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Diagnostic testing for bloodborne viruses

Ian Collacott
Virology Scientific Advisory Panel
Institute of Biomedical Science

Bloodborne viruses (BBVs) are associated with illnesses transmitted by blood and body fluids that may be given therapeutically, or exchanged by sharing needles. They are also transmitted sexually. Accidental infections may occur from sharing razors, toothbrushes, and by contact between broken skin or mucous membranes and virus-laden fluids. The viruses commonly considered as BBVs include:

  • Hepatitis B virus (HBV) and the associated hepatitis D virus (HDV).
  • Hepatitis C virus (HCV).
  • Human immunodeficiency virus (HIV).

All these viruses cause significant morbidity and mortality. An important factor in the probability of severe long-term consequences of infection is the propensity of BBVs to establish a carrier state in hosts. HBV infection of adults may lead to chronic carriage in 5–10% of individuals, but HBV may also be transmitted vertically (mother to baby) and chronic infection occurs in up to 90% of children infected around the time of birth. HDV depends on HBV for replication, and may infect victims at the same time as HBV or infect HBV carriers  – this superinfection carries a very high risk of chronicity and severe liver damage. HCV causes chronic infection in around 80% of victims, and HIV seems to invariably establish a chronic infection.

Numbers of infected grows
Recent figures for the global burden of disease caused by BBVs may be found on the World Health Organisation (WHO) website ( topics/ en). HBV is estimated to infect around 5.7 million people per annum, causing up to one million deaths each year. Around 350 million people are chronically infected, and an estimated two billion people show evidence of past infection. Worldwide, vertical transmission is probably the most important source of chronic infections. There are an estimated 180 million carriers of HCV, with three to four million new infections each year, and the virus is believed to be responsible for up to 75% of all liver cancers.

The ongoing HIV epidemic imposes a disproportionate burden on developing countries, particularly in sub-Saharan Africa, with around 40 million infected worldwide (2.5 million aged less than 15), five million new infections and three million deaths each year.

There is an effective immunisation available against HBV, and this vaccine also protects against HDV infection. The main obstacles to universal childhood immunisation – and therefore global control or eradication of HBV – are economic. The countries where HBV is most common are mainly developing nations that lack the resources to initiate and support vaccination programmes. However, there are no effective vaccines available against HCV and HIV, and the genetic variability of these viruses means that development of effective vaccines is complex and may lie in the distant future.

Laboratory testing
Testing for BBV is an important area of work for laboratories. Tests are used for screening purposes; for example, in blood donors and antenatal patients, or for diagnosis in patients with symptoms such as liver disease, immunodeficiency or an appropriate exposure history. In resource poor countries, laboratory testing may be very limited. There are two main technologies used in testing laboratories. Serological immunoassays are used to detect protein antigens (present on virus particles), or antibodies produced by patients in response to infection. Molecular methods amplify viral genetic material to detect BBV with exquisite sensitivity, and also allow quantitative estimates of the amount of virus circulating in the blood (“viral load”). The underlying technological principles are very similar for each virus.

At present, molecular assays are significantly more expensive and complex than immunoassays. They are less easily and less highly automated, and test throughput is smaller. However, these assays are essential for effective treatment and monitoring, and will become far more widely used than at present. Some blood transfusion services are now using molecular diagnostics to screen pooled blood donor samples, in addition to performing immunoassays.

Detection of infection
For HBV diagnosis, the most commonly employed and most useful assay detects the viral coat protein, hepatitis B surface antigen (HBsAg). This protein is a marker of active infection and potential infectivity. Other assays, chiefly for different antibody markers, allow indirect assessment of infectivity and past infection or immunity. Figure 1 shows a typical acute infection with clearance of virus. Chronic HBV infection is defined as the presence of HBsAg in a patient’s blood for more than six months. In such cases, HBsAg does not decline – as shown in the diagram – but remains detectable. Anti-HBs, which correlates with recovery from infection, does not develop.


Graph reproduced courtesy of CDC

HCV is normally diagnosed by detection of antibodies against the virus. An antigen assay is available, but virus detection by molecular methods is much more sensitive. Figure 2 shows the most common serological pattern of infection, with HCV leading to chronic infection and possible cirrhosis or hepatocellular carcinoma years later.


Graph reproduced courtesy of CDC

HIV is now most frequently diagnosed with “combo” assays, which detect the presence of both antibodies against HIV and HIV antigens in blood samples. These “fourth-generation” HIV assays are now the norm, replacing older antibody-only assays and detecting HIV infections earlier.

The most widely used immunoassay format is enzyme-linked immunosorbent assay, known as EIA. A variety of commercial assays are available, ranging from simple rapid test devices aimed at near-patient settings or developing countries, to microtitre plate methods and automated random access analysers able to test large numbers of samples in a short time.

The basic principle of most immunoassays is very similar. A “solid phase”, which may be a microparticle or microwell, is coated with antibodies (in assays detecting antigen) or antigens (in assays detecting antibodies). Patient serum samples are added, and antigen or specific antibodies are bound by the solid phase. Unbound reactants are washed away and a conjugate in the form of an enzyme-labelled antibody or antigen is added. If the desired analyte was bound in the first reaction, the conjugate will specifically bind during this second stage. Finally, an enzyme substrate is added. Specific reactions in the first two stages of the assay will result in metabolism of the substrate with a colour change or light emission, which can be measured and recorded. Many laboratories now use sophisticated instrumentation to allow automation with high sample throughput and positive sample identification and host computer interfacing. Such instruments are available from a range of manufacturers, including Abbott, Bayer and Beckman Coulter. These instruments cut across traditional disciplines by offering assays for biochemistry, virology and haematology on a single machine.

Nuclear acid test
Molecular assays, or nucleic acid tests (NATs), use differing methods but all are essentially variations on a theme. A sample extraction procedure purifies nucleic acid from patient samples and removes potentially inhibitory substances. Extraction procedures may be done manually  – which is very labour-intensive for anything other than a small number of samples – or may be automated. If the organism has an RNA genome such as HCV or HIV, an initial “reverse transcription” stage that copies RNA to DNA is necessary. If the organism has a DNA genome, such as HBV, amplification may be started directly from an extracted sample.

In the polymerase chain reaction (PCR), a reaction mix consisting of the extracted patient sample, nucleotides and enzymes is repeatedly cycled through a range of temperatures to allow amplification of a “target” sequence. Extracted samples are heated to denature DNA and cause the normal double-stranded structure to split into two single strands. The complementary primers in the reaction mix bind to the target sequences, and DNA polymerase extends the double-stranded sequence. Repeated cycles of heating to denature, cooling, and holding at an intermediate temperature – usually around 60°C – to allow extension, result in an increase in target sequences. This amplification is logarithmic and, after a number of cycles, the vast majority of DNA sequences present in the reaction mix are copies of the target sequence.

This sequence may be detected by various methods, such as fluorescence (in real-time PCR) or on a gel. Successful testing depends on a number of factors. Because amplification methods are exquisitely sensitive, stringent attention to good technique is essential. Contamination, leading to false-positive reactions, is well documented. The target sequence must be well chosen. Ideally, it will represent a highly conserved genome sequence that is present in all variants of the organism being sought, and significantly different to sequences present in other organisms. Primers are commonly designed using software that can search for appropriate genomic sequences.

Molecular methods allow efficient monitoring of disease progression, and response to treatment. In the case of HIV infection, treatment using highly active antiretroviral therapy (HAART) depends on regular monitoring of the viral load by NAT methods. Increased use of NATs and technological innovation is likely to reduce test costs and simplify test procedures. Future challenges are twofold: to maximise the use of automation and NATs, and to deliver the necessary level of testing in resource-poor countries as well as in wealthy countries.