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 misidentifications 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.
Showing posts with label The Virus and the Test. Show all posts
Showing posts with label The Virus and the Test. Show all posts
Tests for anti-HIV-1 and HIV-2
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The Virus and the Test
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 pidemiologically to measure incidence. The IgA anti-HIV response is a feature of infection in infancy.
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 pidemiologically to measure incidence. The IgA anti-HIV response is a feature of infection in infancy.
The Virus and the Test
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The Virus and the Test
Although it is clear that HIV is the underlying cause of AIDS and AIDS-related disease, its origin remains obscure. There is firm serological evidence of infection on the east and west coasts of the USA from the mid 1970s, and HIV infection in central Africa may have antedated infection in North America.
Phylogenetic analysis of the HIV-1 genome has suggested an origin in chimpanzees while, in the case of HIV-2, similarity to the simian immunodeficiency virus (SIV) genome may point to an origin in sooty mangabey monkeys. In both cases the butchery and consumption of these “bush meats” has been incriminated in transmissions to the human host. Like some other RNA viruses, HIV appears to have mutated and shifted its host range and virulence, explaining how a new pathogenic retrovirus could arise in man. Its virulence may since have been amplified as a result of travel, population dislocation and promiscuous sexual contact, with rapid passage of the virus.
Retroviruses are so named because their genomes encode an unusual enzyme, reverse transcriptase, which allows DNA to be transcribed from RNA. Thus, HIV can make copies of its own genome, as DNA, in host cells such as the human CD4 “helper” lymphocyte. The viral DNA becomes integrated in the lymphocyte genome, and this is the basis for chronic HIV infection. Integration of the HIV genome into host cells is a formidable obstacle to any antiviral treatment that would not just suppress but also eradicate the infection. Nevertheless, modern treatment with combinations of nucleoside analogues and protease inhibitors has transformed the prognosis for carriers of HIV, usually achieving a sustained fall in virus concentration in blood and restoration of the main target cell (CD4 lymphocyte) to near normal levels.
By contrast, the inherent variability of the HIV genome and the failure of the human host to produce neutralising antibodies to the virus, as well as technical difficulties and concerns about safety, have continued to frustrate attempts to make an effective vaccine. This must not, however, allow efforts to develop and evaluate candidate vaccines to slacken. A particular concern is that a useful candidate vaccine (probably a recombinant envelope vaccine developed in North America or Europe against the locally prevalent HIV-1 B subtype) would be ineffective in those parts of the world where other subtypes predominate.
WHO estimates that in the year 2000 there are 36 million carriers of HIV worldwide, and only a small fraction of them have access to suppressive treatment. Both their contacts, their dependants and possibly they themselves would have their life prospects transformed by an effective, or even partially effective, vaccine, and successful application of antiviral treatment in developed countries should in no way be allowed to deflect attention from the necessity of developing and delivering an effective vaccine and of promoting “safe sex” behaviour.
Phylogenetic analysis of the HIV-1 genome has suggested an origin in chimpanzees while, in the case of HIV-2, similarity to the simian immunodeficiency virus (SIV) genome may point to an origin in sooty mangabey monkeys. In both cases the butchery and consumption of these “bush meats” has been incriminated in transmissions to the human host. Like some other RNA viruses, HIV appears to have mutated and shifted its host range and virulence, explaining how a new pathogenic retrovirus could arise in man. Its virulence may since have been amplified as a result of travel, population dislocation and promiscuous sexual contact, with rapid passage of the virus.
Retroviruses are so named because their genomes encode an unusual enzyme, reverse transcriptase, which allows DNA to be transcribed from RNA. Thus, HIV can make copies of its own genome, as DNA, in host cells such as the human CD4 “helper” lymphocyte. The viral DNA becomes integrated in the lymphocyte genome, and this is the basis for chronic HIV infection. Integration of the HIV genome into host cells is a formidable obstacle to any antiviral treatment that would not just suppress but also eradicate the infection. Nevertheless, modern treatment with combinations of nucleoside analogues and protease inhibitors has transformed the prognosis for carriers of HIV, usually achieving a sustained fall in virus concentration in blood and restoration of the main target cell (CD4 lymphocyte) to near normal levels.
By contrast, the inherent variability of the HIV genome and the failure of the human host to produce neutralising antibodies to the virus, as well as technical difficulties and concerns about safety, have continued to frustrate attempts to make an effective vaccine. This must not, however, allow efforts to develop and evaluate candidate vaccines to slacken. A particular concern is that a useful candidate vaccine (probably a recombinant envelope vaccine developed in North America or Europe against the locally prevalent HIV-1 B subtype) would be ineffective in those parts of the world where other subtypes predominate.
WHO estimates that in the year 2000 there are 36 million carriers of HIV worldwide, and only a small fraction of them have access to suppressive treatment. Both their contacts, their dependants and possibly they themselves would have their life prospects transformed by an effective, or even partially effective, vaccine, and successful application of antiviral treatment in developed countries should in no way be allowed to deflect attention from the necessity of developing and delivering an effective vaccine and of promoting “safe sex” behaviour.
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