The HIVs are retroviruses; their genetic information (genome) is composed of RNA rather than the usual DNA. To reproduce, the virus uses an enzyme known as reverse transcriptase to produce a DNA copy of its genetic RNA and incorporates this material into the host cell genetic material. Some of the copied viral genome also exists in the host cell without being incorporated into host chromosomes. Thus, the host cell nucleus reproduces the virus as well as itself. The HIVs have an unusual property, similar to the herpesvirus group, that active viral reproduction and infection can coexist with presence of antibodies against the virus. In most other virus infections, appearance of specific antibody marks the end of the infection and confers partial or complete protection against subsequent infection by that virus. The HIVs attack a subgroup of T-lymphocytes known as helper (inducer) T-cells (CD4 cells). Helper T-cells are important in cell-mediated immunity (delayed hypersensitivity), which is the immunologic mechanism classically defending against chronic lower virulence infections such as tuberculosis, fungus, and parasites. Monocytes, macrophages, and possibly Langerhans cells also become infected.
The first HIV to be discovered was isolated from patients with the acquired immunodeficiency syndrome (AIDS) by three different groups of investigators who each gave the virus a different name (human T-cell lymphotropic virus type III, or HTLV-III; lymphadenopathy-associated virus, or LAV; and AIDS-associated retrovirus, or ARV). The current terminology for this virus is human immunodeficiency virus type 1 (HIV-1). This virus is present endemically in Central Africa. A related virus that produces a syndrome similar to AIDS is found in West Africa and has been named HIV-2 (originally called HTLV-IV). The HIV viruses are related to a similar virus found in African green monkeys. They are also related, but less closely, to certain animal viruses called lenteviruses (“slow viruses”), of which the most well known is the visna virus of sheep. Besides the HIV virus group that injures or destroys helper T-cells, there is another group of viruses that affects T-cells but that causes excessive T-cell proliferation rather than destruction. This group has retained the name of HTLV and includes HTLV-I (which causes human T-cell leukemia) and HTLV-II (which may be associated with hairy cell leukemia). Similar to the HIV viruses, the HTLV virus group is related to a monkey T-cell leukemia virus and more distantly to a T-cell leukemia virus of cattle.
Clinical findings
HIV-1 can be isolated from many body fluids (including blood or blood products, semen, cervical secretions, saliva, tears, cerebrospinal fluid, breast milk, urine, and various tissues including the cornea). However, urine and saliva appear to have relatively little infectious capacity. HIV-1 is predominantly transmitted in three ways: by sexual intercourse (heterosexual or male homosexual), by transfusion or inoculation of infected blood or blood products, and by mother to fetus through the placenta. After exposure, there is an incubation period that typically lasts 2-6 weeks (range, 6 days-8 weeks, but sometimes lasting several months or years). In about 50% of patients (range, 4%-70%) this is followed by an acute viral type of illness (sometimes called the “acute seroconversion” or “acute HIV syndrome”) resembling infectious mononucleosis or CMV infection that usually lasts 2-3 weeks (range, 3 days-several weeks). Symptoms usually include fever, sore throat, and lymphadenopathy; often include skin rash, myalgias, diarrhea, vomiting, and aseptic meningitis; and sometimes thrombocytopenia. Some patients never develop the initial acute febrile illness or any clinical infection; they may recover completely from the initial exposure (although this is probably uncommon) or may become an asymptomatic carrier. Those who develop the initial acute illness exhibit a wide spectrum of possible outcomes. After recovery they may become asymptomatic carriers; may have defective immunologic responses without clinical disease; may develop persistent generalized lymphadenopathy (PGL); may develop a variety of non-life-threatening fungal, bacterial, or viral infections (e.g., oral Candida) as part of the so-called AIDS-related complex (ARC); or may develop the AIDS syndrome.
AIDS is the most severe manifestation of HIV-1 infection, defined briefly as serologic evidence of HIV antigen or antibody plus certain opportunistic infections or Kaposi’s sarcoma (a malignant tumor of fibroblasts and capillary-sized blood vessels) in a patient who is immunocompromised without known cause. The most frequent opportunistic organism producing active infection in AIDS is pneumocystis carinii (about 60% of cases; range, 35%-80%); other common agents include Cryptococcus neoformans (4%-13% of cases), Candida albicans esophagitis (14%-25%), “atypical” mycobacteria of the Mycobacterium avium-intracellulare complex (22%-30%), and protozoans such as Toxoplasma(3%-12%) and Cryptosporidium (4%-13%). Other organisms with evidence of frequent past or recent infection include CMV (66%94%) and HSV (4%-98%, the lower figures being active infection). Incidence of Kaposi’s sarcoma varies according to risk group; in male homosexuals with AIDS the incidence is about 35% (range, 25%-50%) in clinical studies and 30%-75% in autopsy studies; but in drug abusers and hemophiliacs it is found in less than 5%. Some 50%-80% of patients with AIDS develop various types of neurologic disorders with or without dementia, which may precede other evidence of AIDS in 25% of patients. In one series, 66% of these patients had elevated CSF protein levels (42-189 mg/100 ml; 0.42-1.89g/L); 20% had a small degree of mononuclear cell count elevation (4-51 WBCs); and three of seven patients tested had oligoclonal bands detected in their CSF. Cerebral abnormalities were found in two thirds of the patients with AIDS who were autopsied. There is an increased incidence of B-cell lymphoma, especially primary CNS lymphoma (2%-6% of patients).
In the United States as of 1992, about 58% of AIDS patients were male homosexuals, usually those who had multiple sex partners. About 23% were intravenous drug abusers; about 6% were persons infected heterosexually; and about 4% were of undetermined etiology. However, incidence of heterosexual infection (as opposed to current incidence of AIDS, a late-stage development of HIV-1 infection) is becoming more frequent. Infection has been reported after a single heterosexual encounter, although more commonly it takes more than one episode. After an infected person develops detectable antibody, the current approximate calculated progression to AIDS per year is about 2.5% for asymptomatic patients, 3.5% for PGL patients, and 8.5% for ARC patients. Progression to AIDS is highest among infected male homosexuals (4%-10%/year) and low among transfusion-infected hemophiliacs. About 20%-40% (range, 10%-73%) of infected mothers transmit the virus to the fetus during pregnancy. A few infants appear to become infected during delivery and some during breast feeding.
Laboratory findings
In the few small studies in AIDS patients that contain hematologic data, anemia was present in about 80% (range, 45%-95%), leukopenia in about 65% (range, 40%-76%), thrombocytopenia in about 25%-30% (range, 3%-59%; about 5%-10%, range 3%-15% in HIV-infected non-AIDS patients) and pancytopenia in 17%-41%. Lymphocytopenia was reported in about 70%-80% (range, 30%-83%).
Diagnosis of HIV-1 infection
Culture. HIV-1 can be isolated from concentrated peripheral blood lymphocytes and less frequently from body fluids. Isolation rates in already seropositive patients average about 50%-60% (range, 8%-100%; more likely positive just before or during the first half of the acute HIV syndrome). Culture is difficult, is expensive, takes several days, is available only at a relatively few laboratories, and is positive more often in early stages of infection than in later stages. Culture may be the only method that can confirm infection in the first 2-3 weeks after exposure. Culture detects only about 50% of neonates infected in utero in the newborn period up to the first month of life (range, 30%-50%) but a greater percentage at 3 and 6 months. Culture is positive in CSF from about 30% (range, 20%-65%) of seropositive adult patients whether or not CNS symptoms are present, but about 20% more often in more advanced states of disease.
Antigen detection. Viral antigen may become detectable as soon as 2 weeks after infection (in one report it was detected 4 days after a transplant operation). Antigenemia (viremia) lasts roughly 3 months (range, 1 week-5 months). In several reports, antigen could be detected from a few days to as many as 6-9 months before first-generation ELISA antibody test results became positive. Several methods have been used, including RIA, fluorescent antibody, and ELISA. Until about 1990, sensitivity was usually less than 50% and varied considerably between kits of different manufacturers. It was discovered that varying amounts of the circulating p24 antigen were bound to immune complexes. Methods are now available that break up (dissociate) the bound complexes before testing. In one study this increased test sensitivity to 60%-65% in patients without symptoms and 80%-90% in patients with symptoms.
Nucleic acid probe kits with PCR amplification (NA-PCR) have become available. These detect HIV antigen within infected peripheral blood lymphocytes. Sensitivity appears to be about 40%-60% in the first 1-2 weeks of life and up to 98% by age 3 months. NA-PCR detects about 96%-100% of seropositive pediatric patients over age 6 months or adult patients with CD4 counts over 800/mm3 and about 85%-97% of those with CD4 counts below 200/mm3. NA-PCR is more sensitive than culture in HIV-infected but seronegative patients and can detect HIV in CSF from about 60% of seropositive patients. As with all laboratory tests, all manufacturer’s NA-PCR probes are not identical in sensitivity.
Antibody detection. Seroconversion occurs on the average about 6-10 weeks after infective exposure (range, 12 days-5 years), which corresponds to the last part of the acute HIV syndrome stage or up to several weeks afterward (in some degree depending on the sensitivity of the test). Two types of antibodies are produced, IgM and IgG. IgM antibodies are detectable first, and several studies report IgM antibody present in some patients 1-10 weeks before IgG antibody (using first-generation IgG ELISA methods). In general, IgM antibody becomes detectable about 1-2 weeks after onset of the “acute HIV syndrome” (about 5-6 weeks after infection), peaks about 2-3 weeks after first detection, and becomes nondetectable 2-4 months after first detection. IgG antibody becomes detectable 1-2 weeks after IgM antibody, peaks several weeks later, and persists for life (there is controversy over whether a few patients lose antibody shortly before death from AIDS). However, one recent study using a second-generation IgG ELISA found little difference. Commercial ELISA IgM, second-generation IgG, and rapid slide LA methods are now available. Many of these use antibody against one (sometimes more) selected protein components of HIV-1 obtained through recombinant DNA techniques (see Confirmatory Methods section). Test results in HIV-1 infection are shown in Fig. 17-10.
Fig. 17-10 Tests in HIV-1 infection.
The bulk of published test kit evaluations involve first-generation ELISA methods, which are based on crude extracts of the whole virus. There were a considerable number of such methods commercially available, but even the first was introduced in only mid-1985. These tests detect antibody in 94%-99.5% of patients with confirmed AIDS, depending on the particular assay and the investigator. Positive tests in random blood donors have averaged about 0.9% (range, 0.2%-2.56%). However, in some (not all) of these first-generation kits only about 25%-35% (range, 17%-44%) of initially positive ELISA test results on random blood donors remain reactive when retested with the same kit. Of those whose results were repeatedly positive, only about 20%-35% (range, 15%-62%) were positive on confirmatory tests. This means that only about 10%-15% (range, 3%-22%) of the initial positive results on random blood donors from these particular kits eventually were confirmed positive. Some manufacturer’s kits were shown to be more sensitive than others, and some produced more false positive results than others. Some of this discrepancy is explained on the basis of different appearance times or quantity present of different viral antigens being detected by the different kit antibodies being used. There is also controversy whether reactivity against only a single antigen or a certain type (e.g., the core proteins [“group-specific antigen” or gag] p24 and p55 or the envelope glycoproteins gp 120/160 and gp 41) is sufficient to consider the test truly reactive and thus indicative of HIV-1 infection in the absence of reactivity against any other important structural protein. When this happens, it is often considered a false positive or an “indeterminant” reaction, although its significance has not yet been definitely established. In addition to these controversies, definite false negative results and false positive results may occur. Previously mentioned have been false negative results due to variable time periods before antibody is produced and variation in sensitivity of different methods and different manufacturers kits. Also, in the late or terminal stages of AIDS, antibody may disappear from patient serum in about 2%-4% (range, 0%-7%) of those who previously had antibody.
When HIV infection is acquired in utero, IgM (and IgA) antibody is slow to rise until 3-6 months after birth. In several reports, IgA was detectable in 6%-17% at one month of age, 57%-67% at 3 months, and 77%-94% at 6 months. IgG antibody was not helpful for the first 6 months of life (range, 3-18 months) because it may be acquired from the mother through the placenta. False negative and positive results can be due to technical and clerical errors. False positive results in some kits may be due to patient antibodies against certain HLA antigens (most often, DR4) in antigenic material from infected H9 cells used in the kits to capture the patient antibodies. Antigenic material from different cell lines or synthetic (recombinant) antigen does not have this problem. Some kits, but not others, have an increased incidence of false positive results in active alcoholic cirrhosis, renal failure, and autoimmune diseases. Gamma globulin used for HBV prophylaxis may contain antibody against HIV-1, although the gamma globulin is noninfectious due to certain steps in its manufacture. This passively transferred antibody may be detectable for as long as 3 months. Antibody detection methods for urine have been developed with sensitivity reported to be comparable to serum tests.
Confirmatory antibody detection methods. Until 1988 these tests consisted of Western blot and immunofluorescent methods. Western blot is an immunochromatographic technique in which virus material from cell culture is separated into its major component proteins by electrophoresis, transferred (“blotted”) onto a chromatography support medium, and exposed to patient serum. Antibody in patient serum, if present, attaches to whatever virus component proteins it recognizes. Then the preparation is stained to display visually what protein areas had reacted. Not all the virus proteins are considered specific for HIV-1. There is some controversy as to what specific proteins must be present for the results to be considered definitely positive. This affects sensitivity of the test. The two most specific proteins are the virus envelope glycoprotein gp41 and the group-specific antigen (gag) core protein p24 (the numbers refer to molecular weight). However, other proteins, particularly a precursor envelope protein called gp160 (from which gp41 is derived), often appears before either of the more specific proteins. Western blot in general has been shown to detect antibody earlier than most of the first-generation ELISA tests but not as early as IgM or antigen-detection methods (or “second-generation” IgG tests). Unfortunately, Western blot is time consuming, takes 2 days to complete, and reliable results are considerably technique dependent. False negative and false positive results have been reported, although the exact incidence is currently unknown, due to lack of quality control surveys. The test is currently available only in large medical centers and reference laboratories. Immunofluorescence procedures are also available and are claimed to produce results equivalent to Western blot. Immunofluorescence is easier to perform and produces same-day results. However, a minority of investigators found Western blot to be more reliable. Both of these techniques are generally considered suitable to confirm screening test results. The Western blot, however, is currently considered the gold standard. There is also a radioimmunoprecipitation (RIPA) technique that has also been used as a confirmatory procedure. This method is slightly more sensitive than Western blot. However, it is technically difficult and currently is used only in research laboratories.
Recently, tests have become available based on genetically engineered HIV proteins, most often gp160, gp120, gp41, and p24. One or more of these are used in “second-generation” ELISA or LA tests. In general, these tests are somewhat more sensitive and specific than the “first-generation” tests. One kit (HIVAGEN) consists of separate ELISA tests for antibody against several of these antigens, thus becoming, in effect, a sort of ELISA version of the Western blot.
Tests for immunologic status. As noted previously, HIV-1 selectively infects T-lymphocyte CD4 cells (also called helper/inducer, Leu3, or OKT4 cells; CD means cluster designation), which eventually leads to defective immune function. CD8 T-cells (suppressor/cytotoxic or OKT8 cells) are normal or become increased. The 1993 CDC revised classification system for HIV infection considers 500 CD4 T-cells/mm3 or more to be normal; 200-499, moderately decreased; and less than 200, severely decreased. CD4 absolute or relative counts are considered to be the best index of HIV disease severity. Eighty percent to 95% of AIDS patients have a decreased absolute number of helper T-cells (<400/mm3) and a reversed (inverted) helper T-cell/suppressor T-cell ratio, with a T4/T8 ratio less than 1.0. One possible cause of false T4 decrease is the recent report that about 20% of African Americans have helper T-cells that fail to react with the OKT4 antibody but do react with the Leu3 and certain other helper T-cell antibodies. A lesser but substantial number of AIDS patients display more nonspecific immune system abnormalities, such as lymphocytopenia (<1,500/mm3) and nonreactivity to skin test challenge by standard delayed sensitivity antigens such as Candida, mumps, or Trichophyton. Tests of immune function usually do not become abnormal until relatively late stages of HIV-1 infection. These tests are not tests for HIV infection, nor are they diagnostic of AIDS. CD4 cell levels are currently considered the best overall indicator of HIV-1 disease severity and prognosis.
Beta-2 microglobulin. Beta-2 microglobulin (B2M) is a small polypeptide that forms the light chain of the class I histocompatibility complex antigen (HLA) molecules present on the surface of many nucleated cells, including lymphocytes. It is released into serum by cell destruction or membrane turnover and is filtered by the renal glomerulus, after which it is more than 99% reabsorbed and metabolized by the proximal renal tubules. About 50% of serum B2M is derived from lymphocytes. Therefore, B2M levels have been used as a nonspecific marker for lymphocyte proliferation or turnover, as seen in immunologic stimulation or lymphoproliferative disorders. Since it is excreted by the kidney, it has been used to estimate renal function. Since CD4 (T-helper) lymphocytes are affected in HIV infection, B2M is reported to be elevated in about 85%-90% (range, 68%-100%) of patients with AIDS or ARC, 45% of patients with PGL, in smaller numbers of other persons with HIV infection but few or no symptoms, and in varying numbers of clinically healthy male homosexuals (20%-44% in two studies). Some investigators have used B2M as a marker for progression to AIDS, because in general the degree of B2M elevation corresponds roughly with degree of HIV illness severity and inversely with CD4 count. B2M can be assayed by RIA, EIA, or immunodiffusion.
B2M may also become elevated in persons with poor renal function; in various lymphomas and leukemias, especially (but not exclusively) those of B-cell origin, in myeloma, in nonlymphoid malignancies, in sarcoidosis, in infectious mononucleosis and certain other viral infections, in various chronic inflammatory diseases including active liver disease, and in autoimmune disorders.
Neopterin. Neopterin is an intermediate substance in the biopterin synthesis pathway. It is produced by macrophages when stimulated by gamma interferon released by activated T-cells (lymphocytes also produce neopterin but to a minor degree). Therefore, neopterin is an indirect indicator of increased T-cell activity. Neopterin is excreted by the kidney. Plasma or urine neopterin levels can be elevated in acute or active chronic infections or noninfectious inflammatory conditions, similar to B2M or C-reactive protein (CRP). The neopterin level is likely to be elevated in both viral and bacterial infection, whereas the CRP level is more likely to become elevated in bacterial than in viral infection. Also, the neopterin level is more likely than the CRP level to be elevated in immunologic graft rejection, whereas both become elevated in graft infection. In general, like B2M, the neopterin level becomes elevated in HIV infection; and the incidence and degree of elevation have a rough correlation to degree of HIV severity and inverse correlation to CD4 count. One reference states that the neopterin level is elevated in about 90% of seropositive but asymptomatic HIV-infected persons. Another found B2M elevated in 75% of these asymptomatic HIV patients and the neopterin level elevated in 60%; both were elevated in all ARC patients. B2M thus far seems to have aroused more interest than neopterin as a marker for severity in HIV infection.
Summary of human immunodeficiency virus 1 tests. In summary, ELISA tests for HIV-1 antibody are used as a general screen to detect persons infected with HIV-1. Western blot (or equivalent) tests help establish presence of infection. Culture, tests for HIV-1 antigen, and possibly ELISA-IgM antibody tests, may detect infection earlier than the ELISA screening tests. Tests for decreased immunologic function (especially CD4 lymphocyte absolute counts) are useful to help confirm a clinical impression of advanced-stage HIV-1 and AIDS.