Antiretroviral therapy (ART) is becoming more accessible worldwide, facilitated by reductions in drug prices, increased commitment of many countries to provide treatment to all their citizens, and the emerging global consensus that the HIV pandemic in the developing world requires treatment.1,2 CD4+ lymphocyte counts and HIV RNA levels, the established markers of disease progression that form the basis of the guidelines for ART initiation in the developed3 and developing4 world, remain unavailable for many resource-limited regions because of technical and financial considerations, however.1,5 Therefore, it is a priority to develop simpler tests that can provide a basis for deciding when to initiate ART.5,6
One marker that has been proposed for resource-limited settings is the total lymphocyte count (TLC). World Health Organization (WHO) guidelines for 2002 recommend the use of ART for HIV-infected individuals with a TLC less than 1200 cells/mm3 and for those with clinical disease.4 Although many reports have described associations between TLC and CD4+ cell counts,7 the prognostic value of this recommendation has not been determined using prospectively collected natural history data. Hemoglobin is another marker that has been consistently associated with disease progression independent of CD4+ cell counts and HIV RNA levels.7-9
Evidence supporting these markers for monitoring HIV progression remains largely limited to cross-sectional associations,10-16 although recent work has shown that changes in TLC after ART initiation correlate well with changes in CD4+ cell counts.16,17 Using data collected prospectively before the advent of potent ART, we have demonstrated that these markers do not show progressive changes throughout the entire course of infection but, instead, exhibit rapid declines beginning 1 to 2 years (on average) before the onset of AIDS.18-20 Furthermore, we have investigated the initial prognostic values of these rapid changes and demonstrated a sensitivity and specificity for developing AIDS of approximately 70% for TLC declines and 74% for hemoglobin declines.21 On average, the timing of these declines began within a few months of the first CD4+ lymphocyte count less than 350 cells/mm3 and preceded AIDS by medians of 1.3 and 1.2 years for TLC and hemoglobin, respectively.21
To demonstrate the occurrence of rapid declines in TLC and hemoglobin, we previously used all available TLC and hemoglobin marker data to ensure maximum power to determine longitudinal patterns. This method, however, is not easily applicable for clinical monitoring.18 The aim of this study was to develop a method to simulate the prospective monitoring of HIV-infected individuals and to identify rapid declines in TLC or hemoglobin that maximize the sensitivity and specificity for identifying individuals who developed AIDS.21 We then critically evaluated the prognostic value of these markers and their rapid declines to assess the potential of these markers in prospectively monitoring HIV-infected individuals. For these analyses, we used the extensive longitudinal data from the Multicenter AIDS Cohort Study (MACS) available before the advent of potent ART.
MATERIALS AND METHODS
Study Population and Laboratory Methods
The MACS was initiated in 1983 to study the natural history of HIV infection among homosexual and bisexual men in the United States. The study design has been described,22 and only aspects pertinent to the present study are presented here. In 1984 through 1985, 4954 HIV+ and HIV− men were enrolled in Baltimore and/or Washington, DC, Chicago, Los Angeles, and Pittsburgh, with an additional 625 men enrolled between 1987 and 1991. Men with AIDS and men younger than 18 years were excluded from enrollment. Men were scheduled to return for semiannual visits to provide specimens for laboratory analyses, undergo a physical examination, and complete self-administered data forms and an interviewer-administered questionnaire.
T-cell subset levels on all men at each visit were measured in peripheral blood stained with monoclonal antibodies, using a whole blood lysing method and 2-color flow cytometry with monoclonal antibodies specific for CD3+, CD4+, and CD8+ lymphocytes.23,24 Absolute numbers of cells per microliter of blood were calculated using the complete blood cell count with an automated 10,000-cell differential. Hemoglobin concentrations were measured using standard techniques.
The individuals included in the present study consisted of MACS participants with at least 4 AIDS-free visits through November 1996 (before widespread use of highly active antiretroviral therapy [HAART] in the MACS). The visits occurring in the first year after seroconversion or entry into the study (if the individual was seroprevalent for HIV) were omitted from the analysis to minimize the variability that has been demonstrated in marker levels during the acute phase of HIV infection. For individuals developing AIDS, at least 2 visits within 1.5 years preceding AIDS were required to ensure that the marker patterns would be discernable at the time nearest to AIDS.
To determine the occurrence and timing of onset of rapid declines in TLC and hemoglobin, we modified methods previously applied to retrospective identification of inflections in CD3+ cell counts.25 Beginning with the first 4 visits, the rates of declines were calculated as the difference between the mean of the 2 most recent marker values from the mean of all previous marker values, standardized by the time between markers. If a rapid decline for the marker was not found, the algorithm was repeated with markers obtained during subsequent visits. This was repeated until a rapid decline was identified or the individual had no further marker visits to contribute, thus reproducing the prospective monitoring of individuals. For each individual, we determined the first time that TLC levels declined by more than 33% per year and separately determined the first time that hemoglobin levels declined by more than 11.6% per year. These cutoffs for categorizing rapid declines were chosen to be consistent with our previous studies in which these critical values had maximal sensitivity and specificity for distinguishing individuals who did or did not develop AIDS.21
Our analyses consisted of 2 components. First, we evaluated the association of TLC and hemoglobin markers with the incidence of AIDS among eligible MACS seroconverters. Time at risk accumulated from each individual's fourth visit (the minimum AIDS-free time required by the inclusion criteria) until the date of clinical AIDS, November 1, 1996 (if the participant had not reached the end point on that date and remained under follow-up), or the date when the patient was last known to be alive and free from AIDS (if the participant had left the study). Cox proportional hazards models with time-varying covariates were used to examine the relative hazard (RH) for progressing to AIDS. These models assessed the short-term prognostic values of marker levels and occurrence of rapid declines using data updated at each semiannual visit. The impact of the variables was evaluated independently after adjustment for time-varying CD4+ cell counts and HIV RNA levels and after adjustment for a time-varying indicator of whether the TLC was ≤1200 cells/mm3 (WHO criteria). We also computed positive predictive values (PPVs) and negative predictive values (NPVs) for progression to AIDS.
Second, we described the relative timing of rapid marker declines and the onset of clinical AIDS or CD4+ cell counts less than 200 or 350 cells/mm3 in 2 ways. First, we examined timing at the population level by computing Kaplan-Meier estimates of the time from seroconversion to the first occurrence of each decline in the markers (TLC and hemoglobin separately). These were compared with the time to clinical AIDS, time to CD4+ lymphocyte counts ≤200 cells/mm3, and time to CD4+ cell count ≤350 cells/mm3. Second, we also examined the timing at the individual level by estimating the proportion of individuals who demonstrated a rapid decline in their markers; separate proportions were estimated for those who did and did not progress to CD4+ cell counts less than 200 cells/mm3.
Association of Markers With Clinically Defined AIDS
Of the 511 seroconverters, 297 fulfilled the selection criteria, contributing a total of 946 person-years of observation to the TLC analysis and 1048 person-years to the hemoglobin analysis (numbers differ slightly for TLC and hemoglobin analyses because of data availability). Slightly fewer than half developed AIDS over the course of follow-up, with approximately 80% and 88% of these men showing rapid declines in TLC and hemoglobin levels, respectively. At the first visit, the median (interquartile range [IQR]) level that individuals contributed for this study was 1976 (IQR: 1536-2460) cells/mm3 for TLC and 15.2 (IQR: 14.5-15.9) g/dL for hemoglobin.
The results of the Cox proportional hazard analyses are presented in Table 1. The unadjusted results demonstrate the important prognostic value of TLC and hemoglobin levels and rapid declines for predicting AIDS; the incidence of AIDS was approximately 5-fold greater in individuals who showed rapid declines in markers than in those who did not have rapid declines. After simultaneously adjusting for the TLC or hemoglobin level, the magnitude of the risk of AIDS for marker declines was attenuated to between 2.5 and 2.8, but both remained highly statistically significant. After adjusting for CD4+ lymphocyte count, the relationship of TLC decline remained elevated but was no longer significant (P = 0.12). Rapid declines in hemoglobin had a uniformly stronger relation whether or not the model was adjusted for HIV RNA and CD4+ lymphocyte counts, suggesting that a rapid decline of this marker may be of more prognostic value. For both markers, a faster time to AIDS was seen among those with more rapid marker declines (data not shown).
By itself, a TLC less than 1200 cells/mm3 was strongly predictive of AIDS (RH = 6.14, 95% confidence interval [CI]: 4.33-8.71). Table 2 presents an analysis examining the combined effect of a TLC level ≤1200 cells/mm3 and a rapid decline in TLC or hemoglobin levels for predicting the time to AIDS. Importantly, our results demonstrate the increased risk of AIDS among those with marker declines but with a TLC >1200 cells/mm3 (RH = 2.53 for declines in TLC and RH = 5.28 for declines in hemoglobin). A formal test of interaction between these variables was not statistically significant.
Given the prognostic value of these markers, the PPVs and NPVs for having a rapid decline in these markers before AIDS were determined (Table 3). A rapid decline in TLC resulted in a PPV of 0.691 and an NPV of 0.756, and a rapid decline in hemoglobin resulted in a PPV of 0.694 and an NPV of 0.787. This was an improvement over the PPV and NPV of a TLC ≤1200 cells/mm3 alone (0.623 and 0.698, respectively). When a rapid decline in hemoglobin or TLC occurred, the PPV decreased to 0.648 and the NPV increased to 0.861, which were comparable to those obtained for a CD4 count ≤350 cells/mm3 alone (PPV = 0.622 and NPV = 0.865). Furthermore, using the combined criteria of a CD4 count ≤350 cells/mm3 or a rapid decline in hemoglobin or TLC resulted in a significant improvement in the NPV (0.957 vs. 0.865; see Table 3) compared with using only a CD4 count ≤350 cells/mm3, with only a slight impairment in the PPV.
Timing of Rapid Marker Declines
Kaplan-Meier survival estimates in Figure 1 portray the cohort-level timing of rapid declines in TLC and hemoglobin relative to declines to low CD4+ lymphocyte counts (≤350 and ≤200 cells/mm3) and clinical AIDS. The median time from seroconversion to the time of rapid decline was 6.9 years (95% CI: 6.2-7.8) for hemoglobin and 7.3 years (95% CI: 6.4-8.3) for TLC. The onset of these rapid declines preceded the onset of clinical AIDS and occurred consistently between the time CD4+ lymphocyte counts fell to less than 350 cells/mm3 (median = 6.1 years, 95% CI: 5.5-6.7) and the time when CD4+ lymphocyte counts fell to less than 200 cells/mm3 (median = 8.4 years, 95% CI: 7.3 to >10.0).
When evaluating the timing of rapid declines in TLC and hemoglobin relative to low CD4+ levels at the individual level, we found that 82% and 78% of individuals with CD4+ lymphocyte counts less than 200 cells/mm3 showed rapid declines in TLC and hemoglobin, respectively, whereas only 29% of individuals whose CD4+ lymphocyte counts remained greater than 200 cells/mm3 showed rapid declines in either marker. Among those with declines, 59% of the declines in TLC and 55% of the declines in hemoglobin occurred before the first time that CD4+ lymphocyte counts fell to less than 200 cells/mm3.
In this study, there was significant prognostic value to identifying a rapid decline in TLC or hemoglobin concentration. A rapid decline in either of these markers was strongly associated with progression to clinically defined AIDS. This was independent of the corresponding marker levels and HIV RNA. Regardless of whether TLC levels were greater than 1200 cells/mm3, knowledge of a rapid decline in TLC or hemoglobin was informative, provided good operating characteristics (PPV and NPV), and occurred at a time when therapy may be indicated by CD4+ cell counts.
Absolute levels of TLC and hemoglobin concentration also contributed prognostic information for risk of clinical progression, supporting their use in evaluating patients who do not have longitudinal data. In this report, we have presented analyses of data from MACS participants within known seroconversion windows. We have also evaluated the predictive ability of these markers among seroprevalent individuals (ie, those whose date of seroconversion is not known) and have obtained results that are qualitatively and quantitatively similar to those presented here (data not shown). These analyses confirm the usefulness of these markers for monitoring patients with heterogeneous stages of disease, an important consideration for use in developing countries, where the time of HIV infection is generally unknown.
Our previous work with TLC and hemoglobin concentration focused on demonstrating that rapid declines in these markers occur after a period of stability. We have shown that these declines occur at a time that roughly coincides with the decline in CD3+ lymphocytes, which signals the failure of T-cell homeostasis, approximately 1.6 years before AIDS.18,19,25-27 Furthermore, we have previously defined what constitutes a rapid decline in a prospectively monitored individual.21 Although T-cell homeostasis failure has been linked to clinical progression of HIV infection,28 the present study is the first to show that rapid declines in TLC or hemoglobin predict progression to AIDS.
A limitation of this study was that the definition of a rapid decline of the markers (33.0% per year for TLC and 11.6% per year for hemoglobin) was based on the same data from individuals included within this study. This may have biased our results toward finding a significant association with progression to AIDS. Therefore, our results need to be replicated in other natural history studies. The association of more rapid declines with shorter times to AIDS provides added credibility to our analysis, however. Additionally, there are many methods for determining declines in marker values that may be more prognostic. The advantage of the method used in this study is its simplicity, because it entails a difference in 2 means normalized by the time interval separating the measurements. Furthermore, visits within the MACS are approximately 6 months apart, and it is likely that with more frequent monitoring, the prognostic value of the markers may be improved.
Biologically, the rapid decline in TLC is likely the result of T-cell homeostasis failure, because most lymphocytes in HIV-infected men are T cells.19 The reason for a decline in hemoglobin around this time is less clear, however. Several causes of HIV-associated anemia have been hypothesized, including effects of plasma HIV on bone marrow stromal cells29,30 and disturbances of cytokines that affect hematopoiesis.31 We have previously shown on the population and individual levels that HIV RNA levels remain stable and then show accelerated increases before AIDS,20,32 possibly because of the expansion of HIV variants that use the CXCR4 coreceptor.27 If the rate of hematopoiesis is affected by HIV RNA levels, hemoglobin decline may be a result of the late increase in HIV RNA levels. Others have demonstrated similar effects of hemoglobin level, and the univariate RH of 1.78 for a reduction of 1-g/dL hemoglobin concentration was almost identical to that observed in the study8 that has examined hemoglobin as a continuous variable (RH = 1.81, 95% CI: 1.70-1.94).
Our results contribute to the increasing evidence of the utility of TLC and hemoglobin markers for assessing clinical prognosis when CD4+ lymphocyte counts and HIV RNA levels are not available. We found that a TLC ≤1200 cells/mm3 was predictive of AIDS, supporting WHO guidelines. Furthermore, these data support supplementing the WHO guidelines so that individuals are monitored not only for low TLC or hemoglobin levels but for marker declines. Determining TLC and hemoglobin levels is much less expensive and requires less training than ascertaining CD4+ counts and HIV RNA levels. Nevertheless, it is critical that consideration be given to the endemic diseases of a region that might affect TLC or hemoglobin levels, because these results based on data from a US population might not be applicable. Therefore, validation of these results may be warranted in such regions. Other markers, including albumin levels, have also demonstrated strong associations with clinical progression.33-35 Given the strong predictive value of TLC and hemoglobin concentrations and the relative inexpensiveness of measurement of these markers, further studies are warranted to evaluate these markers in countries with limited resources. Finally, because a rapid decline in hemoglobin was prognostic for progression to AIDS independent of CD4+ counts and HIV RNA levels, monitoring patients for a decline may be useful even in the developed world.
Data in this manuscript were collected by the MACS, with centers (Principal Investigators) located at the Johns Hopkins Bloomberg School of Public Health (Joseph Margolick); Howard Brown Health Center and Northwestern University Medical School (John Phair); University of California, Los Angeles (Roger Detels); University of Pittsburgh (Charles Rinaldo); and Data Analysis Center (Lisa Jacobson).
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