Introduction
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 no 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 neutralizing 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” behavior.
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 no 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 neutralizing 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” behavior.
HIV was discovered by Barré-Sinoussi, Montagnier, and colleagues at the Institut Pasteur, Paris, in 1983 and given the name lymphadenopathy associated virus (LAV). In 1984 Popovic, Gallo, and co-workers described the development of cell lines permanently and productively infected with the virus.In line with two previously described retroviruses, HTLV-I and HTLV-II, they designated this virus HTLV-III. Other virus isolates from patients with AIDS and AIDS-related disease in America, Europe and Central Africa have proved to be all the same virus, now referred to as HIV-1. Eight subtypes of HIV-1, alphabetically designated, have so far been described.
Around 1985 another human retrovirus, different from HIV- 1, was recognised in patients from West Africa. This virus, referred to by the Paris investigators as LAV-2 and more recently as HIV-2, is also associated with human AIDS and AIDS-related disease. It is closely related to the simian retrovirus, SIV, carried by healthy African green monkeys, and the cause of an AIDS-like disease in captive rhesus monkeys.Though potentially important worldwide, HIV-2 infections remain uncommon outside West Africa and they have proved far less virulent than HIV-1 infections.
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 milliliter. At these times viraemia can be recognized 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.
The virus and the tests
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.
The virus and the tests
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 recognize 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. 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 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.
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 recognize 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. 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 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.
Test for the virus: antigen, viral DNA and RNA, subtypes, mutants Viral antigens are present in serum, in particular the HIV core antigen, p24. This is only detectable for as long as it is in excess of antibody to p24, 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 sub typed-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.
analysis of the sequences of amplified sections of the envelope gene, HIV-1 has been sub typed-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.
Sequencing of PCR “amplicons” is also the basis for proving HIV transmission events in special settings, for example, health care.
The growing use of antiretroviral drugs, especially singly, has encouraged the emergence of resistance. This is usually associated with point mutations in the HIV genome. As the common resistance mutations have become better known, testing for them has begun to be used to guide changes in therapy. There is also growing interest in the epidemiology of those mutations that confer resistance for the obvious reason that a highly transmissible resistant mutant might beuntreatable and assume an epidemic character.
Testing of patients and blood donors
Tests for anti-HIV-1 and -2, HIV-1 antigen and HIV-1 genome are widely available in the UK. Anti-HIV tests are carried out daily in most public health laboratories and in blood transfusion centres. The facilities in transfusion centres emphatically do not exist to provide testing for those at risk, however. The primary means by which the blood supply is protected from contamination with HIV is through those individuals at increased risk of HIV infection refraining from volunteering to give blood (see chapter 16). Those who wish to be tested for anti-HIV should instead consult their general practitioner or
attend a sexually transmitted diseases (genitourinary medicine) clinic, where the advisability of HIV testing can be discussed. If a decision to test is made the necessary investigations are readily and freely available. In some localities “open access” facilities exist to encourage self-referral for counselling and testing. Other innovations, such as home testing on the patient’s own initiative, are being considered in the USA and might be introduced into the UK.As testing becomes more common, and as kits with which people can test themselves are now technically feasible and might be introduced in the future, it is important to be aware of the psychological impact of test findings on those who are tested. While the emergence of effective drug treatment for HIV carriers makes testing for anti-HIV desirable for those who think they may have been put at risk, there should remain an element of medical supervision to respond to patients’ questions and anxieties. Telephone helplines have been proposed to provide this support.
attend a sexually transmitted diseases (genitourinary medicine) clinic, where the advisability of HIV testing can be discussed. If a decision to test is made the necessary investigations are readily and freely available. In some localities “open access” facilities exist to encourage self-referral for counselling and testing. Other innovations, such as home testing on the patient’s own initiative, are being considered in the USA and might be introduced into the UK.As testing becomes more common, and as kits with which people can test themselves are now technically feasible and might be introduced in the future, it is important to be aware of the psychological impact of test findings on those who are tested. While the emergence of effective drug treatment for HIV carriers makes testing for anti-HIV desirable for those who think they may have been put at risk, there should remain an element of medical supervision to respond to patients’ questions and anxieties. Telephone helplines have been proposed to provide this support.
Important precautions
The desirability of discussing investigations for HIV infection with patients beforehand and of interpreting the results to them afterwards is discussed in Chapter 13. When patients are tested for anti-HIV in a healthcare setting, permission to collect a sample should always have been sought by the doctor and given by the patient. An exception to this is when serum residues, already irreversibly anonymised, are tested for anti-HIV as part of an epidemiological study. Such studies have become a basis for monitoring the epidemic and predicting future trends and resource needs. They have shown, for instance, that in the UK approximately a third of the HIV infected population (total about 30 000 in year 2000) are unaware of their infection or have not disclosed it at the time of the medical contact.
Clotted blood for testing should be obtained by careful venepuncture without spillage or risk of inoculation accident The needle and syringe should be disposed of safely and the blood placed in a leak proof container, properly identified, and sent by a secure route to the laboratory. PCR testing requires a fresh EDTA specimen such as commonly used for hematological investigations. Oral fluid can be collected from the gum/tooth margin and anti-HIV detected in this fluid. Anti-HIV can also be detected in urine. The patient’s identity and the suspected diagnosis should not be exposed to public gaze, and use of numbers or codes rather than names may be preferred. However, the risk of misidentification may thereby be increased. Patient information should only be shared over the telephone between individuals who know each other, and written reports should be sent to named members of staff, under confidential cover. Positive results should be checked on a fresh newly-drawn specimen.
The consequences of breaches of these well-tried procedures may be very serious for patients and damaging to the reputation of doctors. Because of the implications of positive laboratory findings for the health of the patient and his or her family and contacts, and for the patient’s social and professional life, a high level of competence and sensitivity is to be expected from all who are concerned in instigating investigation for HIV infection. Testing patients without their informed consent is unacceptable.
Laboratory tests for HIV have increased understanding of AIDS and greatly facilitated diagnosis, management, treatment and control measures. However, to derive most benefit from them and do least harm, tests must be used wisely, with proper regard to all the possible consequences for those who are being tested. Any changes to what are now well-established procedures must be carefully considered, piloted, evaluated for cost effectiveness, and, if introduced, periodically audited to ensure that they are yielding the benefits promised
Laboratory tests for HIV have increased understanding of AIDS and greatly facilitated diagnosis, management, treatment and control measures. However, to derive most benefit from them and do least harm, tests must be used wisely, with proper regard to all the possible consequences for those who are being tested. Any changes to what are now well-established procedures must be carefully considered, piloted, evaluated for cost effectiveness, and, if introduced, periodically audited to ensure that they are yielding the benefits promised
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