|Year : 2004 | Volume
| Issue : 1 | Page : 7-15
Laboratory markers associated with progression of HIV infection
V Gupta, S Gupta
Department of Microbiology, Dayanand Medical College and Hospital, Ludhiana - 141 001, Punjab, India
Department of Microbiology, Dayanand Medical College and Hospital, Ludhiana - 141 001, Punjab, India
Infection with HIV may develop to AIDS at different rates in different individuals, with a spectrum varying from rapid progression to long term non-progression. The variable course of HIV-1 infection causes emotional trauma for the infected person and complicates the design and interpretation of therapeutic trials because of unrecognized differences in prognosis. Thus it is essential to have tests which can accurately assess the stage of infection in an individual, as well as predict its course and monitor its progression. These laboratory tests are very valuable during the period of clinical latency and subsequently supplement various clinical parameters.
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Gupta V, Gupta S. Laboratory markers associated with progression of HIV infection. Indian J Med Microbiol 2004;22:7-15
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Gupta V, Gupta S. Laboratory markers associated with progression of HIV infection. Indian J Med Microbiol [serial online] 2004 [cited 2014 Dec 20];22:7-15. Available from: http://www.ijmm.org/text.asp?2004/22/1/7/8055
Although the median interval between HIV-1 infection and the development of the Acquired Immuno Deficiency Syndrome (AIDS) in adults is 10 to 11 years, some infected persons rapidly progress to AIDS in less than 5 years. Still others remain asymptomatic without evidence of immunologic decline for more than 6 years. The biological basis of this variability is unknown, but differences in viral strains, host immune responses, and exposure to microbial or environmental cofactors, probably contribute. Clinicians, dealing with symptomatic HIV infected patients, draw on a number of indicators of immunologic function that are associated with an increased probability of disease progression. These laboratory tests are very valuable during the period of clinical latency and subsequently supplement various clinical parameters. Response to antiretroviral therapy is also monitored using these prognostic tests. Identification of laboratory tests that help predict progression to AIDS in people infected with HIV is desirable because of the implications for both clinical management and counselling of the patient.
Many clinical and laboratory markers have been used to estimate prognosis in patients with HIV-1 infection. Markers of AIDS development include HIV related symptoms,, depletion of CD4+ T cells, cutaneous anergy,, elevated serum b2-microglobulin (b2-m) and neopterin levels, HIV-1 p24 (core) antigenaemia,, and syncytium inducing HIV-1 phenotype. None of these markers are ideal; all have limitations in sensitivity, specificity, or predictive power. The single best predictor of AIDS onset identified thus far, is the percentage or absolute number of circulating CD4+ T cells, but less variable and earlier markers of risk for AIDS are needed.
Assays used in monitoring HIV infected individuals have relied predominantly on detecting the effects of HIV on the immune system rather than directly measuring HIV load. There is importance of both immune deficits (decreased number of CD4 + T cells) and immune stimulation (increased levels of neopterin, b2-m, soluble IL-2 receptors and IgA) in predicting prognosis. The levels of soluble IL-2 receptors reflect the activation of T cells and the number of CD8 + T cells reflect the stimulation of that population. The level of IgA reflects B cell activation and that of b2-m reflects lymphoid activity more generally. Recently, data on the high viral turnover in HIV infection at all stages of disease and the value of viral load measurements in antiretroviral drug trials have led to the use of markers that directly measure viral load or other viral characteristics in clinical practice.,
The CD4 cell count, plasma viral load and other markers should not be used alone in deciding patient care, nor is it advisable to base decisions on a single test result. The patient's clinical condition must be taken into account in selecting which tests to perform, how often to perform them and the significance of abnormal results. Laboratory markers can be classified into : 1) Viral markers, 2) Surrogate markers, and 3) Non-specific markers.
| ~ Viral Markers|| |
Many viral assays [including DNA polymerase chain reaction (PCR), p24 antigen tests and HIV isolation] detect but do not quantify the virus, and are therefore usually used in diagnostic situations.
| ~ Quantitative viral load assays|| |
Quantitative assays used in clinical trials are based either on HIV isolation or genome detection.
| ~ Plasma HIV RNA load|| |
Plasma viral load (HIV RNA) quantification is presently considered the most representative and sensitive laboratory test for monitoring progression of HIV infection and response to antiretroviral therapy. ,, Active replication of virus occurs in all clinical stages of infection. It is possible to detect and quantify virus through out the course of HIV infection. The viral load usually ranges between 102 and 107 HIV RNA copies/mL in untreated individuals though it may be lower in those on treatment. The techniques available for quantifying viral RNA are quantitative RNA-PCR, branched DNA (bDNA) assay and nucleic acid sequence based amplification (NASBA). The RNA-PCR assay can detect as few as 40 copies of HIV RNA per milliliter and is positive in >98% of patients. The bDNA assay can detect as few as 500 copies of HIV RNA per milliliter and is positive in >90% of patients. Measurement of levels of HIV RNA over time have been of great value in delineating the relationship between levels of virus and rates of disease progression, the rates of viral turnover, the relationship between immune system activation and viral replication, and the time to development of antiviral drug resistance. While p24 antigen capture assays reflect changes in total viral burden over an extended period, the HIV RNA assays allow an evaluation of the changes in viral burden that occur over a matter of hours. Measurements of HIV RNA levels should be made approximately every six months and more frequently in a setting where changes are made in anti-retroviral therapy.
Plasma HIV RNA levels are more than 10 fold lower in long term non-progressors (mean value 70,000 copies/mL) than in individuals with progressive disease (mean value 1,000,000 copies/mL). A viral load of >100,000 copies/mL by bDNA assay with in six months of seroconversion increases the risk of progression to AIDS with in 5 years by more than 10 fold. Persistently detectable viraemia and high base line levels carry a poor prognosis, while risk of progression is low at copy numbers <10,000/mL. Viral load measurements can be performed at all stages of HIV disease, and high viral load correlates with a lower baseline CD4 cell counts, quicker decline in CD4 cell counts and faster disease progression.,, Viral load measurements can provide prognostic information about time to AIDS and death, and is independent of the CD4 cell count.
Viral loads regress rapidly with antiretroviral therapy and can be used to monitor it. While precise guidelines have yet to be established, a level of HIV RNA of >20,000 copies per milliliter is felt by many experts to be an indication for antiretroviral therapy. Viral load is the test of choice in deciding when to start or change anti-retroviral therapy, or assessing the relative effect of different regimens., Monotherapy with zidovudine reduces HIV RNA levels by about 0.7 log, compared with nucleoside combination (~1.5 log) and protease inhibitor -nucleoside combination (~2.0 log). Significant reductions are associated with clinical benefit. A number of issues still need to be considered, including choice of assay, cost, quality control, testing strategies and acceptable post therapy levels of viral load. Some interim recommendations for viral load assays are given in [Table - 1].
[Table - 1] : Interim recommendations for viral load assays
| ~ Plasma HIV RNA level that suggests initiation of treatment:|| |
More than 5000-10000 copies/mL and a CD4 cell count or clinical status suggestive of progression.
| ~ Target level of HIV RNA after initiating treatment:|| |
Undetectable; < 5000 copies/mL is an acceptable target.
| ~ Minimal decrease in HIV RNA indicative of antiviral activity:|| |
>0.5 log decrease
| ~ Change in HIV RNA that suggests drug treatment failure:|| |
Return to (or within 0.3 to 0.5 log of) pretreatment value, or a significant rise (0.7-1.0 log) from the treatment nadir
| ~ Suggested frequency of HIV RNA measurement:|| |
- At baseline: 2 measurements, 2-4 weeks apart
- Every 3 to 4 months or in conjunction with CD4 cell counts
- Shorter intervals when critical decision points are closer
Serum p24 antigen
HIV antigen detection tests were made available commercially in 1986 for detection of HIV antigen in serum, plasma and CSF using EIA technology.,,, It detects the viral protein p24 in the blood of HIV infected individuals, where it exists either as free antigen or complexed to anti p24 antibodies. Overall, approximately 30% of the individuals with HIV infection have detectable levels of free p24 antigen. The sensitivity increases to up to 50%, when samples are treated with a weak acid to dissociate antigen antibody complexes prior to assay. Serum immune complex dissociated (ICD) p24 antigenaemia was also associated with AIDS development, but only in univariate analysis. ICD p24 antigenaemia was a less sensitive marker than plasma HIV-1 RNA; only 36% of samples from persons developing AIDS have detectable serum ICD p24 antigen within the first year of infection compared with 73% for HIV-1 RNA. In addition, ICD p24 antigen could not discriminate persons at risk for a significant decline in the CD4 + T cell count from those in whom CD4 + T cell counts remained stable. p24 antigen may be detectable transiently during primary HIV infection and again after years of HIV infection often in association with clinical or laboratory evidence of immunodeficiency. HIV antigenaemia occur during window period, in newborn and during late disease when the patient is usually symptomatic. Antigen detection indicates active HIV infection as opposed to past exposure to HIV proteins, diagnosis of HIV infection prior to antibody seroconversion and has prognostic significance. Throughout the course of HIV infection an equilibrium appears to exist between p24 antigen and anti p24 antibodies. During the first few weeks of infection, before an immune response develops, there is brisk rise in p24 antigen levels. After the development of anti p24 antibodies these levels decline. Late in the course of infection, when circulating levels of free virus are high, p24 antigen levels also increase. The sensitivity of the test improves with clinical progression of disease showing 4% positivity in asymptomatic patients, 56% in AIDS related complex patients and 70% in AIDS patients.
Studies during primary HIV infection have shown that subjects with higher concentrations of p24 antigen tend to have higher plasma HIV titres. Several studies have shown a greater likelihood of progression to AIDS for HIV infected subjects who are positive for serum p24 antigen.,, Though precise significance of p24 antigenaemia is unclear, it has been shown that for a group of asymptomatic HIV infected patients having similar CD4 + T cell counts, those with detectable levels of p24 antigen, are three times more likely on an average to show progression to AIDS, over a period of 3 years, than those in whom p24 antigen levels cannot be detected. Among HIV antigen positive, asymptomatic, antibody positive homosexual males, 23.9% progressed to AIDS over a 21 month period, compared with 1.3% of HIV antigen negative, asymptomatic, antibody positive homosexual males. In the San Francisco cohort study, p24 antigen levels was shown to correlate with disease progression, with 59% of p24 antigen positive patients progressing to AIDS over three years compared to 15% of patients who were p24 antigen negative. In this study, rising level of circulating p24 antigen preceded a fall in the percentage of CD4 cells.
In addition, multiple clinical trials have demonstrated that patients receiving anti-retroviral therapy show a decline in their circulating levels of p24 antigen. Some workers have advocated the use of this assay for monitoring the effectiveness of such treatment but the validity of this assay has yet to be established.
Until the availability of tests of HIV load, p24 antigen assay was the main assay available. The antigen is poorly quantifiable, may not be detectable in many individuals, and does not change very well with anti-retroviral therapy. It has a limited role in diagnosis and is not considered as a useful prognostic marker.
| ~ Serum p24 antibody|| |
Antibodies to HIV p24 antigen begin to appear within several weeks of acute infection. Once present, p24 antibodies tend to persist unless levels of p24 antigen rise during periods of active viral replication. The level of p24 antibody declines because of formation of circulating immune complexes with p24 antigen and depression of immune function. Thus a drop in the titre of p24 antibody predicts an increased viral load, and an increased rate of disease progression to AIDS. This is not routinely measured in clinical practice.
Assays for qualitative viral change
| ~ Syncytium inducing strains|| |
Some HIV strains may form syncytia, or giant cells, in certain continuous cultured cell lines. Syncytium inducing (SI) strains are more likely than non syncytium inducing (NSI) strains to be seen at end stage infection, and are associated with increased viral load, a blunted CD4 cell response to antiretroviral therapy, more rapid decline in CD4 cell counts, drug resistance and progressive clinical deterioration. Not all AIDS patients develop SI strains, but a switch from NSI to SI isolates may predict clinical deterioration., This assay is expensive and is restricted to laboratories with HIV isolation facilities.
| ~ Surrogate Markers|| |
Virus specific markers
HIV specific antibodies have also been used as markers of progression., Though they are not considered particularly sensitive. Decline in, or absence of antibodies to various HIV antigens including p24, p17, gp120, gp 41 and nef gene product have been used as surrogate markers in the past. Diagnostic potential of HIV-I nef gene derived protein also attracted attention, as antibodies to it are found in 54% of known HIV infected individuals. Antibodies to nef encoded protein can be detected before the appearance of antibodies directed against viral structural proteins. In addition, absence or disappearance or intermittent presence of nef specific antibodies have been related to unfavorable outcome in individual infected with HIV-1. Nef specific antibodies have been evaluated by Western Blot (WB) technique with a recombinant p27 nef. It would be premature to consider anti p27 nef as a useful marker in HIV Infection.
| ~ Non-Specific Markers|| |
Cellular Markers / Markers of immune function
CD4 cell count
A number of non HIV specific cellular markers, have been used for staging, monitoring progression of HIV infection and assessing response to therapy but the most commonly used cellular marker is the CD4 lymphocyte count. CD4 is one of the several glycoproteins termed “cluster of differentiation (CD) antigens,” expressed on the surface of lymphocytes. Some of these antigens can be used to classify lymphocytes into subpopulations that correlate with their function (e.g. CD3 is expressed on all T cell and CD4 and CD8 on T helper and T cytotoxic lymphocytes, respectively). CD4 count can be measured by flow cytometry, microsphere assay, and Enzyme immunoassay (EIA). Antibodies to these antigens allow a rapid and accurate measurement by flow cytometry of the number of cells expressing each antigen. CD4 also serves as a receptor for HIV, and cells expressing this protein usually decline in number with progressive HIV infection. The number of cells that express the CD4 antigen is therefore a useful guide to the pathological effects of HIV on the immune system. Its decline is the hallmark of HIV infection and the rate of loss in each person is unique. Patients with an initial diagnosis of HIV infection should have CD4 + T cell measurements performed approximately every six months and more frequently if a declining trend is noted.
CD4 cell count is extremely important in staging of HIV infection and a revised classification of the centre for disease control (CDC), Atlanta, USA (1993) divides HIV positive persons into three CD4 count categories: 1) >500/mL; 2) 200-499/mL, 3) <200/mL (along with 3 parallel clinical stages A, B, and C). A low CD4 count (less than 10%), a number less than 100/mL and a low CD4/CD8 ratio (<0.2) are highly predictive for death from AIDS-related complications. Many studies of HIV infection have shown a relation between a low CD4 count and the subsequent development of AIDS.,, Other studies have shown that subjects with low CD4 counts are at risk of specific AIDS related illnesses such as Pneumocystis carinii pneumonia., The introduction of prophylaxis against various opportunistic infections is based primarily on the CD4 cell count; when to begin antiretroviral therapy is based on both CD4 cell count and viral load estimations. Patients with CD4 + T cell counts below 200/mL are at high risk of infection with Pneumocystis carinii while patients with CD4 + T cell counts below 100/mL are at high risk of infection with Cytomegalovirus and the Mycobacterium avium-intracellulare complex.
The clinical benefit of anti-retroviral drugs is not well indicated by CD4 counts (unlike viral RNA load) and it is less effective in monitoring therapy. Antiretroviral therapy is generally indicated when the CD4 + T cell count falls below 500/µL. Pneumocystis carinii prophylaxis should start when the CD4 cell count is less than 200-250/mL. Once the count has fallen below 50/mL, there is probably little benefit in continuing to monitor it unless a rise is expected with the introduction of new anti-retroviral drugs. All effective forms of antiretroviral therapy to date have been associated with at least a transient increase in either CD4 + T cell count or CD4 proportion. Use of zidovudine in symptom free HIV infected subjects with CD4 counts below 0.5 x 109/litre has been reported to slow the rate of progression to AIDS or advanced AIDS related complex. In the Los Angeles Multicentre AIDS cohort study, HIV infected patients with CD4 cell counts of 350-500/mL had a rate of progression to AIDS in two years of 8%, while for those with CD4 counts of 250-350/mL, the rate was 24%, and 50% if the CD4 cell count was below 250/mL. Because of its prognostic significance, a CD4 cell count below 250/mL is now included as an AIDS defining condition by the CDC.
Though CD4 cell count is widely used by clinicians it is a crude predictor of progression and is expensive to perform. A single abnormal result is not usually a sufficient reason to introduce or change treatment as there are many physiological variables which may affect the count, including the time of day the sample is collected, concurrent infections, and recent exercise. It is much more important to follow CD4 cell counts serially and to observe emerging trends.
| ~ Percentage of CD4 cells|| |
Despite the good correlation between clinical progression and absolute CD4 cell count, there are some limiting factors. The total number of CD4 cells in any one sample is influenced by specimen handling, age of the patient, time of sampling (low counts in early morning), use of pharmacological agents and the presence of infection. Hence some clinicians place more value on the percentage of CD4 cells in the total lymphocyte count as a marker of immune function. Several studies have shown that this is a more accurate predictor of progression to AIDS.
In general a CD4 cell count of 200/mL is roughly equivalent to 20% of the lymphocyte count. The normal range for CD4 cell values vary between laboratories but they are around 500-1300/mL for the absolute count and 38%-65% for the percentage. Clinical decisions should not be based on a single CD4 cell count but rather on the trend over two to three counts. While most physicians rely on CD4 cell numbers, and the CD4 cell count varies unexpectedly, or the clinical stage does not correlate with the CD4 cell count, then the CD4 percentage and its variation overtime should be monitored.
| ~ CD 8 cell count|| |
It is not as useful as the CD4 cell count in most circumstances, usually remaining elevated for many years after infection. However, in advanced immune deficiency (CD4 cell count <200/mL), a marked decline in the CD8 cell count indicates a poor prognosis. Again the trend in CD8 cell count is of greater value than a single determination.,
| ~ Multitest, delayed type hypersensitivity (DTH) skin test|| |
It is the semiquantitative measurement of immune function. This is a practical and simple way to assess the degree of HIV induced damage., Lack of response (anergy) is associated with poorer prognosis.
In vivo DTH skin testing, by means of the Multitest device (Pasteur Merieux, Lyon), provides a quantitative measure of the host's cell mediated immune function (both T cell and monocyte/macrophage function). Testing involves the placement of a panel of seven purified recall antigens epidermally on the volar aspect of forearm, with the diameter of induration of each reaction measured 48 hrs. later. Reactions over 2 mm in diameter are considered positive and the average total diameter of all positive reactions in the normal man is 20mm and in normal woman is around 14 mm. Failure to respond to any antigen (Multitest score of 0) is termed anergy and is an indicator of poor cell mediated immune function. Although published data are few, a poor Multitest score (below 5 mm) appears to have prognostic value independent of CD4 cell count or percentage.
Multitest DTH skin testing is not routinely used in HIV clinics partly due to the inconvenience of a return visit after 48 hrs. and partly due to a paucity of data demonstrating benefit over and above the CD4 cell count or percentage. It appears to have an increasing role, however, in determining when to start antiretroviral therapy and in recruitment for early intervention trials.
The use of HIV antigens such as recombinant gp 160 in a DTH skin testing panel is presently being investigated and may prove a more specific marker of immune response to HIV.
| ~ Phenotypic markers of lymphocyte activation|| |
These include an increase in indicators of immune activation on T lymphocytes like CD38, HLA-DR, IL-2R, CD45RO and markers of apoptosis (e.g. Fas). A flow cytometer with at least >2 colour analysis facility is a necessary requirement for these markers.
| ~ Neopterin|| |
Neopterin (6-D-erythrotrihydroxypropylpterin) is a low molecular weight compound derived from an intermediate product of the denovo biosynthesis of tetrahydrobiopterin from guanosine triphosphate (GTP).,,, It is produced by activated monocytes and B cells. It is an early marker of HIV infection. The levels rise further on progression from pre AIDS to clinical AIDS. Since it is a nonspecific marker besides HIV infection, high neopterin levels may be found in numerous viral infections, inflammatory disorders, collagen vascular diseases and in advanced stages of certain malignancies, atypical phenylketonuria and in patients receiving immunostimulant therapy. Neopterin assay can be done by RIA or ELISA.
Since neopterin levels are stimulated by HIV infection, measurement of neopterin levels can be useful in monitoring progression and evaluating antiviral therapy. Like b2-m, serum or urine levels of neopterin are highest in patients with advanced HIV disease and in asymptomatic patients higher levels are associated with increased progression to AIDS that is independent of CD4+ T cell count .
Serum and urinary neopterin levels have been shown in several studies to be elevated in patients with HIV infection and to correlate well with prognosis.,, Further more, the combination of CD4 + T cell and neopterin measurements is a better indicator of prognosis than either used alone. In the San Francisco cohort study, patients with neopterin levels below 12nmol/L, between 12 and 17nmol/L and above 17nmol/L, had three years rates of progression to AIDS of 10%, 20% and 45% respectively.
b2-m is an 11 kDa protein that is expressed on surface of most nucleated cells. It forms a heterodimer with class I major histocompatibility complex molecules present on the surface of most nucleated cells and exhibits amino acid homology with the constant region of immunoglobulin.,,, Free b2-m can be measured in both serum and urine levels of b2-m correlate with the degree of progression of HIV disease. It spikes in acute infection, declines and then rises during the infection. Levels of b2-m are elevated in a variety of conditions characterized by lymphocyte activation and/or lymphocyte destruction; e.g. lymphoproliferative syndromes, auto immune diseases, viral infections and in patients with renal diseases. Elevated levels are also seen in certain HIV high risk groups such as haemophiliacs and drug abusers. High levels of b2-m have also been reported in tuberculosis, cytomegalovirus infection and lymphomas which may be present in the late stage of HIV infection making it important to interpret the data on serum b2-m levels in the clinical context. It can be measured in serum or plasma by using RIA or competitive ELISA based tests. b2-m measurement has several advantages as a laboratory assay to help determine prognosis. By contrast with CD4 cell counts which require special procedures for specimen handling and processing, b2-m can be measured with a serological assay and equipment available in many laboratories.
Several studies have shown that high concentrations of b2-m in people with HIV infection are predictors of AIDS.,, Hofmann et al found that higher b2-m concentrations predicted subsequent decline in CD4 counts. This finding suggests that changes in b2-m occur sooner after HIV seroconversion than changes in CD4 count. It is independent of its inverse relationship to CD 4+ T cell count. In a study, 34% of patients with b2-m level of >3.8 µg /mL went on to develop AIDS over a 3 year period, compared to only 7% of patients with a level of <2.9 µg/mL. In the San Francisco cohort strudies, patients with b2-m levels below 3mg/L, between 3.0 and 5.0 mg/L and above 5mg/L had three years rate of progression to AIDS of 12%, 33% and 69% respectively. b2-m levels appear to be a marker of disease progression independent of CD4 cell count, p24 antigenaemia and zidovudine therapy. Levels of b2-m have been shown to decrease in a dose dependent fashion in patients treated with zidovudine.
| ~ Other Markers|| |
Other surrogate makers of immune function, such as soluble interleukin-2 receptor levels, serum IgA levels and serum cytokine levels have been studied. However, their predictive value is less than that of the markers already described. Other potentially promising immunologic prognostic markers include percentage of CD38+,CD4+ cells and CD4 +, CD29 “bright” memory cells.
Serum IgA levels
Generally elevated in HIV infected persons. Serum IgA can be measured by simple immunodiffusion method using reference anti IgA.
Serum cytokine levels
| ~ Soluble receptors for TNF a and acid labile endogenous IFN|| |
While each of these quantities may have some value as a marker of disease activity, they have not been evaluated as carefully as the parameters discussed above and at present do not play a major role in laboratory monitoring of patients with HIV-I infection.,
| ~ Soluble Interleukin -2- receptor levels|| |
Levels are generally elevated in AIDS and data suggest a good correlation with the disease stage as indicated by CD4 + T cell level. Assay can be done by EIA which uses standard anti - IL2 receptor capture antibody and a peroxidase coupled secondary anti soluble IL2 receptor antibody.
| ~ Summary|| |
Plasma HIV RNA load, though most representative and sensitive laboratory test for progression of HIV infection which respond to antiretroviral therapy, is not practical in monitoring progression of HIV because of cost, quality control issues, testing strategies and acceptable post therapy levels of viral load.
In practice, the clinician should use at least two markers to monitor disease progression. CD4 cell count, CD4 cell percentage of the total lymphocyte count and b2-m levels are collectively the best markers of the risk of disease progression. Recent findings indicate that the progression to AIDS can be best predicated by measuring the level of T4 subset in combination with serum neopterin level, followed in descending order by b2-m, IgA and IL-2 receptors. However, these indirect predictors are of little value if direct tests for HIV antigen or antibody show a negative test result. The clinical disease spectrum, the risk of opportunistic infections, and the decision to start antiretroviral therapy and antimicrobial chemoprophylaxis are all dependent on the stage of disease as indicated by these surrogate markers. In addition, the response to antiretroviral therapy is often reflected in improvement in surrogate markers.,
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