Lawrence A. Kingsley

Prognosis in HIV-1 Infection Predicted by the Quantity of Virus in Plasma

The relation between viremia and clinical outcome in individuals infected with human immunodeficiency virus-type 1 (HIV-1) has important implications for therapeutic research and clinical care. HIV-1 RNA in plasma was quantified with a branched-DNA signal amplification assay as a measure of viral load in a cohort of 180 seropositive men studied for more than 10 years. The risk of acquired immunodeficiency syndrome (AIDS) and death in study subjects, including those with normal numbers of CD4+ T cells, was directly related to plasma viral load at study entry. Plasma viral load was a better predictor of progression to AIDS and death than was the number of CD4+ T cells.

Quantitation of HIV-1 RNA in Plasma Predicts Outcome after Seroconversion

The course of infection with human immunodeficiency virus type 1 (HIV-1) varies considerably. Although the median interval between HIV-1 infection and the development of the acquired immunodeficiency syndrome (AIDS) in adults is 10 to 11 years [1], some infected persons rapidly progress to AIDS in less than 5 years [2]. Still others remain asymptomatic without evidence of immunologic decline for more than 6 years [3]. The biological basis of this variability is unknown, but differences in viral strains, host immune responses [4], and exposure to microbial [5] or environmental cofactors probably contribute. The variable course of HIV-1 infection causes uncertainty for the infected person and complicates the design and interpretation of therapeutic trials because of unrecognized differences in prognosis. Many clinical and laboratory markers have been used to estimate prognosis in patients with HIV-1 infection [6]. Markers of AIDS development include HIV-related symptoms [7, 8], depletion of CD4+ T cells [9], cutaneous anergy [7, 10], elevated serum 2-microglobulin and neopterin levels [9], HIV-1 p24 (core) antigenemia [11, 12], and syncytium-inducing HIV-1 phenotype [13]. None of these markers is 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 [9], but less variable and earlier markers of risk for AIDS are needed. Several new methods have been developed to directly measure HIV-1 nucleic acid in body fluids. One of these technologies is the branched-DNA (bDNA) signal amplification method for quantitating HIV-1 RNA in plasma [14]. Although less sensitive than RNA detection by the polymerase chain reaction (PCR), the bDNA method has the advantage of large sample capacity, speed, reproducibility, and a format similar to an enzyme-linked immunosorbent assay. The ability of the bDNA assay or other HIV-1 RNA detection methods to predict clinical outcome in HIV-1 infection has not been clearly defined in appropriate cohorts or been compared with the ability of other predictive markers. Previous studies have shown a strong correlation between disease stage and the amount of circulating HIV-1, whether measured as cell-free infectious virus [15, 16], viral proteins [11, 12], or RNA [17, 18]. Recent studies have shown that an increase in HIV-1 expression in peripheral blood mononuclear cells can precede immunologic deterioration by 1 to 2 years [17, 18]. Our objective, therefore, was to compare plasma HIV-1 RNA with determinations of serum p24 antigen, neopterin, and 2-microglobulin levels and CD4+ T-cell counts as predictors of outcome in a cohort of homosexual men with documented HIV-1 seroconversion. Methods Study Populations The initial pilot study population consisted of 10 seroprevalent men (unknown date of seroconversion) enrolled in the Pittsburgh portion of the Multicenter AIDS Cohort Study (MACS). Five of these men developed AIDS (Centers for Disease Control and Prevention [CDC] 1987 definition) after 35 to 74 months of follow-up (median, 59 months), and five remained asymptomatic with stable CD4+ T-cell counts after a similar follow-up interval (median, 56 months). The second study population consisted of 62 homosexual men enrolled in the MACS who had documented seroconversion (change from negativity for HIV-1 antibody to positivity). Eighteen of these men progressed to AIDS (CDC 1987 definition) by a median of 3.8 years after seroconversion (maximum, 6.5 years), and 44 did not develop AIDS after a median follow-up of 5.4 years (maximum, 8.3 years). Details about the recruitment and characteristics of the MACS cohort have been described previously [19]. All participants gave written informed consent, and the MACS protocol was approved by the Internal Review Board of the University of Pittsburgh. Study Samples The study samples were selected from stored ( 70C) longitudinal plasma and serum samples obtained from enrollees at 6-month intervals as part of the MACS protocol. In patients who developed AIDS, the samples tested were obtained from the seroconversion visit (first visit at which the patient was positive for the HIV-1 antibody), the most recent visit before AIDS diagnosis, and equally spaced visits in between. In patients without AIDS, the samples tested were obtained from the seroconversion visit; visits 1, 2, and 3 years after seroconversion; and the last available visit, which occurred as long as 8.3 years after seroconversion. Definition of Outcomes Study patients were classified into one of three outcome groups: 1) AIDS; 2) decline in the CD4 count; and 3) stable CD4 count. Patients in the AIDS outcome group (n = 18) met the CDC 1987 case definition for AIDS. For each patient who had seroconversion and did not develop AIDS, we used linear regression to fit a line through prospective CD4+ T-cell measurements (minimum of three measurements per patient). We calculated the slope of each line and determined the statistical significance of the negative slopes. Patients with declining CD4 counts (n = 21) had statistically significant (P < 0.05) negative slopes but did not develop AIDS during follow-up. Patients with stable CD4 counts (n = 23) had no significant decline in the CD4+ T-cell count during follow-up, and 6 of 23 patients (26.1%) had a positive slope, that is, an increasing linear trend in the number of CD4+ T cells. Measurement of T-Lymphocyte Subsets We measured T-lymphocyte subsets in whole blood by staining them with fluorescent dye-conjugated monoclonal antibodies specific for CD3, CD4, and CD8 (Becton Dickinson, Mountain View, California) as previously described [20]. The total number of CD4+ T cells was determined by multiplying the percentage of lymphocytes that were CD4+ T cells by the total lymphocyte count. Serum 2-Microglobulin and Neopterin Assays We measured serum 2-microglobulin (Kabi Pharmacia, Uppsala, Sweden) and serum neopterin levels (Henning, Berlin, Germany) with commercial radioimmunoassays and standards provided by the manufacturers. Four replicates of normal control serum were included in each assay to assess variability. The coefficient of variation for control samples was 15% or less. Serum Immune Complex Dissociated p24 Assay Immune complex dissociated (ICD) p24 antigen levels were measured with a commercial enzyme immunoassay (Dupont, NEN Products, Wilmington, Delaware). The ICD p24 antigen levels in serum were interpolated from a standard curve provided by the manufacturer. The assay has a sensitivity of 12 pg of p24 antigen/mL. The interassay coefficient of variation for the p24 standards was less than 10%. Plasma and Cellular HIV-1 RNA Assays Levels of HIV-1 RNA in plasma samples were quantitated with the Quantiplex HIV-1 RNA assay, which is based on bDNA signal amplification technology (Chiron Corp., Emeryville, California). This assay measures HIV-1 RNA associated with viral particles that are pelleted from 1.0-mL plasma samples (23 500 g for 1 hour at 4 C). The assay has a quantitation limit of 1 104 HIV-1 genome equivalents per mL of plasma (Eq/mL) and is linear at levels as high as 1.6 106 Eq/mL. For this study, the interassay coefficient of variation for the positive control samples run with each assay was 11.2%. Additional details about the assay procedure and its performance characteristics have been described previously [14]. We categorized longitudinal plasma HIV-1 RNA results from individual patients into one of four groups: 1) detection of HIV-1 RNA (>1 104 Eq/mL) in all samples tested [n = 9]; 2) detection in most ( 50%) samples [n = 24; mean percentage of positive samples, 67.3%]; 3) detection in fewer than 50% of samples [n = 16; mean percentage of positive samples, 29.3%]; and 4) detection in none of the samples tested (n = 13). We identified an additional subgroup (n = 6) that showed evidence for clearance of detectable HIV-1 RNA from plasma, that is, two or more consecutive negative samples and no further positive samples after one or two initial positive samples. Assays for neopterin, 2-microglobulin, ICD p24, and HIV-1 RNA were done in duplicate on coded serum or plasma samples. Samples from a given patient were batch-tested to minimize the potential effect of interassay variability. Semi-quantitative PCR-based assays for cellular HIV-1 gag RNA were done on stored peripheral blood mononuclear cell samples as described previously [17]. Statistical Analyses The pilot study data are shown in the tables and figures to familiarize the reader with the raw data obtained from the bDNA assay. All cellular PCR results were adjusted per million CD4+ T cells. Analyses of the data set from patients with HIV-1 seroconversion were similarly stratified by outcome group. The Fisher exact test, chi-square test, and Wilcoxon rank-sum test were done where noted in the text. We estimated the association between progression to AIDS and laboratory covariates at seroconversion by multiple logistic regression analysis using BMDP statistical software (BMDP Statistical Software, Inc., Los Angeles, California). Results HIV-1 Quantitation by Branched DNA and Polymerase Chain Reaction in Seroprevalent Patients An initial pilot study of plasma HIV-1 RNA quantitation was done in 10 seroprevalent men enrolled in the MACS. Five of the men developed AIDS after 35 to 64 months of follow-up (progressors), and 5 remained asymptomatic with stable CD4+ T-cell counts (nonprogressors) after 38 to 74 months of follow-up. The median duration of follow-up for progressors and nonprogressors was similar (59 and 56 months, respectively). The bDNA assay was done on stored longitudinal plasma samples from 4 to 6 time points for each patient. Figure 1 shows the plasma HIV-1 RNA levels in the nonprogressors and progressors. Levels of HIV-1 RNA in all five nonprogressors were less than the limit of quantitation (<1 104 Eq/mL) at each time point dur

Seroconversion to Antibodies against Kaposi's Sarcoma–Associated Herpesvirus–Related Latent Nuclear Antigens before the Development of Kaposi's Sarcoma

BACKGROUND If Kaposis sarcoma-associated herpesvirus (KSHV) is the cause of Kaposis sarcoma, serologic evidence of infection should be present in patients before the disease develops. METHODS Using an immunoblot assay for two latent nuclear antigens of KSHV, we tested serum samples from homosexual male patients with the acquired immunodeficiency syndrome (AIDS) with and without Kaposis sarcoma (HIV-infected men with hemophilia), HIV-seronegative blood donors, and HIV-seronegative patients with high titers of antibodies against Epstein-Barr virus (EBV). Serial serum samples obtained from patients with Kaposis sarcoma before the diagnosis of the disease were tested for evidence of seroconversion. RESULTS Of 40 patients with Kaposis sarcoma, 32 (80 percent) were positive for antibodies against KSHV antigens by the immunoblot assay, as compared with only 7 of 40 homosexual men (18 percent) without Kaposis sarcoma immediately before the onset of AIDS. Of 122 blood donors, 22 EBV-infected patients, and 20 HIV-infected men with hemophilia, none were seropositive. When studied by the immunoblot assay over a period of 13 to 103 months, 21 of the 40 patients with Kaposis sarcoma (52 percent) seroconverted 6 to 75 months before the clinical appearance of Kaposis sarcoma. The median duration of antibody seropositivity for KSHV-related latent nuclear antigens before the diagnosis of Kaposis sarcoma was 33 months. CONCLUSIONS In most patients with kaposis sarcoma and AIDS, seroconversion to positivity for antibodies against KSHV-related nuclear antigens occurs before the clinical appearance of Kaposis sarcoma. This supports the hypothesis that Kaposis sarcoma results from infection with KSHV.

Kaposi's sarcoma-associated herpesvirus infection prior to onset of Kaposi's sarcoma

Objectives:Kaposis sarcoma-associated herpesvirus (KSHV), a newly discovered human gammaherpesvirus, is found in the majority of KS lesions from patients with and without AIDS. Peripheral blood mononuclear cells (PBMC) were examined for KSHV DNA to determine whether viral infection precedes onset of this neoplasm. Design:Randomized and blinded case–control study of prospectively collected PBMC samples from ongoing cohort studies. Methods:Paired PBMC drawn before and after KS onset from 21 AIDS-KS patients were compared to paired PBMC from 23 high-risk HIV-infected homo-/bisexual patients who did not develop KS and to a single PBMC sample from 19 low-risk, HIV-infected hemophiliac patients. Extracted DNA samples were amplified by polymerase chain reaction (PCR) using two non-overlapping nested primer sets to control for potential PCR contamination. Results:In all comparisons, patients who went on to develop KS were significantly more likely to show evidence of KSHV infection prior to onset of KS than either control group. Of PBMC samples from AIDS-KS patients drawn prior to KS, 52% were positive for KSHV DNA whereas both high- and low-risk control groups had lower rates of PBMC infection (9–13%). KSHV infection can precede KS onset by up to 21 months among AIDS-KS patients. Conclusions:AIDS-KS patients are significantly more likely to show evidence of KSHV infection in PBMC prior to KS onset than control HIV-infected patients. Because identical PBMC samples from cases and controls were examined blindly, these results are not caused by a bias in tissue sampling. Homo-/bisexual and hemophiliac AIDS patients who do not develop KS appear to have a low prevalence of infection. These findings indicate that KSHV infection is specifically associated with the subsequent development of KS in AIDS patients.

RISK FACTORS FOR SEROCONVERSION TO HUMAN IMMUNODEFICIENCY VIRUS AMONG MALE HOMOSEXUALS: Results from the Multicenter AIDS Cohort Study

2507 homosexual men who were seronegative for human immunodeficiency virus (HIV) at enrollment were followed for six months to elucidate risk factors for seroconversion to HIV. 95 (3.8%) seroconverted. Of men who did not engage in receptive anal intercourse within six months before baseline and in the six-month follow-up period, only 0.5% (3/646) seroconverted to HIV. By contrast, of men who engaged in receptive anal intercourse with two or more partners during each of these successive six-month intervals, 10.6% (58/548) seroconverted. No HIV seroconversions occurred in 220 homosexual men who did not practise receptive or insertive anal intercourse within twelve months before the follow-up visit. On multivariate analysis receptive anal intercourse was the only significant risk factor for seroconversion to HIV, the risk ratio increasing from 3-fold for one partner to 18-fold for five or more partners. Furthermore, data for the two successive six-month periods show that men who reduced or stopped the practice of receptive anal intercourse significantly lowered their risk of seroconversion to 3.2% and 1.8%, respectively. Receptive anal intercourse accounted for nearly all new HIV infections among the homosexual men enrolled in this study, and the hazards of this practice need to be emphasised in community educational projects.

Determinants of heterogeneous adherence to HIV-antiretroviral therapies in the multicenter AIDS cohort study

Summary: Assessment of adherence to HIV antiretroviral therapy (ART) is required for studying therapeutic effectiveness and identifying subgroups needing focused education. The studys goals were to describe the level of ART adherence using selfreported recall over a 4‐day period and to characterize determinants of lower adherence. The interaction between adherence and drug holidays on level of HIV RNA also was investigated. Perfect self‐reported adherence was defined as taking all doses and numbers of pills as prescribed for current HIV medications. Independent predictors of <100% adherence were determined using multivariate logistic regression. Among 539 men, 419 (77.7%) were 100% adherent by the algorithm using self‐reported data. HIV‐1 RNA was <50 copies/ml in 48.2% of the adherent group versus 33.7% in the less adherent group (p = .015). This proportion dropped to 28% if a drug holiday was reported in addition to lower adherence. A drug holiday was not virologically detrimental if the participant was otherwise adherent. Determinants of lower adherence included African American race (odds ratio [OR], 2.4; p = .008), income <U.S.

Cumulative exposure to nucleoside analogue reverse transcriptase inhibitors is associated with insulin resistance markers in the Multicenter AIDS Cohort Study

50,000 (OR, 2.2; p = .002), no outpatient visits (OR, 3.6; p = .003) and increasing numbers of ART medications (OR, 4.5; p = .001). These data support the validity of using a questionnaire to assess adherence in observational studies. Identification of individuals with characteristics associated with lower adherence provides the basis for interventions to enhance adherence and optimize effective therapies.

Low CD4+ T cell count as a major atherosclerosis risk factor in HIV-infected women and men

Objective:To estimate insulin resistance and its relationship to antiretroviral therapy (ART) in a cohort of HIV-infected persons with comparison to HIV-seronegative controls. Design:Prospective cohort of 533 HIV-infected and 755 HIV-seronegative men in the Multicenter AIDS Cohort Study evaluated at 6-month intervals between 1999 and 2003. Methods:Recent ART exposure was assessed by type of treatment in the preceding 6 months [i.e., no ART, monotherapy, combination ART, or highly active antiretroviral therapy (HAART) with and without a protease inhibitor (PI)]. Cumulative exposure was determined for the three major ART classes and for individual medications within each class. Two endpoints, a modified QUICKI index, 100 × 1/[log10(glucose) + log10(insulin)] and fasting hyperinsulinemia (insulin > 15 μU/ml), were assessed. All statistical models were adjusted for age, body mass index, race, nadir CD4 cell count, hepatitis C serostatus and family history of diabetes mellitus. Results:Each of the HIV-infected groups had higher odds of hyperinsulinemia and lower mean QUICKI than the HIV-seronegative men. Each additional year of exposure to nucleoside analogue reverse transcriptase inhibitors (NRTI) was associated with increased odds of hyperinsulinemia [odds ratio (OR), 1.08; 95% confidence interval (CI), 1.02–1.13) and a lower QUICKI (−0.04; 95% CI, −0.07 to −0.01). Cumulative exposure to non-nucleoside analogue reverse transcriptase inhibitors or PI drugs was not associated with either insulin resistance marker. Of individual medications examined, stavudine was associated with the highest risk of hyperinsulinemia (OR, 1.2; 95% CI, 1.2–1.3). Conclusions:Fasting surrogate markers suggest increased insulin resistance in HIV-infected men, which is related to cumulative NRTI exposure.

Ten-Year Predicted Coronary Heart Disease Risk in HIV-Infected Men and Women

Objective:To assess the association of HIV infection, HIV disease parameters (including CD4+ T-cell counts, HIV viral load, and AIDS) and antiretroviral medication use with subclinical carotid artery atherosclerosis. Design:Cross-sectional study nested within a prospective cohort study. Methods:Among participants in the Womens Interagency HIV Study (1331 HIV-infected women, 534 HIV-uninfected women) and Multicenter AIDS Cohort Study (600 HIV-infected men, 325 HIV-uninfected men), we measured subclinical carotid artery lesions and common carotid artery intima-media thickness using B-mode ultrasound. We estimated adjusted mean carotid artery intima-media thickness differences and prevalence ratios for carotid lesions associated with HIV-related disease and treatments, with multivariate adjustment to control for possible confounding variables. Results:Among HIV-infected individuals, a low CD4+ T-cell count was independently associated with an increased prevalence of carotid lesions. Compared with the reference group of HIV-uninfected individuals, the adjusted prevalence ratio for lesions among HIV-infected individuals with CD4+ T-cell count less than 200 cells/μl was 2.00 (95% confidence interval, 1.22–3.28) in women and 1.74 (95% confidence interval, 1.04–2.93) in men. No consistent association of antiretroviral medications with carotid atherosclerosis was observed, except for a borderline significant association between protease inhibitor use and carotid lesions in men (with no association among women). History of clinical AIDS and HIV viral load were not significantly associated with carotid atherosclerosis. Conclusion:Beyond traditional cardiovascular disease risk factors, low CD4+ T-cell count is the most robust risk factor for increased subclinical carotid atherosclerosis in HIV-infected women and men.

Weight Loss Prior to Clinical Aids as a Predictor of Survival

BACKGROUND Highly active antiretroviral therapy (HAART), in addition to traditional vascular risk factors, may affect coronary heart disease (CHD) risk in individuals with human immunodeficiency virus (HIV) infection. METHODS Among HIV-infected (931 men and 1455 women) and HIV-uninfected (1099 men and 576 women) adults, the predicted risk of CHD was estimated on the basis of age, sex, lipid and blood pressure levels, the presence of diabetes, and smoking status. RESULTS Among HIV-infected men, 2% had moderate predicted risk of CHD (10-year CHD risk, 15%-25%), and 17% had high predicted risk (10-year CHD risk of > or = 25% or diabetes). Among HIV-infected women, 2% had moderate predicted CHD risk, and 12% had high predicted CHD risk. Compared with users of protease inhibitor-based HAART, the adjusted odds ratio (OR) for moderate-to-high risk of CHD was significantly lower among HAART-naive individuals (OR, 0.57; 95% confidence interval [CI], 0.36-0.89). Users of HAART that was not protease inhibitor based (OR, 0.74; 95% CI, 0.53-1.01) and former HAART users (OR, 0.68; 95% CI, 0.46-1.03) were also less likely than users of protease inhibitor-based HAART to have moderate-to-high CHD risk, although 95% CIs overlapped the null. Low income was associated with increased likelihood of moderate-to-high CHD risk (for annual income <