Longitudinal Study of Hepatitis A Infection by Saliva Sampling: The Kinetics of HAV Markers in Saliva Revealed the Application of Saliva Tests for Hepatitis A Study.

Despite the increasing numbers of studies investigating hepatitis A diagnostic through saliva, the frequency and the pattern of hepatitis A virus (HAV) markers in this fluid still remains unknown. To address this issue, we carried on a longitudinal study to examine the kinetics of HAV markers in saliva, in comparison with serum samples. The present study followed-up ten patients with acute hepatitis A infection during 180 days post diagnosis (dpd). Total anti-HAV was detected in paired serum and saliva samples until the end of the follow-up, showing a peak titer at 90th. However, total anti-HAV level was higher in serum than in saliva samples. This HAV marker showed a probability of 100% to be detected in both serum and saliva during 180 dpd. The IgM anti-HAV could be detected in saliva up to 150 dpd, showing the highest frequency at 30th, when it was detected in all individuals. During the first month of HAV infection, this acute HAV marker showed a detection probability of 100% in paired samples. The detection of IgM anti-HAV in saliva was not dependent on its level in serum, HAV-RNA detection and/or viral load, since no association was found between IgM anti-HAV positivity in saliva and any of these parameter (p>0.05). Most of the patients (80%) were found to contain HAV-RNA in saliva, mainly at early acute phase (30th day). However, it was possible to demonstrate the HAV RNA presence in paired samples for more than 90 days, even after seroconversion. No significant relationship was observed between salivary HAV-RNA positivity and serum viral load, demonstrating that serum viral load is not predictive of HAV-RNA detection in saliva. Similar viral load was seen in paired samples (on average 104 copies/mL). These data demonstrate that the best diagnostic coverage can be achieved by salivary anti-HAV antibodies and HAV-RNA tests during 30-90 dpd. The long detection and high probability of specific-HAV antibodies positivity in saliva samples make the assessment of salivary antibodies a useful tool for diagnosis and epidemiological studies. The high frequency of HAV-RNA in saliva and the probability of detection of about 50%, during the first 30 dpd, demonstrate that saliva is also useful for molecular investigation of hepatitis A cases, mainly during the early course of infection. Therefore, the collection of saliva may provide a simple, cheap and non-invasive means of diagnosis, epidemiological surveys and monitoring of hepatitis A infection purposes.

Cross-Sectional Study of Hepatitis A Virus Infection in the Pantanal Population before Vaccine Implementation in Brazil: Usage of Non-Invasive Specimen Collection.

Population-based prevalence studies are essential tools for screening of hepatitis A and provide important data on susceptible groups. However, surveillance in isolated communities is difficult because of the limited access to these areas and the need for blood sample collection. This study aimed to determine the anti-HAV prevalence using oral fluid samples to provide an alternative tool for epidemiological studies that might be useful for vaccination-related decisions. The study population was composed of 224 volunteers from South Pantanal, aged 3 to 86 years old. This study was performed using oral fluids, previously standardized for anti-HAV antibody detection, which were collected using a ChemBio device. Eluates were tested using modified commercial EIA to detect anti-HAV antibodies. The overall prevalence was 79.1%, corresponding to 178 reactive EIA tests out of 224 samples. The age stratified data revealed a prevalence of 47.8% between 0-10 years, 84% in 11-20 years and 91.9% in subjects older than 21 years. Results indicate that hepatitis A prevalence was higher in adolescents and adults, corroborating the literature reports. Thus, oral fluid samples could replace serum in HAV epidemiological studies in isolated communities as they are efficient at detecting anti-HAV antibodies.

Could oral fluid be used to evaluate anti-hepatitis A virus status in individuals living in difficult-to-access areas?

A strategy adopted by different countries to reduce the number of new cases of hepatitis A is the vaccination. However, the mosaic of the epidemiological profile in developing countries has hampered the establishment of a unified nationwide vaccination program. To determinate national vaccination policies, the results of epidemiological studies need to be carefully considered. For this monitoring, the use of oral fluid is very important due to the painless and non invasive collection characteristics. There are few studies investigating which oral fluid collection device is optimal to detect low antibody levels and its use in selecting individuals for vaccination. So, the present study aimed to evaluate different oral fluid collection devices to detect humoral immune response against hepatitis A virus and its application in epidemiological studies. Therefore, 90 matched serum and oral fluid samples were collected from volunteers with different immune status, under ideal conditions of collection (optimization panel); and 224 matched samples in difficult-to-access areas (epidemiological study). Serum was collected by venipuncture and the oral fluid was obtained using three commercial devices: Salivette(®), OraSure(®) and ChemBio(®). Serum and oral fluid were submitted to a commercial immunoblot to detect total anti-HAV antibodies. The optimization panel demonstrated that ChemBio(®) device had the best performance (100% agreement), followed by OraSure(®) (95.4%) and Salivette(®) (90.8%). The optimal collection device (ChemBio(®)), tested in a difficult-to-access area and evaluated under precarious conditions of collection, showed similar prevalence of total anti-HAV between serum and oral fluid, 80.8% and 79%, respectively. A follow-up was performed to evaluate the stability of oral fluid and it was observed that 210 days after the collection it was possible to detect anti-HAV antibodies. Oral fluid can be used to detect low levels of specific-antibody, being important to select age groups to be vaccinated. Therewith, the choice of proper collection device is essential to evaluate HAV antibodies in the epidemiological scenario.

Importance of the cutoff ratio for detecting antibodies against hepatitis A virus in oral fluids by enzyme immunoassay.

Multiple studies have examined the use of oral fluids in modified serum-based assays aiming to replace serum in antibody detection for hepatitis A. However, the reliable detection of HAV immunity in oral fluid requires an extremely sensitive assay; most immunoassays designed for serum antibody determination lack sufficient sensitivity for this purpose. Consequently, an "in-house" competitive enzyme immunoassay (EIA) designed specifically for use with oral samples collected using a ChemBio(®) device was developed to detect total anti-HAV antibodies (IgG and IgM). This system was compared to an in-house competitive EIA and a commercial EIA considered to be the "gold standard" using corresponding serum samples (n=225) to determine the accuracy of the assay and to evaluate the importance of the cutoff ratio for the detection of anti-HAV antibodies in oral fluids. When the median serum cutoff and the optimal oral fluid cutoff (ROC analysis) obtained from the in-house competitive EIA were compared, the oral fluid cutoff was found to be 28.8% higher than the serum cutoff. When different oral fluid cutoff values were compared, a reduction of about 17% was shown to be essential to increase test accuracy. At an oral fluid cutoff value of 0.351, sensitivity and specificity were higher, reaching 91.7% and 86.2% (p<0.001, AUROC=0.915), respectively. The convenience, accuracy and non-invasive nature of the developed method make it a useful alternative to serum-based assays for discriminating between HAV-immune and non-immune individuals.