Received 1 September 1998/Returned for modification 28 October
1998/Accepted 6 February 1999
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INTRODUCTION |
Both direct and indirect markers
have been used to define clinical manifestations of human
immunodeficiency virus type 1 (HIV-1) infection. The use of indirect
markers of infection, for example, determination of neopterin levels,
attempts to correlate perturbations of physiological processes that
occur as a result of HIV-1 infection with patient clinical status
during disease progression. Use of direct markers of HIV-1 infection
attempts to measure either viral infectious titer, various constituent
proteins, or reverse transcriptase (RT) activity associated with
retroviruses. The most commonly assayed HIV-1 constituent protein is
the p24 core protein, which is commonly referred to as HIV-1 p24
antigen. Often multiple markers of HIV-1 infection are used to assess
patient status, although the correlation of different markers has been
inconsistent. Some investigators have reported a correlation of p24
with other markers for HIV-1 infection (30), while other
investigators have not (3, 10). The utilities of individual
surrogate HIV-1 markers described in various studies can appear to be
conflicting (5), indicating a limitation for clinical relevance.
With the complexity of HIV-1 pathogenesis in vivo and the variability
associated with commonly used surrogate markers, the development and
commercialization of assays designed to detect and quantitate HIV-1 RNA
in plasma or serum, referred to as the viral load, have been considered
a significant advance. Thus, determination of viral load has rapidly
become accepted as an integral component of care for patients infected
with HIV-1. Viral load measurements provide a means to characterize
progression of disease (18-20) and to estimate the efficacy
of antiviral therapy (21). The correlation between HIV-1 RNA
concentrations in plasma specimens and pathogenesis has resulted in the
formulation of specific antiviral treatments that, together with the
use of CD4+-lymphocyte counts as a measure of immune
function, afford distinct medical strategies for individual patient
care. Thus, determination of viral load has emerged as the major
clinical marker for HIV-1 disease (1, 2, 24).
The purpose of this study was to investigate the correlation of viral
load testing with another direct marker of HIV-1 infection, p24. As the
determination of viral load is based on viral RNA, presumably present
as mature virions in the cell-free plasma, we examined the correlation
of the presence of the virion constituent p24 with viral load
determinations in clinical specimens. To gain further insight into the
relationship between HIV-1 markers, studies were also conducted with
four in vitro laboratory strains of HIV-1.
(This study was presented in part at the 97th General Meeting of the
American Society for Microbiology, Miami Beach, Fla., 4 to 8 May 1997.)
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MATERIALS AND METHODS |
Cell culture.
Four laboratory-adapted HIV-1 (group M, clade
B)-infected human cell lines (MN/H9, LAV1/H9, ARV2, and HTLV-IIIB/H9)
were propagated in Dulbecco's high-glucose medium supplemented with
10% fetal bovine serum by using techniques previously described
(7). The cultured cell lines were maintained at equivalent
cell densities; the doubling time of the cells was approximately 4 days. The kinetics of HIV-1 markers in the cell lines were assessed
over time by daily removal of serial specimens from each cell line over
a total time period of 7 days. The cells were separated from the
culture medium by centrifugation at 1,500 × g for 5 min. The supernatant was removed and stored at 
70°C until use.
Dilutions of each specimen were made in Dulbecco's high-glucose medium
prior to analysis in order to obtain results within the dynamic range
of the p24 enzyme-linked immunosorbent assay (ELISA) (described below).
Clinical specimens.
HIV-1-infected patients from the North
Shore University Hospital Center for AIDS Research and Treatment
(Manhasset, N.Y.) were recruited for the study over a 27-month period
from August 1994 to November 1996. Demographic information concerning
the subjects' overall health status and medication was obtained from the subjects; there were no specific exclusionary criteria for subject
participation. Informed consent was obtained from each subject prior to
specimen donation. Peripheral blood was collected by venipuncture from
each subject into a VACUTAINER (Becton Dickinson, Franklin Lakes, N.J.)
tube with potassium EDTA. The specimens were processed by standard
techniques and stored at 70°C until use. For HIV-1 RNA testing,
specimens were added to NASBA lysis buffer (Organon Teknika Corp.,
Durham, N.C.) and stored as described above.
CD4+-cell concentration determination.
CD4+-lymphocyte concentrations were determined by standard
flow cytometry techniques.
RT determination.
Each specimen from the infected-cell
cultures was evaluated with an RT assay as previously described
(7).
HIV-1 p24 determination.
For determination of HIV-1 p24
levels, an ELISA based on analyte capture with p24-specific monoclonal
antibodies (Organon Teknika Corp.) was used according to the
manufacturer's directions. Cell culture-derived specimens were tested
directly in the assay, whereas the clinical specimens were treated with
a reagent designed to disrupt immune complexes (Organon Teknika Corp.).
Following addition of the specimens to the microtiter plate and
incubation at 37°C for 1 h, the microtiter plate was washed four
times with a phosphate-buffered solution. An anti-p24
antibody-horseradish peroxidase conjugate was added to the microtiter
plate and, following a second 1-h incubation at 37°C, was again
washed four times. Addition of tetramethylbenzidine·2 HCl substrate
for 30 min resulted in the formation of color if p24 was present in the
microtiter wells. The enzymatic reaction was stopped with the addition
of 2 N H2SO4, at which time the optical density
was determined with a microtiter plate reader at 450 nm. The amount of
p24 present was determined from a standard curve which was derived from
the absorbances of individual p24 standards (concentration range, 5 to
80 pg/ml) included in each microtiter plate. The disruption of
potential immune complexes present in the clinical specimens was
effected by a 16- to 24-h incubation of the specimens at ambient temperature with Base Dissociation Reagent (Organon Teknika Corp.) (11). The subsequent steps of the assay were identical to
those described above. The presence of HIV-1 p24 antigen in
ELISA-reactive specimens was confirmed with an antibody neutralization
assay specific for HIV-1 p24 (Organon Teknika Corp.).
HIV-1 RNA concentration determination.
HIV-1 RNA present in
both cell culture specimens and clinical specimens was quantitated with
the NASBA HIV-1 RNA QT System (Organon Teknika Corp.) according to the
manufacturer's directions (28). Each specimen was tested
with the NASBA HIV-1 RNA QT assay, using a 0.1-ml volume. The cutoff
used for the assay was 400 copies. Results for HIV-1 RNA copies are
presented here as copies per 0.1-ml input volume.
Data analysis and statistical methods.
Results for HIV-1 RNA
and p24 from the cell culture studies were calculated as a function of
the dilution of the specimen tested. Data from the individual HIV-1
markers were transformed to base 10 logarithms for statistical
analysis. Correlation coefficients were derived with PROM GLM within
the SAS/STAT module (SAS Institute, Cary, N.C.). Linear regression was
estimated by the least-squares method.
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RESULTS |
HIV-1 markers in vitro.
For specimens from each of the
HIV-1-infected cell lines, a general increasing trend for each of the
three viral markers studied, i.e., HIV-1 RNA, HIV-1 p24, and RT, was
observed over the 7-day observation period (Table
1). Each viral marker for the four HIV-1-infected cell lines was detected at each sampling time.
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TABLE 1.
Kinetics of accumulation of extracellular HIV-1 markers
from chronically infected human T-cell lymphocytes over a 7-day period
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Differences in the kinetics of marker accumulation over time were
observed in each of the four HIV-1-infected cell lines. An increase in
viral RNA was evident for each observation interval for each cell line
(15 of 16 observations [94%]), with the exception of day 7 for the
ARV2-infected cells, at which time an 88% reduction from the
concentration at day 6 was observed. For RT, an increase in activity
was observed for each cell line for 14 of 16 (88%) of the total
observations; a decrease in activity was observed at the final time
point (day 7) for the LAV1- and ARV2-infected cells. The accumulation
of p24 during the observation period appeared to be the most variable
of the three markers evaluated for these cell lines. An increase in the
p24 concentration was observed in 12 of 16 (75%) of the observations.
For the MN- and LAV1-infected cell lines, increasing p24 accumulation
was observed through the entire observation period, whereas for the
HTLV-IIIB-infected cells, a narrow range of increased concentration of
p24 was observed (range, 24,500 to 44,000 pg/ml) through the
observation period. The accumulation of p24 in ARV2-infected cells
appeared as a stepwise increase, with the first two observations being
similar (5,500 and 5,000 pg/ml), as were the next two time points
(22,000 and 19,000 pg/ml), until the highest concentration (31,000 pg/ml) was reached at day 7.
Differences in the overall kinetics describing the increase in the
concentration or activity for each viral marker were further demonstrated for the four cell lines, as evidenced by the calculated regression slope for each marker (Table
2). LAV1-infected cells demonstrated a
consistent linear increase for each of the three markers, with a mean
regression slope for the three markers of 0.123. The regression slopes
of the three markers were also similar in the ARV2-infected cells when
the last time point for HIV-1 RNA was excluded from the calculation.
HTLV-IIIB- and MN-infected cells demonstrated greater variability in
the rates of accumulation among the three markers. In these cell lines,
the increases of RT were similar (2.05 and 2.09, respectively). For
HTLV-IIIB-infected cells, the rate of p24 accumulation was
approximately 50% of that of HIV-1 RNA (0.0379 and 0.089, respectively), whereas for the MN-infected cell line, the rate of
accumulation for p24 was slightly greater than that for HIV-1 RNA
(0.081 and 0.070, respectively).
As shown in Table 2, comparison of the regression line slopes for the
individual HIV-1 markers in each of the four cell lines indicated that
the greatest variability in the rate of accumulation was observed for
p24 (range, 0.09805), while RT demonstrated the least variability
(range, 0.0115). The variability of HIV-1 RNA accumulation was
intermediate, with a range of 0.0434.
HIV-1 markers in clinical specimens.
Previous studies have
indicated the occurrence of immune complexes of p24 and p24-specific
antibodies in HIV-1-infected individuals that lead to a decrease in the
amount of free p24 antigen that can be detected with an ELISA
(16). Disruption of HIV-1 immune complexes by acidic
(13) or basic (11) pH treatment results in
greater availability of p24 for reaction in an ELISA. To determine the
efficacy of the p24 immune complex dissociation procedure with the p24
ELISA used in the present study, 235 clinical specimens from
HIV-1-seropositive individuals were analyzed with and without a base
specimen dissociation reagent. Use of the base dissociation reagent
resulted in a significant increase in the number of clinical specimens
reported to be reactive for HIV-1 p24 (119 of 235 [51%]) compared to
the number (69 of 235 [29%]) reported to be reactive with the
standard ELISA procedure without the reagent. There was no specimen
positive with the standard ELISA that was nonreactive in the ELISA
after treatment with the base dissociation reagent. Agreement with both
the standard technique and the base dissociation technique was seen for
69 positive specimens (29%) and 116 nonreactive specimens (49%).
Thus, this result indicated that the dissociation of immune complexes
with the basic pH reagent was more efficacious in detection of p24
antigenemia in clinical specimens from HIV-1-seropositive individuals
than was the standard technique. A 72% increase in the number of
specimens with reported positive p24 results was observed following
dissociation treatment, which was then subsequently used for the
analysis of additional clinical specimens.
Upon testing of 244 individual clinical specimens from HIV-1-infected
subjects collected at single time points, a reported HIV-1 RNA copy
number was obtained from 177 specimens (73%) with the NASBA HIV-1 RNA
QT System, while only 109 (45%) were reported to be reactive for p24
antigen. Overall, the number of RNA copies reported from the clinical
specimens was inversely proportional to the CD4+-cell
count, as the largest amounts of HIV-1 RNA were observed in subject
specimens with the lowest CD4+-cell counts and the smallest
amounts of HIV-1 RNA were observed in specimens with the highest
CD4+-cell counts (Table 3). A
similar relationship was observed with p24 antigen.
Within this population of clinical specimens, agreement between the
status of the reported viral markers (HIV-1 RNA and p24) was observed
with 164 specimens (67%). Discordant results between the two markers
were observed with 80 (36%) of the specimens. There were approximately
8 times more specimens with only an HIV-1 RNA value than with only a
p24 value (Table 4).
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TABLE 4.
Agreement of reported assay results for HIV-1 RNA and p24
in clinical specimens from collections at a single time point
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Correlation analysis indicated a higher association between the HIV-1
RNA levels and CD4+-cell counts (correlation coefficient,
0.426) than between the p24 levels and CD4+-cell counts
(correlation coefficient, 0.059). A low level of correlation between
the HIV-1 RNA levels and the p24 levels was observed when all of the
data were analyzed (correlation coefficient, 0.278), and this
association did not appear to be consistent for the levels of these
markers in the individual specimens (Fig. 1 to
3).
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FIG. 1.
Correlation between HIV-1 p24 antigenemia and HIV-1 RNA.
Individual values from clinical specimens were transformed to base 10 logarithms and analyzed by least-squares regression. Least-squares
regression line:  = 0.35 + 0.32x.
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FIG. 2.
Correlation between HIV-1 RNA and CD4+-cell
counts. Individual HIV-1 RNA values from clinical specimens were
transformed to base 10 logarithms and analyzed by least-squares
regression with the square root value for the corresponding
CD4+-cell count. Least-squares regression line:
= 32.31 4.51x.
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FIG. 3.
Correlation between HIV-1 p24 antigenemia and
CD4+-cell counts. Individual HIV-1 p24 values from clinical
specimens were transformed to base 10 logarithms and analyzed by
least-squares regression with the square root value for the
corresponding CD4+-cell count. Least-squares regression
line: = 13.49 0.61x.
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As antiviral regimens might be expected to affect the level of HIV-1
marker expression, further analysis of the viral marker results and the
medication regimen was done from available records for 230 subjects
(Table 5). The majority of the specimens
evaluated (180 of 230 [78%]) in the study were obtained prior to the
widespread availability of HIV-1 protease inhibitors and the following
initiation of highly active antiretroviral therapy regimens, which are
in current use with about 70% of the clinic's patients. The results of this analysis indicated that a majority of the subjects with CD4+-cell counts of <200 or of 200 to 499 were receiving
antiretroviral medication at the time of sampling (approximately 70%
for each group), whereas for the group with CD4+-cell
counts of >500, 42.4% of the subjects were receiving antiretroviral treatment. The antiviral treatment regimens for the three groups ranged
from single therapy to triple therapy with HIV-1 protease inhibitors
(Table 5). In the subject group with CD4+-cell counts of
<200, 95% (21 of 22) of the subjects with no reported antiretroviral
medication and 88% (49 of 56) of the subjects with reported
antiretroviral medication were positive either for HIV-1 RNA and p24 or
for HIV-1 RNA only. In none of these subjects was p24 present without a
positive viral load result. In the subject group with
CD4+-cell counts of 200 to 499, 84% (26 of 31) of the
subjects not receiving antiretroviral treatment were positive for viral
load and p24, while 65% (48 of 74) among those receiving antiviral treatment were marker positive. In the latter group, 6 of 48 (12.5%) were positive for p24 only, with a range of 3 to 78 pg/ml. Five subjects from this group were receiving dual therapy (zidovuAdine [AZT] and lamivudine [3TC], n = 1; AZT and
dideoxycytosine, n = 2; 3TC and dideoxyinosine,
n = 1; and stavudine [d4T] and crixivan, n = 1), and one subject was receiving triple therapy (crixivan, 3TC,
and d4T). In the subject group with CD4+-cell counts of
>500, positive marker results were reported for 63% (17 of 27) of the
subjects receiving no antiviral treatment and for 55% (11 of 20) of
the subjects receiving medication. In this group, three subjects
receiving antiretroviral treatment were positive for p24 only; one
subject was receiving monotherapy (AZT), and the other two were
receiving triple therapy (crixivan, d4T, and 3TC). These results
indicate that the presence of a viral load and p24 is partially related
to antiretroviral treatment but is also strongly affected by the immune
status of individual subjects as evidenced by the CD4+-cell
count.
To determine the expression of individual markers over time in
HIV-1-infected individuals, specimens from six different subjects were
collected sequentially and analyzed for the presence of HIV-1 markers.
More reportable results were obtained with the viral load measurement
(33 of 39 [85%]) than with p24 (23 of 39 [59%]). No p24 was
detected in any of the specimens from two of the individuals, while a
viral load value was reported for 10 of 16 (63%) specimens from these
subjects. With the specimens from the other four subjects, both HIV-1
RNA and p24 were detected in each of the specimens (Table
6). The levels of each marker, when
reported, tended to fluctuate during the study period. In some cases
(for example, with subject 1), the observed levels of either of the
markers appeared to correlate with an active antiretroviral treatment regimen, while in the other subjects this correlation was not observed.
| |
DISCUSSION |
In this study, the frequencies of several HIV-1 markers present in
chronically infected cell lines and in clinical specimens were
determined by different measures. The in vitro systems also served as a
control to characterize the performances of the two main viral marker
assays for p24 antigenemia and viral load in the absence of the effects
of the in vivo host immune system. Moreover, the characteristics of
HIV-1 marker expression observed in vitro were distinctly different
from those of expression in clinical specimens.
Direct comparison of the extracellular prevalences of the three HIV-1
markers present in the cell-free medium from infected-cell-line cultures indicated a consistent expression over a 7-day period. This
result was expected in the absence of host immune regulation and
indicated the utility of the assays used in the study for detection of
the HIV-1 markers. Further, the levels of the three HIV-1 markers (RNA,
p24, and RT) observed with the laboratory strains of the virus
correlated in that an increase in each marker was observed during the
culture period. In contrast, the results from clinical specimens
demonstrated that the reported frequencies of two major viral markers,
HIV-1 RNA and p24 antigen, were different. This observation emphasizes
that differences in viral marker expression between the cell lines and
the human subjects make direct comparisons between the two types of
host tenuous. In clinical specimens, HIV-1 RNA was detected at a
greater frequency (approximately 30%) than p24, indicating a higher
level of clinical sensitivity for the viral load test. This result is
in agreement with previous results comparing these two markers
(15, 17, 23, 27). However, in the clinical specimen
population the more sensitive nucleic acid amplification test gave a
reportable result for only 72% of the specimens. This observation is
in part related to the lower limit of detection of the assay. As the
HIV-1 RNA copy number in a clinical specimen diminishes to the 400-copy
reportable threshold for this assay, a commensurate decrease in the
probability of reporting would be expected. This phenomenon is in part
due to the variability of the assay at low HIV-1 RNA concentrations, for which a low copy number may or may not be reported. However, this
circumstance was not demonstrated when testing of the serial clinical
specimens with RNA copy numbers below the lower limit of the assay was
repeated, and none of the specimens were again reported with a HIV-1
RNA copy number. The use of more-sensitive HIV-1 RNA amplification
assays (see, e.g., references 8 and 25) with enhanced detection capabilities at a copy
level threshold of <100 should further improve the clinical utility of
viral load measurements (9, 22). As an alternative, the use
of larger specimen input volumes may also have utility in increasing
the frequency of detection of HIV-1 RNA in specimens with low copy numbers (14).
In the clinical specimens from HIV-1-infected individuals, HIV-1 RNA
levels correlated better with CD4+-cell counts than did p24
levels, which was the result of greater p24 variability in this
population. The levels of HIV-1 RNA present in the clinical specimens
were inversely proportional to the CD4+-cell counts
reported, an observation in agreement with the results of other studies
(17). Some correlation was observed between HIV-1 RNA levels
and p24 levels, which is in agreement with previous studies
(29) which also described a correlation of proviral DNA
levels with viral RNA levels. A low but significant correlation among
markers including the HIV-1 p24 level in plasma, the HIV-1 RNA level in
plasma, and the infectious HIV-1 titer in peripheral blood mononuclear
cells has been reported (17). In our sequentially collected
clinical specimens, the HIV-1 RNA levels and CD4+-cell
counts were correlated; the relationship between these markers was
again inversely proportional. In four of the six specimen series
evaluated, the HIV-1 RNA levels appeared to increase over the
observation period, as did the p24 levels. These results suggest that
both markers can indicate changes in patient status over time. In the
specimens from two subjects, no p24 was detected, while HIV-1 RNA was
detected in each of the specimens from these subjects. Thus, while p24
trend results may suggest changes in patient status, the low
correlation between p24 and HIV-1 RNA limits the utility of p24 testing
in clinical situations where monitoring of drug efficacy is vital
(4, 12). In both of these earlier studies (4,
12), a significant proportion of study subjects did not have
measurable concentrations of p24, a result consistent with observations
in the present study.
In contrast to the consistent detection of HIV-1 p24 and RNA in the
infected-cell cultures, the detection of these markers in clinical
specimens was distinctly different. The inconsistency of reported p24
in clinical specimens might be attributable in part to the inherent
limitations of the assays used. Thus, the static nature of the
traditional ELISA capture detection system for p24 detection compared
to the HIV-1 RNA amplification capability of the NASBA system, which
increases the concentration of the target analyte, could account for
the difference in clinical sensitivity observed between the two assays.
This general lack of sensitivity for HIV-1 p24 detection in clinical
specimens may be a characteristic of the analyte concentration in
typical clinical specimens, as polyethylene glycol precipitation of p24
complexes from clinical specimens results in significantly increased
sensitivity of detection (6). Further complicating the
detection of HIV-1 p24 is the formation of immune complexes that bind
free p24 and HIV-1 virions. These immune complexes might be refractory
to dissolution with the reagent used in the present study for this
purpose, which could offer some explanation for the lower frequency of
p24 reporting compared to that of HIV-1 RNA. Other factors that may
influence the detection of a specific HIV-1 marker involve the specific stage of disease progression, the presence of opportunistic disease agents (5), and use of an antiviral drug regimen
(26). In the case of individuals receiving antiretroviral
therapy, the cessation of active replication with a concomitant
decrease in HIV-1 RNA present for detection might be expected prior to
clearance of residual p24 immune complexes. This sequence of HIV-1
marker repression following antiviral treatment could account for the observation of nine subjects with detectable p24 but no reported RNA.
Further, results from our in vitro studies suggest that differences in
HIV-1 marker concentrations might also be attributable to different strains of the virus and may be related to the variability among HIV-1
strains in extracellular excretion of virions. Further analysis of
viral products from both intracellular and extracellular fractions from
infected-cell lines is necessary to more fully understand these
relationships among different HIV-1 strains.
In conclusion, determination of the HIV-1 RNA viral load provides more
information about virologic status than does p24 antigenemia and,
in conjunction with the CD4+-cell count as a measure of
immune function, affords greater clinical utility.
We thank the nurses and patients of the North Shore Center for
AIDS Research and Treatment (CART), whose generosity made these studies
possible; Dana Gallo (Viral Rickettsial Disease Laboratory, Berkeley,
Calif.) for assistance with the cell culture study; and Andrew Stead
(Organon Teknika Corp., Durham, N.C.) for assistance with the
statistical analysis of the data.
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Abstract
Introduction
