Symptomatic HIV infection before the development of AIDS

Group IV Symptomatic HIV infection before the development of AIDS

    The progression of HIV infection is a result of a decline in immune competence that occurs due to increased replication of HIV from sites where it has been latent. The exact triggers for this reactivation are poorly understood.
    As the disease progresses, infected persons may suffer from constitutional symptoms, skin and mouth problems and haematological disorders, many of which are easy to treat or alleviate. A decrease in viral load in response to the introduction of antiviral therapy often corresponds to a complete or partial resolution of these symptoms.

Indications for lymph node biopsy

 Indications for lymph node biopsy

 • Constitutional symptoms
• Painful nodes
• Asymmetrical enlargement
• Sudden increase in size
• Hilar lymphadenopathy

Persistent generalised lymphadenopathy

   Group III Persistent generalised lymphadenopathy

    Persistent generalised lymphadenopathy may be a presenting feature of HIV infection in a person who is otherwise well. HIV-related lymphadenopathy persists for at least three months, in at least two extra-inguinal sites and is not due to any other cause.

    A lymph node biopsy in HIV disease is not recommended as a routine procedure as the findings are non-specific and the presence of lymphadenopathy due to HIV alone does not worsen the prognosis. The indications for a biopsy are the same in HIV and non-HIV-related conditions.

Asymptomatic infection

     Group II  Asymptomatic infection

     After PHI, HIV antibodies continue to be detectable in the blood. The amount of virus in blood and lymphoid tissues falls to very low levels and the rate of HIV replication is slow although it does not cease. CD4 lymphocyte counts are within normal limits or generally above 350 cells/mm3. This phase may persist for 10 years or more.

     The decision to treat is made on the basis of the CD4 count and the viral load. The aim of therapy is to maintain immune function by suppressing viral replication to prevent further damage to the immune system. As for PHI treatment, the potential gain of therapy must be weighed against the potential risks and uncertainties.

Group I Primary HIV infection

Group I   Primary HIV infection

      Primary HIV infection (PHI) is also called the seroconversion illness or acute HIV infection. It represents the stage of infection after the acquisition of the virus when antibodies are developing as shown in Figure 4.1. Between 25% and 65% of people have been found to present with symptoms at the time of seroconversion. These can range from a mild, glandular feverlike illness to an encephalopathy. Common symptoms and signs are shown in Box 4.2. The severe symptoms are rare. The differential diagnosis of the mild seroconversion illness is protean and, without a high index of suspicion and a history indicating relevant risk behaviours or factors, the diagnosis may be missed.

     The appropriate diagnostic tests for PHI, which should be carried out on serial blood samples, include tests for HIV antibodies and antigen. If these are negative and PHI is suspected, the definitive test is an HIV RNA PCR, which is the most sensitive test for the detection and quantification of the virus. Some of these assays are not routine and the interpretation of investigation results during PHI is difficult, therefore close consultation with colleagues in virology is strongly advised. At the time of PHI there is sometimes a high rate of viral replication, leading to a transient rise in HIV viral load and concomitant immunosuppression due to a short-lived fall in the CD4 count. This may result in manifestations of HIV disease which are normally seen later in the infection, for example oral candida. Diagnostic confusion as to the stage of HIV infection may arise, which can only be resolved by following up the patient for long enough to see the symptoms and signs resolve, HIV antibodies appear, the viral load fall and the CD4 count rise.

     Treatment should be directed at alleviating any symptoms, and there is considerable interest in the possible use of antiretroviral agents at this time because the virus may be more susceptible due to the relatively low numbers of virus particles which can replicate, the reduced ability of the predominantly non-syncytium-inducing strains of virus to infect a wide variety of cell types and the enhanced immune response seen in PHI. Such treatment may decrease long-term damage to the immune system and delay or even prevent the development of AIDS.

      However, if not started within 12–18 months of PHI the theoretical advantage may be lost and, in any case, has to be balanced against the uncertain outcome, drug toxicity, adherence difficulties and the possibility of developing resistant virus, limiting future treatment options.

Summary of CDC 1992 classification system for HIV disease

Group I        Primary HIV
Group II     Asymptomatic infection
Group III    Persistent generalised lymphadenopathy
Group IV     Symptomatic infection
Group IVA   HIV wasting syndrome (AIDS) and constitutional disease
Group IVB   HIV encephalopathy (AIDS) and neurological disease
Group IVC1 Major opportunistic infections specified as AIDSdefining
Group IVC2 Minor opportunistic infections
Group IVD  Cancers specified as AIDS-defining
Group IVE   Other conditions

Possibilities for immunotherapy

        Attempts at immune reconstitution have been made using interleukin 2, interferons, thymic factors or bone marrow transplantation.

        These have not been notably successful and remain potentially harmful, since the very factors which activate T-cells will also activate HIV replication. In vivo, activation of CD4 cells is caused by stimulation with antigens in the form of micro-organisms or vaccines.

      This suggests that it is sensible to treat intercurrent infections promptly and provides a rationale for prophylactic chemotherapy for pneumocystis. In some studies, vaccination (for example with influenza vaccine) has been shown to be enough of an antigenic stimulus to increase HIV replication. The advent of highly activated antiretroviral therapy (HAART) has enabled the viral load to be enormously reduced, but the difficulty of maintaining this type of therapy over long periods has led to a search for strategies to complement drug treatment.

      Two observations are pertinent, the first is that even after 2–3 years of HAART treatment, latent virus can still be detected and the second is that antiviral immune responses decline during treatment. It has therefore been suggested first that latent virus should be “flushed out” by activation of the immune system with anti-CD3 antibody or interleukin 2 while still continuing drug treatment. Secondly vaccination against HIV should be instituted to prevent recrudescence of low level infection. Both strategies are being actively investigated.

Strategies for vaccine development

• A good vaccine should induce neutralising antibody, helper T-cells and cytotoxic T-cells.

• Since antibodies bind to three dimensional structures, induction of neutralising antibody requires native envelope.
Problem: Native envelope is trimeric.

 • T-cells recognise 8–15 amino acid-long peptides bound to Major Histocompatibility Complex (MHC) class I and II molecules.
Problem: Antigen needs to enter antigen presenting cells, usually dendritic cells, to be broken down to peptides.

• Peptides with novel adjuvants can generate good T-cell responses.
 Problem: Different peptides bind to each MHC allele so a large cocktail of different peptides may be needed.

 • Adjuvants are needed to induce large responses.
Problem: There are very few adjuvants available for unrestricted use in humans. Alum is mainly good for induction of antibody responses.

• DNA immunisation can generate antibody, helper and cytotoxic responses and allows incorporation of adjuvant molecules into the vaccine. Problem: So far DNA vaccination has not proved as effective in man as in experimental animals.

• HIV is very variable and escape variants arise rapidly in infected individuals. Prophylactic immunisation may tip the balance in favour of the host and prevent escape. Some parts of the virus sequence are relatively invariant, these should be targeted if possible.

• In experimental animals immunisation with different immunogens appears promising. DNA vaccination followed by immunisation with antigen in a recombinant viral vector seems particularly effective. This is now under trial in man.

Vaccine development

          Immunisation against an organism whose target is an important component of the immune system presents particular difficulties.
         In addition, HIV has already been shown to be perhaps the most variable virus yet discovered, and HIV-2 differs greatly from all HIV-1 isolates. So far, efforts to immunise against the virus have concentrated on the use of cloned gp120 because all strains of virus so far tested use gp120 to bind to the CD4 molecule, implying that a part of the envelope is similar in all strains. In experimental animals gp120 does induce a neutralising antibody response to the virus but restricted to the immunising strain of virus (type specific immunity) and these neutralising sera do not provide reliable protection against virus challenge in vivo in animal experiments. More recently it has been shown that gp120 and its anchor gp41 exist in the viral envelope as a trimer of heterodimers. Because of this and because gp120 is heavily glycosylated, much of the antibody response is to the variable V2 and V3 loops. Furthermore, primary isolates have been shown to be less susceptible to neutralisation than the tissue culture-adapted strains, from which the recombinant gp120 used as immunogen in most experiments derives. Thus new immunogens are needed to raise broadly reactive neutralising antibody and a variety of oligomeric and deglycosylated forms of gp120, lacking the V2 and V3 loops, are being tried.

        High levels of CTL are seen in the early stages of HIV infection and the demonstration of CTL escape mutants suggests that they play a role in controlling the virus. That individuals exposed to HIV but with no evidence of infection exhibit CTL responses, reinforces the view that this type of response is important in protection. An effective vaccine might therefore contain components able to stimulate both neutralising antibody, CD4 T-cells and strong CTL responses.

     A key factor in generating immune responses is the way in which the antigens are presented to the immune system. For the generation of effective CTL responses attenuated live viruses are effective and attenuated (nef deleted) simian immunodeficiency virus (SIV) has been shown able to protect monkeys against challenge with virulent virus. While such a strategy is unlikely to be used in humans because of worries about the safety of such a virus, it suggests that live viral vectors may be an effective means of immunising against HIV. HIV genes have been inserted into several possible vectors (vaccinia, canary pox, adenovirus) and a number of phase 1 trials are in progress. Alternate means of delivery capable of inducing both antibody and cellular immunity, such as peptides or proteins in novel adjuvants, naked DNA, or the use of different methods of antigen administration in sequence (prime/boost regimes) are under active investigation.

Causes of CD4 lymphopenia

• HIV infection: seroconversion illness and during disease progression • Acute viral infections* • Tuberculosis* • Sarcoidosis* • Corticosteroid therapy • Purine metabolism defects; ADA and PNP deficiency • SLE * Reduce CD4 counts when not associated with HIV and can further reduce levels in HIV infection. ADA, Adenosine deaminase; PNP, Putine nucleoside phosphorylase

Monitoring HIV infection

     Counting CD4 lymphocyte numbers (the “CD4 count”) is an important part of monitoring HIV infection. A progressive downward trend in CD4 cells reflects disease progression and decreased life expectancy, even in the absence of symptoms.

    Epidemiological studies have firmly correlated distinct ranges of CD4 cell counts with risk of particular opportunist infections. Recent data show that monitoring either the absolute CD4 lymphocyte count or the ratio of CD4 to CD8 cells, the 4:8 ratio, are both equally good at monitoring progression in HIV
infection. 2 microglobulin and neopterin are molecules shed from activated lymphocytes; serum levels increase with progressive HIV infection and can be a useful adjunct to CD4 counts in monitoring.

   CD4 lymphocyte numbers have a diurnal variation and delays in the sample reaching the immunological laboratory (for example, when a sample is held overnight) also cause profound changes. Because CD4 lymphocyte counting is a lengthy process, most consistent results are obtained when samples are taken at a set time in the morning and sent straight to the lab. In case of unavoidable hold ups, samples should not be
refrigerated.

Positive and negative effects of immune responses

    Antibody
    Beneficial effects
• Neutralising antibody (demonstrated in vitro only) might prevent primary infection and destroy some infectious particles
• Evidence for beneficial effect of passive transfer of antibody in man requires confirmation
Harmful effects
• Antibody may also help the virus to enter cells with Fc receptors
• Immune complexes may cause tissue damage, anemia and neutropenia

Cellular immune responses
Beneficial effects
• A strong CD8 response is correlated with primary resistance in some individuals and with long-term survival
• Cytotoxic T-cells may delay the progress of disease by killing infected cells.
• They produce CD8 T-cell anti-viral factor (CAF) which inhibits viral replication and may be important in slowing disease progression

Harmful effects
• They may kill uninfected cells which take up shed gp120
• Abnormal cytokine secretion may cause immunopathology (perhaps including encephalopathy)

Causes of CD4 lymphopenia

• HIV infection: seroconversion illness and during disease
progression
• Acute viral infections*
• Tuberculosis*
• Sarcoidosis*
• Corticosteroid therapy
• Purine metabolism defects; ADA and PNP deficiency
• SLE
* Reduce CD4 counts when not associated with HIV and can
further reduce levels in HIV infection. ADA, Adenosine
deaminase; PNP, Putine nucleoside phosphorylase

HIV infection antibody responses

     Early after HIV infection antibody responses are not impaired; indeed, development of antibodies to the virus envelope and core proteins is the principal evidence for HIV infection and persists until death. In adults, massive activation of B lymphocytes is manifested by a rise in serum immunoglobulin concentration, perhaps due to direct activation of B cells by HIV. This polyclonal activation explains why a variety of false positive serological tests are seen in HIV infection. In young children, the reverse pattern may be seen, with extremely low levels of immunoglobulin sometimes requiring intravenous replacement therapy. Within days or weeks after infection there may be a transient fall in CD4 lymphocyte numbers and a more sustained rise in the number of CD8 cytotoxic/suppressor cells. Among the CD8 cells, expanded oligoclonal populations are frequently seen and as in other acute virus infections, some of these represent a specific response to HIV.

   Following this acute reaction, healthy seropositive individuals may have normal numbers of lymphocytes, although the numbers of CD8 cells frequently remain high. Even at this stage, however, in vitro testing may show a lowered response to previously encountered (recall) antigens (tetanus toxoid or purified protein derivative, for example). This seems to be due to poor production of the lymphokine interleukin 2. Individuals may remain healthy for long periods, but a hallmark of disease progression, often prior to the development of new clinical symptoms, is a fall in the number of CD4 lymphocytes. In AIDS the number of CD8lymphocytes also falls.

    Biopsy of the lymph nodes in patients with persistant generalised lymphadenopathy shows many enlarged follicles, often infiltrated by CD8 lymphocytes, with depletion of CD4 cells. Even in clinically silent HIV infection, lymph nodes are the site of remarkably active HIV replication. Uninfected cells may also die by apoptosis, initiated by unexplained mechanisms. In the later stages lymph nodes return to normal size and
follicles become “burnt out”, with loss of normal architecture and progressive cellular depletion.

Infection with HIV
Many of the clinical features of HIV infection can be ascribed
to the profound immune deficit that develops in infected
individuals. HIV is immunosuppressive because it infects cells of
the immune system and ultimately destroys them. An
understanding of this process is helpful in interpreting tests
used in monitoring the disease and may explain the failure of
immunotherapy and the difficulties in developing vaccines for
HIV.
The most obvious target of the virus is a subset of thymusderived
(T) lymphocytes carrying the surface molecule CD4,
which has been shown to bind the envelope glycoprotein of
HIV (gp120). CD4 is also present on a large proportion of
monocytes and macrophages, Langerhans’ cells of the skin and
dendritic cells of all tissues. More recently it has also become
clear that virus entry also requires co-receptors, most of which
are members of the seven transmembrane-spanning G proteincoupled
receptor family. In the immune system these principally
function as receptors for chemokines that orchestrate the
migration, differentiation and function of leucocytes during
immune responses. Two receptors, CCR5 and CXCR4, are
particularly important. CCR5 (R5) is widely expressed on
lymphocytes, macrophages, dendritic cells and cells of the
rectal, vaginal and cervical mucosae. Virus strains able to infect
primary macrophages (macrophage (M) or R5 tropic viruses) use
CCR5 as a co-receptor. Only R5 strains are detected early after
infection, while both R5 viruses and strains that infect T cells
and use CXCR4 (T or X4 tropic viruses) are found late in
infection. These data suggest that R5 strains are important for
transmission of HIV while X4 variants arise during the course
of infection and may be responsible for T-cell loss and disease
progression. Even stronger evidence that CCR5-using M tropic
viruses transmit infection, comes from the observation that
individuals homozygous for a 32 base pair deletion of CCR5
show greatly increased resistance to HIV infection. Several
other chemokine receptors have been shown capable of acting
as co-receptors in vitro and polymorphisms in CCR2 as well as
CCR5 and SDF1 (the ligand for CXCR4), are associated with
different rates of progression to AIDS.

Immunopathology

     CD4 lymphocytes (T helper cells) have been termed “the leader of the immunological orchestra” because of their central role in the immune response, and their destruction accounts at least in part for the immunosuppressive effect of the virus. When these cells are stimulated by contact with an antigen they respond by cell division and the production of lymphokines, such as interferons, interleukins, tumour necrosis factor and the chemoattractant chemokines. Lymphokines act as local hormones controlling the growth, maturation and behaviour of other lymphocytes, particularly the cytotoxic/suppressor (CD8) T-cells and antibody-producing B lymphocytes. Lymphokines also affect the maturation and function of monocytes, tissue macrophages and dendritic cells.

    Macrophages and particularly dendritic cells are important antigen-presenting cells for initiating immune responses of lymphocytes. Not only do they act as a reservoir for the virus but their antigen-presenting function is impaired, with secondary effects on lymphocytes. Monocytes are the precursors to some glial cells and abnormal lymphokine production after HIV infection may have harmful effects on neural tissue and
result in HIV encephalopathy.

Specific immune responses to HIV

    In spite of the fact that HIV-infected individuals show the gross abnormalities of immune function described above, they are able to mount a specific immune response to HIV itself. Although serum reactivity to all the viral proteins is detectable, virus neutralising titres are generally low and directed against
the immunising virus strain (type specific immunity). Passive transfer of antibody from asymptomatic to symptomatic patients is claimed to be beneficial, but this requires confirmation. Antibodies to HIV may even facilitate infection of cells bearing immunoglobulin (Fc) receptors, such as monocytes. In AIDS a fall in the titre of antibodies to core protein (p24) is often associated with disease progression. p24 antigen, which is
detectable in the serum of some patients, may show a rise at the same time and has been used as a marker of disease progression.

      CD8 cytotoxic lymphocytes (CTL) capable of killing HIVinfected targets are detected in most HIV-infected individuals and may be beneficial. This is suggested by the observation that viraemia declines at the time that CTL are first detected following infection, and in patients with stable disease, a high frequency of CTL is detectable in the peripheral blood. In addition, in individuals who have been regularly exposed to
HIV while remaining seronegative and without detectable virus, HIV-specific CTL have been detected. As well as killing infected cells directly, CD8 lymphocytes may contribute to protection by producing several chemokines and CAF (CD8 T-cell antiviral factor), which strongly inhibit viral replication in CD4 cells. All
this has led to the suggestion that CTL are an effective protective mechanism. However, because reverse transcription is an error-prone process, virus mutants arise, which evade the CTL response (escape mutants). These mutants may not only evade recognition themselves but also inhibit recognition of unmutated
virus.

    There is some evidence to suggest that a minority of patients mount a specific CD4 T-cell response to HIV and that this is associated with effective control of virus replication. In animal experiments CD4 cells have been shown to be important for the maintenance of an effective CTL response, which may explain
this association.

Important precautions

    The desirability of discussing investigations for HIV infection with patients beforehand and of interpreting the results to them afterwards. 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 HIVinfected 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 leakproof container, properly identified, and sent by a secure route to the laboratory. PCR testing requires a fresh EDTA specimen such as commonly used for haematological 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 costeffectiveness, and, if introduced, periodically audited to ensure that they are yielding the benefits promised.

Prevalent HIV infection diagnosed/undiagnosed

     30 000 people living with HIV and AIDS in the UK
     34% undiagnosed:
                                 Homosexual men 28%
                                 Heterosexual men/women 49%
                                 Injecting drug users 6%

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.

      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.

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.

HIV and related viruses

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.

The virus and the tests

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.

AIDS - USA, UK and Europe

By June 1999, 702 748 adult cases of AIDS had been reported in the USA. In addition there were 8596 paediatric cases (<13 years old). Most of the cases in children (91%) occur because a
patient suffered from HIV or belonged to a group at increased risk of HIV; 4% occurred through blood transfusion; 3% in children with haemophilia.

Information on risk factors for the remaining 2% of the parents of these children is not complete.
Adult cases in Europe totalled 234 406 by June 2000, and those in the UK 17 151 (December 2000). There are five times more people infected with HIV at any one time than have AIDS. The rate for AIDS cases varies throughout Europe, with particularly high rates in Italy, Portugal, Spain, France and Switzerland, where the commonest mode of infection is through intravenous drug use and the sharing of needles and equipment.

In North America and the UK the first wave of the epidemic occurred in homosexual men. In the UK, proportionally more homosexual men have been notified than in America: 67% of cases compared with 48% respectively. Even though infections amongst men who have sex with men still arise, an increasing proportion of new infections in the USA is occurring amongst intravenous drug users sharing needles and equipment. There is also an increase amongst heterosexuals in both the USA and the UK. Currently in the USA, 16% of cases of AIDS have occurred amongst women, and although the commonest risk factor amongst such women is injecting drug use (42%), the next most common mode of transmission is heterosexual contact (40%).
The nature of the epidemic within the UK is changing with more heterosexual transmission. In the UK 12% of adult cases of AIDS have occurred in women, 70% of which have resulted
from heterosexual intercourse. In 2000 there were more new annual infections of HIV than ever before and for the first time more occurring as a result of heterosexual sex than men having sex with men. Most heterosexually acquired infections are seen in men and women who have come from or have spent time in Sub-Saharan Africa.

The advent of an effective antibody test in 1984 has allowed for a clearer understanding of the changing prevalence and natural history of HIV infection. Surveys show that the proportion of individuals infected needs to be high before cases of AIDS start to become apparent. It also underlines the importance of health education campaigns early in the epidemic, when the seroprevalence of HIV is low. Once cases of AIDS start to appear the epidemic drives itself and a much greater effort is required in terms of control and medical care.

Within countries one finds considerable variation in seroprevalence levels for HIV. Over 70% of cases of AIDS and HIV infection within the UK occur and are seen in the Thames regions (London and the surrounding area). Among different groups one also finds geographical differences. For example, the rates among drug users is higher in Edinburgh than London,
and for gay men higher in London than anywhere else in the UK. This is also found in the developing world; for example, in Tanzania and Uganda, the urban level of HIV infection in men
and women can be five times higher than rural rates.

The use of highly active antiretroviral therapy (HAART) in resource-rich countries has resulted in an increase in life expectancy. This, in combination with the increase in new HIV infections, means that the prevalent pool of those infected, and potentially infectious, is increasing. This presents a continuing challenge for health promotion and a re-statement of the importance of safe sex techniques, particularly condom use.
AIDS results in a considerable cost not only in human suffering also to health services. Other costs include time off work and the effect of the deaths of young people on national productivity. AIDS represents a major public health problem in the world. A clear understanding of the epidemiology forms the basis of developing a strategy of control ranging from health education to research.

Growth and size of the epidemic AIDS

Even though North America and Europe experienced the first impact of the epidemic, infections with HIV are now seen throughout the world, and the major focus of the epidemic is in
developing/resource-poor countries.

Worldwide
The joint United Nations programme on AIDS (UNAIDS) has estimated that by the end of 2000 there were 36.1 million people living with HIV/AIDS (34.7 million adults and 1.4
million children <15 years). The new infections during that year were 5.3 million, approximately 16,000 new infections per day.

Currently, 95% of all infections occur in developing countries and continents, the major brunt of the epidemic being seen in sub- Saharan Africa and south-east Asia. It is now recognised that cases of AIDS were first seen in Central Africa in the 1970s even though at that time it was not recognised as such. Current surveys from some African countries show that the prevalence of infection is high amongst certain groups – 50–90% of prostitutes, up to 60–70% of those attending departments for sexually transmitted diseases and antenatal clinics. In the developing world, HIV is spread mainly by heterosexual intercourse.

At a family level, UNAIDS estimated that by the end of 1999 the epidemic had left behind a cumulative total of 13.2 million AIDS orphans (defined as those having lost their mother
or both parents to AIDS before reaching the age of 15 years).

Many of these maternal orphans have also lost their father. Orphans in Zimbabwe are expected to total 1 million by 2005 and 2 million in South Africa by 2010. Traditional family structures and extended families are breaking down under the strain of HIV. Population growth and death rates are increasingly affected. Life expectancy in countries with adult prevalences of over 10% (for example Botswana, Kenya, Zimbabwe, South Africa, Zambia, Rwanda) are expected to see
an average reduction in life expectancy of 17 years by 2010–2015. Young, highly productive adults die at the peak of their output, which has a considerable impact on a country’s
economy.

Transmission of the Virus - summary

Sexual intercourse
• anal and vaginal
• Contaminated needles
• intravenous drug users
• needlestick injuries
• injections
• Mother ➝ child
• in utero
• at birth
• breast milk
• Organ/tissue donation
• semen
• kidneys
• skin, bone marrow, corneas, heart valves, tendons etc.

Transmission of the virus

HIV has been isolated from semen, cervical secretions, lymphocytes, cell-free plasma, cerebrospinal fluid, tears, saliva, urine, and breast milk. This does not mean, however, that these
fluids all transmit infection since the concentration of virus in them varies considerably. Particularly infectious are semen, blood, and possibly cervical secretions. The commonest mode of transmission of the virus throughout the world is by sexual intercourse. Whether this is anal or vaginal is unimportant.

Other methods of transmission are through the receipt of infected blood or blood products, donated organs, and semen. Transmission also occurs through the sharing or reuse of contaminated needles by injecting drug users or for therapeutic procedures, and from mother to child. Transmission from
mother to child occurs in utero and also possibly at birth. Finally, the virus is transmitted through breast milk.

The virus is not spread by casual or social contact. Health care workers can, however, be infected through needlestick injuries, and skin and mucosal exposure to infected blood or body fluids. Prospective studies in health care workers suffering percutaneous exposure to a known HIV seropositive patient indicate a transmission rate of 0.32%. As of December 1999 there have been 96 reported cases of documented seroconversion after occupational exposure in such workers.

The precautions and risks for such groups are covered in detail in chapter 15. Finally, there is no evidence that the virus is spread by mosquitoes, lice, bed bugs, in swimming pools, or by sharing cups, eating and cooking utensils, toilets, and air space with an infected individual. Hence, HIV infection and AIDS are not contagious.

AIDS-defining conditions with laboratory evidence of HIV

Diseases diagnosed definitively

• Recurrent/multiple bacterial infections in child <13 years of age
• Coccidiomycosis – disseminated
• HIV encephalopathy
• Histoplasmosis – disseminated
• Isosporiasis with diarrhoea persisting >1 month
• Kaposi’s sarcoma at any age
• Primary cerebral lymphoma at any age
• Non-Hodgkin’s lymphoma: diffuse, undifferentiated B cell type, or unknown phenotype
• Any disseminated mycobacterial disease other than M. tuberculosis
• Mycobacterial tuberculosis at any site
• Salmonella septicaemia: recurrent
• HIV wasting syndrome
• Recurrent pneumonia within 1 year
• Invasive cervical cancer
• Diseases diagnosed presumptively
• Candidiasis: oesophagus
• Cytomegalovirus retinitis with visual loss
• Kaposi’s sarcoma
• Mycobacterial disease (acid-fast bacilli; species not identified by culture): disseminated
• Pneumocystis carinii pneumonia
• Cerebral toxoplasmosis

AIDS-defining conditions with laboratory evidence of HIV

Diseases diagnosed definitively
• Recurrent/multiple bacterial infections in child <13 years of age
• Coccidiomycosis – disseminated
• HIV encephalopathy
• Histoplasmosis – disseminated
• Isosporiasis with diarrhoea persisting >1 month
• Kaposi’s sarcoma at any age
• Primary cerebral lymphoma at any age
• Non-Hodgkin’s lymphoma: diffuse, undifferentiated B cell type, or unknown phenotype
• Any disseminated mycobacterial disease other than M. tuberculosis
• Mycobacterial tuberculosis at any site
• Salmonella septicaemia: recurrent
• HIV wasting syndrome
• Recurrent pneumonia within 1 year
• Invasive cervical cancer
• Diseases diagnosed presumptively
• Candidiasis: oesophagus
• Cytomegalovirus retinitis with visual loss
• Kaposi’s sarcoma
• Mycobacterial disease (acid-fast bacilli; species not identified by culture): disseminated
• Pneumocystis carinii pneumonia
• Cerebral toxoplasmosis

The definition of AIDS has changed over the years

The definition of AIDS has changed over the years as aиresult of an increasing appreciation of the wide spectrum of clinical manifestations of infection with HIV. Currently, AIDS is defined as an illness characterised by one or more indicator diseases. In the absence of another cause of immune deficiency and without laboratory evidence of HIV infection (if the patient has not been tested or the results are inconclusive), certain diseases when definitively diagnosed are indicative of AIDS. Also, regardless of the presence of other causes of immune deficiency, if there is laboratory evidence of HIV infection, other indicator diseases that require a definitive, or in some cases only a presumptive, diagnosis also constitute a diagnosis of AIDS.

In 1993 the Centers for Disease Control (CDC) in the USA extended the definition of AIDS to include all persons who are severely immunosuppressed (a CD4 count <200 106/1) irrespective of the presence or absence of an indicator disease.

For surveillance purposes this definition has not been accepted within the UK and Europe. In these countries AIDS continues to be a clinical diagnosis defined by one or more of the indicator diseases mentioned. The World Health Organisation (WHO) also uses this clinically based definition for surveillance within developed countries. WHO, however, has developed an alternative case definition for use in sub-Saharan Africa. This is based on clinical signs and does not require laboratory confirmation of infection. Subsequently this definition has been modified to include a positive test for HIV antibody.

Early history of the epidemic AIDS

1981 Cases of Pneumocystis carinii pneumonia and Kaposi’s sarcoma in the USA
1983 Discovery of the virus. First cases of AIDS in the UK
1984 Development of antibody test


The first recognised cases of the acquired immune deficiency syndrome (AIDS) occurred in the summer of 1981 in America.
Reports began to appear of Pneumocystis carinii pneumonia and Kaposi’s sarcoma in young men, who it was subsequently realised were both homosexual and immunocompromised. Even though the condition became known early on as AIDS, its cause and modes of transmission were not immediately obvious.

The virus now known to cause AIDS in a proportion of those infected was discovered in 1983 and given various names. The internationally accepted term is now the human immunodeficiency virus (HIV). Subsequently a new variant has been isolated in patients with West African connections –HIV-2.

AIDS-defining conditions without laboratory evidence of HIV

• Diseases diagnosed definitively
• Candidiasis: oesophagus, trachea, bronchi or lungs
• Cryptococcosis: extrapulmonary
• Cryptosporidiosis with diarrhoea persisting >1 month
• Cytomegalovirus disease other than in liver, spleen, nodes
• Herpes simplex virus (HSV) infection
• mucocutaneous ulceration lasting >1 month
• pulmonary, oesophageal involvement
• Kaposi’s sarcoma in patient <60 years of age
• Primary cerebral lymphoma in patient <60 years of age
• Lymphoid interstitial pneumonia in child <13 years of age
• Mycobacterium avium: disseminated
• Mycobacterium kansasii: disseminated
• Pneumocystis carinii pneumonia
• Progressive multifocal leukoencephalopathy
• Cerebral toxoplasmosis

The logical steps that should be taken to prevent and to cure people with AIDS are:

1. Prevent the causes of AIDS by educating the public about the toxic effects of the illicit drugs and alcohol;
2. Limit the use of glucocorticoids in the treatment of chronic conditions and in the treatment of people with AIDS;
3. Monitor the levels of CD4+ T cells and CD8+ T cells in the blood of patients who are receiving medium or high therapeutic doses of glucocorticoids for significant times;
4. Discontinue the treatment of patients with AIDS and asymptomatic patients with AZT and protease inhibitors immediately. These are very toxic medications;
5. Provide proper clinical support and nutrition to patients with AIDS based on their medical needs. Prior to the development of full blown AIDS in drug users and homosexuals, the damage is caused by the use of drugs.

V1 Evidence that invalidate the HIV-hypothesis. The following is a list of medical facts that invalidate the HIV-hypothesis a claim which states that HIV selectively kills CD4+T cells and causes AIDS.

1. The reduction of CD4+ T cells in HIV positive homosexual men who used steroids was reversed after the cessation of the treatment with corticosteroids. If the HIV is the cause of AIDS in these patients then the cessation of the steroids will not reverse the disease.
2. The reversal of the reduction of CD4+ T cells in HIV-positive pregnant women following the feeding multivitamin and provided balanced diet disprove the idea that HIV is the cause of AIDS. The average CD4+ T cells increase in these patients from 426/ul to 576/ul (Fawzi, et al. 1998).
3. The lymph nodes of majority of the 505 HIV-infected individuals showed lymphoid hyperplasia that include T and B cells (Al-Bayati, 1999).
4. The lymphoid atrophy observed in HIV-infected patients include reduction in T cells (CD4+ and CD+8), B cells, and stroma (Al-Bayati, 1999; Muro-Cacho, et al., 1995).
5. HIV particles were found in CD4+, CD8+ T cells, B cells, and other cells indicating HIV do not need specific receptors as the HIV-hypothesis predict (Al-Bayati, 1999).
6. 90% of AIDS cases were reported to be in drugs users and homosexuals and the changes in the lymphoid organs of HIV-negative drug users or homosexuals were similar to those described in HIV-positive drug users and/or homosexual men ( Fauci, et al., 1998; Al-Bayati, 1999).