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.