Hepatitis B virus (HBV) is one of the major causes of chronic liver disease worldwide; however most of individuals are not aware about the infection. Oral fluid and dried blood spot (DBS) samples may be an alternative to serum to HBV diagnosis to increase the access to diagnosis in remote areas or high-risk groups. The main objective of this review is to give an insight about the usefulness of oral fluid and DBS for detecting HBV markers. Several groups have evaluated the detection of HBsAg, anti-HBc, and anti-HBs markers in oral fluid and DBS samples demonstrating 13 to 100% of sensitivity and specificity according different groups, sample collectors, and diagnosis assays. In the same way, HBV DNA detection using oral fluid and DBS samples demonstrate different values of sensitivity according type of collection, studied group, extraction, and detection methods. Thus, serological and molecular diagnostic tests demonstrated good performance for detecting HBV using oral fluid and DBS according some characteristics and could be useful to increase the access to the diagnosis of HBV.
Hepatitis B virus (HBV) infection is responsible for acute and chronic cases all over the world. Diagnosis of infection is made by detection of serological (HBsAg, HBeAg, anti-HBe, anti-HBc, anti-HBc IgM, and anti-HBs) and molecular markers (HBV DNA) [
Enzyme immunoassay (EIA), electrochemiluminescence (ECLIA), chemiluminescence immunoassay (CLIA), microparticle enzyme immunoassay (MEIA), radioimmunoassay (RIA), and rapid assays have been used for serological diagnosis [
Molecular methods for detection and quantification of HBV DNA are important for (1) diagnosis of hepatitis B infection; (2) evaluation of disease prognostic evaluation the risk to cirrhosis and cancer risk; (3) defining the beginning of antiviral treatment; (4) monitoring antiviral treatment and identifying resistance to nucleus (t)ide analogues [
For the diagnosis of hepatitis B, serological and molecular detection are commonly performed using serum or plasma samples, which can be difficult in remote areas with scarce resources and potentially painful among individuals like drug users and patients under hemodialysis, obese, and elder people. However, alternative body fluids such as oral fluid and dried blood spots (DBS) have been studied as alternative fluids to serum for epidemiological and molecular diagnosis of HBV. Therefore, the objective of this review is to give new insight about serological and molecular diagnosis of HBV using oral fluid and DBS as alternative biological samples.
Saliva is a body fluid comprising secretions from glands fed by the vasculature of the body, has an important role in maintaining oral health, with antiviral and antibacterial activity, in the lubrication and repair of oral mucosa, in the palate, and in a digestion, and is slightly acidic and composed of small organic substances, proteins, peptides, and polynucleotides [
There are two important aspects that should be considered to collect saliva: the type (total or gland-specific) and the level of stimulation (stimulated or unstimulated) [
The diagnosis of some diseases using saliva or oral fluid samples is simple and noninvasive, being safe for both the professional and the patient. These characteristics make the collection of this fluid very attractive in children, the elderly and other individuals with difficult venous access [
The first reports for detecting HBV markers in saliva or oral fluids are from the decade 70’s [
Main Characteristics of studies that detect HBV antigen and antibodies in saliva or oral fluid samples.
Marker | Population | Country | Oral fluid device or sample collection | Assay | Sensitivity | Specificity | Reference |
| |||||||
HBsAg | Local and out-of-state public health clinics | USA | Orasure | Commercial Enzyme immunoassay | 100.0% | 100.0% | [ |
Anti-HBc IgM | Local and out-of-state public health clinics | USA | Orasure | Commercial Enzyme immunoassay | 100.0% | 100.0% | [ |
HBsAg | Patients with and without HBV | Thailand | Whole saliva | Commercial Enzyme immunoassay | 96.5% | 100.0% | [ |
Anti-HBc | Rural community | Ethiopia | Sponge swab | Commercial Enzyme immunoassay | 43.0% | 87.0% | [ |
Anti-HBc | blood donors and injecting drug users | Denmark | Omni-Sal | Commercial Enzyme immunoassay | 85.9% | 100.0% | [ |
HBsAg | Unit of Gastroenterology of three university hospitals | Belgium | Oracol | Commercial Enzyme immunoassay | 90.7% | 100.0% | [ |
Anti-HBc Total | Viral Hepatitis ambulatory | Brazil | Orasure | Commercial Enzyme immunoassay | 13% | 100.0% | [ |
Anti-HBc IgM | Viral Hepatitis ambulatory | Brazil | Orasure | Commercial Enzyme immunoassay | 100.0% | 100.0% | [ |
Anti-HBs | Viral Hepatitis ambulatory | Brazil | Orasure | Commercial Enzyme immunoassay | 8.33% | 100.0% | [ |
HBsAg | Viral Hepatitis ambulatory | Brazil | Salivette | Commercial Enzyme immunoassay | 85.1% | 94.1% | [ |
Whole saliva | 93.6% | 92.6% | |||||
HBsAg | Viral Hepatitis ambulatory and Pantanal of Mato Grosso do Sul | Brazil | Chembio | Commercial Enzyme immunoassay | 95.2% | 100% | [ |
78.3% | 89.9% | ||||||
Salivette | |||||||
HBsAg | Patients with and without HBV | India | Whole saliva | Commercial Enzyme immunoassay | 100% | 100% | [ |
HBsAg | Alcoholics | Brazil | Salivette | Commercial Enzyme immunoassay | Not available | 100% | [ |
HBsAg | Viral Hepatitis ambulatory | Brazil | Salivette | Commercial Enzyme immunoassay | 80.9% | 86.8% | [ |
Anti-HBc | 82.4% | 96.9% |
Some studies evaluated the detection of HBsAg in saliva and found that saliva could be used as an alternative sample for serum to identify HBV carriers [
Spitting samples were evaluated for the presence of HBsAg in acute HBV patients in Russia. Authors showed correlation between HBsAg detection in spitting and serum samples, but HBsAg were not detected earlier in saliva than serum in serial samples. They also observed high frequency of HBeAg in saliva compared to serum suggesting that saliva could be a source of HBV diagnosis and treatment monitoring [
Nowadays some oral fluid collection devices are commercialized, such as: Oracol (Malvern Medical Developments), Chembio (Chembio Diagnostic Systems, New York, USA), OraSure (OraSure Technologies, USA) and Salivette (Sarstedt, Germany). Some studies used these collectors to obtain oral fluid and compared to serum results for detecting HBsAg marker where values of sensitivity varied from 62% to 100% and specificity varied from 78.0% to 100% [
High values of sensitivity and specificity were found for detecting HBV markers using Orasure, Oracol and Chembio collectors. HBsAg detection using oral fluid obtained by Orasure Collector and EIA with adaptations (increase of sample volume) demonstrated sensitivity and specificity of 100% [
Studies evaluating Salivette device observed lower sensitivity and specificity values compared to other devices for detecting HBV markers. Salivette and EIA without modifications demonstrated sensitivity from 78% to 92.0% and specificities from 86.8% to 89.9% [
Anti-HBc IgM to identify acute HBV was also tested in samples obtained by spitting and Salivette device where detectable anti-HBc IgM were found in oral fluid, but the number investigated (8) was insufficient to draw firm conclusions on the usefulness of salivary IgM anti-HBc detection for diagnosing recent HBV infection [
The studies concerning HBV DNA detection in oral fluid samples started in the years 2000. A study conducted by Noppornpanth and colleagues [
Some authors evaluated detection of HBV DNA using in-house real-time PCR methodologies and spitting samples. In the study of Van der Eijk et al. [
More recently, spitting samples from 50 chronic carriers were submitted to a commercial method for HBV amplification and quantification (Roboscreen GmbH/Analytik Jena Group) followed by DNA extraction with QIAamp DNA Kit (Qiagen; Venlo, Limburg, The Netherlands). In this study, the sensitivity of HBV DNA detection in saliva was 68% and mean viral load among oral fluid samples was 3.8x104 ± 5.42x104 copies/mL when viral load is > 107 in paired serum samples [
Some studies suggest that collectors of oral fluid that uses mechanical friction to the mouth mucosa present higher efficiency. In 2010, Heiberg and colleagues [
Oral fluid samples collected with Salivette were also evaluated for detection of HBV DNA among patients with occult hepatitis B infection (OBI) [
Information regarding these studies are compiled and described in Table
Main characteristics of studies that detect HBV-DNA in saliva or oral fluid samples.
Reference | Type of saliva or collection device | Population | Serum HBsAg or HBV | Molecular method | Sensitivity of HBV DNA |
---|---|---|---|---|---|
Detection in oral fluid | |||||
DNA positive subjects (n) | |||||
[ | Whole saliva | Thailand HBV carriers | 23 | In-house qualitative PCR | 47.8% |
[ | Whole saliva | Siberian military men | 42 | In-house qualitative PCR | 46.2% |
[ | Whole saliva | HBV chronic carriers | 147 | In-house real-time PCR | 47% |
[ | Whole saliva | HBV chronic carriers | 23 | In-house real-time PCR | 21.7% |
[ | Whole saliva | HBV chronic carriers | 200 | In-house real-time PCR | 72.5% |
[ | Oracol | Children with chronic HBV | 46 | In-house real-time PCR | 92% |
[ | Whole saliva | HBV chronic carriers | 50 | Commercial PCR | 68% |
[ | Salivette, FTA Cards, DNA-Sal and whole saliva | HBV chronic carriers | 32 | In-house qualitative PCR | 53.12% |
[ | Salivette | HBV chronic carriers | 55 | In-house real-time PCR | 18.2% |
[ | Salivette | Occult HBV carriers | 5 | In-house qualitative PCR | 80% |
Some studies have reported the use of DBS for diagnostic screening tests for human immunodeficiency markers of virus (HIV) and hepatitis virus [
Despite the numerous benefits, it is worth to mention that the use of this type of material present some difficulties. The small amount of sample disposed in the filter paper cards may limit the number of analyzes to be performed on that material if the collection has not been satisfactorily performed. The DBS drying process may interfere with subsequent analyzes of the sample, since dried blood can carry disrupted cells and a large amount of cellular constituents and may modify the biochemical structure of the molecule to be quantified (antibodies, for example). In addition, it is not easy to adapt commercial tests designed to serum or plasma analysis to be used on DBS samples. In addition, the concentration of the substances in serum and whole blood differ, which may raise the need to propose a correction factor that approximates the quantitative results obtained in the DBS from the serum values of paired samples in cases where the serum analysis is the “gold standard” diagnosis [
It is understood that the use of DBS in the diagnosis of infectious diseases is possible but must undergo rigorous studies of validation and evaluation of sensitivity and specificity that also involve a good knowledge of the target microorganism [
Diagnosis of HBV is usually performed on blood samples obtained by venous puncture. However, obtaining this sample is difficult in some cases, as in regions where there is no access to an appropriate laboratory. In addition, the transport of serum or plasma samples from remote areas to central laboratories is also complicated. Because of this, it is important to use alternative biological samples capable of providing an adequate diagnosis.
Initial studies using DBS to detect HBsAg, HBeAg, and anti-HBs occurred in the 1970s. In this period, the positive aspects of the use of this strategy, including results comparable to those obtained from serum samples, were already emphasized [
Detection of HBV antibodies and antigen, such as HBsAg, anti-HBc and anti-HBs by enzyme immunoassays (EIAs) can also be obtained through DBS samples [
Main characteristics of studies that detect HBsAg, anti-HBc, and HBsAg in DBS compared to serum.
Marker | Population | Country | Type of Assay | Specificity | Sensitivity | Reference |
---|---|---|---|---|---|---|
HBsAg | Pregnant women | Brazil | Enzyme Immunoassay | 100% | 100% | [ |
HBsAg | Unclear | UK | chemiluminescent microparticle immunoassay | 100% | 98% | [ |
HBsAg | Inpatients | Germany | Not specified | 99.8% | 91.7% | [ |
HBsAg | Patients attending HCV clinic | Brazil | Enzyme Immunoassay | 97% | 98% | [ |
HBsAg | Unclear | Germany | chemiluminescent microparticle immunoassay | 100% | 99% | [ |
HBsAg | Prospective patients from hepatitis clinic and blood donors | France | chemiluminescent microparticle immunoassay | 98% | 100% | [ |
HBsAg | Attendees of HIV testing center | Burkina-Faso | Rapid test, Enzyme Immunoassay, chemiluminescent microparticle immunoassay | 100% | 96% | [ |
HBsAg | Broad | Gambia | Rapid test | 100% | 96% | [ |
HBsAg | Patients at a tertiary hospital | Malaysia | chemiluminescent microparticle immunoassay | 98% | 97% | [ |
HBsAg | Unclear | US | chemiluminescent immunoassay | 100% | 100% | [ |
Anti-HBc | Unclear | UK | chemiluminescent microparticle immunoassay | 100% | 100% | [ |
Anti-HBc | Patients referred to the Viral Hepatitis Clinic | Brazil | Enzyme Immunoassay | 92.6% | 90.5% | [ |
Anti-HBc | Patients referred to the Viral Hepatitis Clinic | Brazil | Enzyme Immunoassay | NR | 90.4% | [ |
Anti-HBc | Unclear | Germany | chemiluminescent microparticle immunoassay | 100% | 97% | [ |
Anti-HBs | Hospital | Malaysia | chemiluminescent microparticle immunoassay | 86.9% | 74,2% | [ |
Anti-HBs | Prospective patients from hepatitis clinic and blood donors | France | chemiluminescent microparticle immunoassay | 100% | 98% | [ |
Anti-HBs | Unclear | Germany | chemiluminescent microparticle immunoassay | 100% | 97.5% | [ |
Anti-HBs | Patients referred to the Viral Hepatitis Clinic | Brazil | Enzyme Immunoassay | 97.3% | 78% | [ |
The studies for detecting HBV in DBS evaluated several factors, such as: size of disc paper used, ranging from 6mm-12mm, sample amount (50
A factor highlighted in several studies was the cut-off value for determining the positive and negative samples; this value could be higher for DBS samples than for plasma or serum samples. The volume of DBS eluate was also considered, mostly between 50
Several studies have demonstrated agreement in the detection of HBV markers between DBS samples and serum samples in EIAs. Cruz
In a meta-analysis, 23 studies were included and assays such as Enzygnost (Behring) and ARCHITECT (Abbott) showed HBsAg sensitivity and specificity of 98% (95% CI: 95%-99%) and 100% (95% CI: 99-100%), respectively. Other studies demonstrated sensitivity and specificity values to detect HBsAg in DBS from 78.6 to 98.6% and 88.6 to 100.0%, respectively [
In the evaluation of HBsAg in DBS in the presence of HIV coinfection, Flores et al. [
Anti-HBc marker that indicates previous exposure to HBV was also evaluated under various parameters such as disc sample size, sample eluate, cut-off value, and storage temperature [
Other studies also evaluated the detection of anti-HBc in DBS and presented sensitivities and specificities ranging from 90% to 97% and 92.6% to 100%, respectively [
A recent study evaluated the prevalence of anti-HBc in prisoners in Scotland using DBS along to Architect Hepatitis B core II antibody test [
Some studies also evaluated anti-HBs detection in DBS samples and found sensitivity and specificity of 74.2 to 97.5% and 86.9 to 100%, respectively [
Lee et al. [
As previously mentioned, Gupta innovated HBV diagnosis when proposed the role of DBS for detecting HBV DNA [
In 2004, Jardi and colleagues [
In 2009, Lira and colleagues evaluated the utility of DBS samples for monitoring HBV infection in patients from Mexico City [
After 2009, several studies evaluated DBS to detect and quantify HBV DNA [
Two years later, in 2015, a research group evaluated the application of DBS to HBV DNA quantification in coinfected samples (HBV+ and HIV+) [
In 2016, in Ethiopia, Stene-Johansen and colleagues [
In 2016, Mossner and colleagues [
Main characteristics of studies of detection of HBV-DNA from DBS samples compared to serum or plasma.
Reference | Population | Country | DNA Extraction Method (DBS) | Amplification Method | Sensitivity (%) | Specificity (%) |
---|---|---|---|---|---|---|
[ | HBsAg positive serum + negative whole blood | India | Not performed | Simple PCR (core gene) | NR | NR |
[ | HBsAg positive patients (82) | Spain | QIAamp mini columns | Real Time PCR (core gene) | 88 | 100 |
[ | HBsAg positive patients (47) | Mexico | QIAamp® DNA micro kit | Cobas Amplicorv1.5 | 100 | NR |
[ | 60 patients with undetermined serological Status | China | Chelex-100 | Nested PCR (pre-core/core gene) | NR | NR |
[ | HBsAg positive patients (50) | Egypt | Not performed | Direct amplification (KAPA blood PCR Kit) | 100 | 100 |
HBsAg negative patients (10) | ||||||
[ | HBV DNA positive patients (100) | Germany | MagNaPure 96 system using Viral NA Universal kit | Artus HBV LC PCR | 93 | 100 |
HBV DNA negative patients (50) | ||||||
[ | HBV DNA positive patients (50) | France | FTA purification reagent | Cobas Taqman HBV test V2.0 | 98 | NR |
HBV DNA negative patients (10) | ||||||
[ | HBsAg positive patients (26) | Congo | COBAS® | COBAS® AmpliPrep/COBAS® Taqman® HBV test v1.0 | 96 | NR |
[ | HBV DNA positive patients (100) | Germany | Not informed | Not informed | 93 | NR |
[ | HBV DNA positive and HIV-infected patients (68) | Zambia | Pre-extraction buffer | AmpliPrep/COBAS TaqMan HBV test | 91 | NR |
[ | HBV viral load value range from 2.14 log to | Ethiopia | Abbott RealTime HBV assay on sp2000 extractor | rt2000realtime PCR instrument | 88 | NR |
[ | Hepatitis B-infected patients (85) | Denmark | Not performed | Ultrio Elite assay | 97.6 | NR |
Blood donors negative for HBV infection (99) |
HBV antigen, antibodies, and DNA could be detected in DBS and oral fluid samples using different devices and detection methods. Different values of sensitivity and specificity were found for HBV detection in saliva or oral fluid samples according type of collection device, EIA, extraction, and PCR method. It is interesting to observe that HBeAg status and HBV DNA presence in serum are positively related to HBV DNA detection in oral fluid. As well as HIV status or antiretroviral treatment could interfere in the performance of HBsAg and anti-HBc detection in oral fluid.
With respect to DBS, HBV detection was made using different sizes of disc paper, elution volume, and cut-off values. For each marker, it is important to optimize the assay. Sometimes dilution factors were employed to approach quantitative results obtained in DBS from that one acquired at the standard samples. For molecular HBV detection in DBS, high sensitivity and more accurate results were found using commercial methods. However, commercial methods are expensive which can hamper the employment of these tests in low resource areas. Despite this, studies show that HBV DNA could be detected using molecular methods.
The authors did not present conflicts of interest.
This work was supported by the FAPERJ, CAPES, and CNPQ.