ethics Committee Approval: The study was approved by the Institutional Ethical Comittee Dokuz Eylül University (approval number: 200895).

Informed Consent: Prior informed consent was obtained from all participating patients, who were >18 years of age.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Surgical and Medical Practices - O.Ö.E.K., Concept: O.Ö.E.K., A.Y., Design: O.Ö.E.K., Z.K., Data Collection or Processing: O.Ö.E.K., Analysis: O.Ö.E.K., Literature Search: O.Ö.E.K., Writing: O.Ö.E.K.

Conflict of Interest: All authors declare to have no conflict of interest.

Financial Disclosure: This study was funded by Dokuz Eylul University (approval number: 200895).


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