Test for the virus: antigen, viral DNA and RNA, subtypes, mutants

      Viral antigens are present in serum, in particular the HIV core antigen. This is only detectable for as long as it is in excess of antibody, typically at the outset of infection. Tests for this HIV antigen are commercially available, and they assist in the diagnosis of early infection and the recognition of infection in infants. In practice, however, tests for HIV antigen have proved of limited value due to lack of sensitivity, although this
may be enhanced by preliminary acid or alkali dissociation of immune complexes in the specimen. Viraemia may also be recognised by isolation of HIV from plasma in cultured lymphocytes, but this is time consuming and not especially sensitive. Essentially it has become a research tool.
    HIV can also be detected in specimens in the form of genome sequences. Though only rare lymphocytes carry the HIV genome, the polymerase chain reaction (PCR) can be used greatly to amplify chosen HIV genome sequences in those clinical specimens that contain these small numbers of infected lymphocytes. To a large extent, therefore, viral culture has been superseded by PCR amplification of HIV DNA extracted from
mononuclear cells in the circulation. Even more commonly, reverse transcription and amplification of HIV RNA is now being used to detect and quantify virus present in blood. While these procedures are no more accurate than anti-HIV assays and much more expensive, they may be useful in diagnosis, for example in infancy when any anti-HIV detected may be of maternal origin. PCR amplification also provides rapid access to the HIV genome and can lead to characterisation of an HIV isolate to strain level. The (semi) quantification of viraemia (i.e. to within about 0.5 log10 ) is an important determinant of the need for, and the effect of treatment. It is especially useful as the choice of antiviral combinations widens.
    Targets for genome amplification include the genes coding for the main envelope, core and transcriptase proteins. On the basis, particularly, of analysis of the sequences of amplified sections of the envelope gene, HIV-1 has been subtyped – so far from A to K. In some cases the sequences found in the various HIV genes are not concordant, showing that recombination occurs in HIV.

Simple and non-invasive tests, confirmatory tests, follow-up tests

     Simple anti-HIV screening tests have been developed for use in clinics, in unfavourable laboratory conditions and close to the patient. When results are needed urgently, for instance before transplantation procedures and to select a blood donor in the field, they are quick and practical. Saliva (oral fluid) and urine can conveniently be used as specimens to investigate for anti-HIV when venepuncture is difficult, hazardous or unacceptable to the patient. These simple rapid and non-invasive tests are attractive options and may lead to developments such as home testing. However, few of these tests are quite as accurate as the conventional assays on serum, and follow-up confirmatory tests are essential before a positive diagnosis is made by these means.
     In many countries, including the UK, formal procedures have been put in place to secure accurate testing. The most important is that when there is a positive anti-HIV finding the test is repeated and the implicated specimen is tested by other, methodologically independent, anti-HIV assays.

     Another specimen should then be sought. Although this may cause some delay in confirming a positive finding, anti-HIV testing is as a consequence more precise. A few infected individuals may have little or no detectable anti-HIV when first tested or there may have been technical or clerical mistakes, including specimen misidentification and transcription errors. Follow-up at an interval of one to four weeks greatly diminishes the chance of either a false negative or a false positive anti-HIV result, and follow-up specimens are the most important element in the accurate laboratory diagnosis of HIV infection. When newly infected individuals are followed up, they show an increase in the titre and range of HIV antibodies. By contrast, persistently weak anti-HIV reactions are usually non-specific. Sometimes PCR (see below) will resolve a difficult-to-confirm antibody reaction. Follow-up procedures also guard against specimen misidentification and transcription errors.

Tests for anti-HIV-1 and HIV-2

     Anti-HIV tests have transformed our understanding of the epidemiology of AIDS in the years since they were introduced in 1984, and they are still the bedrock of clinical diagnosis and much epidemiological research. Anti-HIV appears three weeks to three months after exposure to HIV and thereafter is invariably detectable in spite of any detrimental effect the virus may have on lymphocyte function and therefore antibody production. Neutralising antibodies to HIV are also measurable, but their titres are low. An inability to mount a neutralising response to HIV antigens together with the mutability of the virus are the most likely reasons why conventional approaches to preparing a vaccine have so far failed.

At first HIV antigen was prepared from infected cell lines. However, antigens can now be made by DNA cloning and expression or by synthesis of viral polypeptides. Several types of anti-HIV test exist, but most use a similar enzyme conjugate and give a colour signal due to the reaction between an enzyme specifically bound onto a polystyrene surface, membrane or inert particles and a substrate that then changes colour. Other tests depend on the binding of a fluorescein or chemiluminescent conjugate, or the visible agglutination of HIV-coated gelatin or latex particles.

Since anti-HIV tests became commercially available in 1985 they have been widely used in diagnostic and transfusion laboratories in the developed world. The accuracy – both sensitivity and specificity – of the antibody assays is continually being improved, and in competent hands the occurrence of false positive and false negative results is less and less frequent. The proportion of true to false positive results depends on the
population studied, but even in low risk groups such as volunteer blood donors it is now very high in well conducted laboratories. Human, not test, errors cause most false results, and the key to avoiding these mistakes is continuous review with repeat testing where necessary. All positive reactions should both be confirmed by additional assays and succeeded by a test on a follow-up specimen (see below). The use of several screening tests in parallel on proven positive specimens also acts as a check on the possibility of false negativity in these assays (which it is otherwise difficult to guard against).

More discriminating tests can recognise the components of the antibody response. The serological response to individual HIV proteins can be studied by Western blot, and the immunoglobulin class response to HIV in blood and other fluids can also be investigated. The IgM response slightly proceeds the IgG response early in infection and is indicative of recent infection. Other test procedures, which employ both a highly sensitive and a “detuned” assay for anti-HIV are designed to detect infection within the previous few months and may
therefore be used epidemiologically to measure incidence. The IgA anti-HIV response is a feature of infection in infancy.

Transmission of HIV infection

HIV-1 and HIV-2, the major and minor human AIDS viruses,
are transmitted in ways that are typical for all retroviruses –
“vertically” – that is from mother to infant, and “horizontally”
through sexual intercourse and through infected blood. The
lymphocytes of a healthy carrier of HIV replicate, and
eliminate, over one billion virions each day and the circulating
virus “load” may exceed ten million virions per millilitre. At
these times viraemia can be recognised by measuring the p24
antigen of HIV in blood and quantifying viral DNA or RNA
(see below). Transmission also depends on other factors,
including the concentration of HIV secreted into body fluids
such as semen, secondary infection of the genital tract, the
efficiency of epithelial barriers, the presence or absence of cells
with receptors for HIV, and perhaps the immune competence of
the exposed person. All infections with HIV appear to become
chronic and many are continuously productive of virus. The
ultimate risk of spread to those repeatedly exposed is therefore
high.
The stage of infection is an important determinant of
infectivity. High titres of virus are reached early in infection,
though this phase is difficult to study because symptoms may be
mild or absent and any anti-HIV response undetectable; it is
nevertheless a time when an individual is likely to infect
contacts. When, much later, the cellular immune response to
HIV begins to fail and AIDS supervenes the individual may
again become highly infectious. In the interval between, there
may be periods when except through massive exposures – for
example blood donation – infected individuals are much less
infectious. Nevertheless, in the absence of reliable markers of
infectivity, all seropositive individuals must be seen as
potentially infectious, even those under successful treatment.
Effective ways are constantly being sought to protect their
contacts and this has led to the development of the concept of
“safe sex”. Ideally, this should inform sexual contact between all
individuals regardless of whether they are known to be infected
with HIV.