Charles R. Rinaldo

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.

Genetic Restriction of HIV-1 Infection and Progression to AIDS by a Deletion Allele of the CKR5 Structural Gene

The chemokine receptor 5 (CKR5) protein serves as a secondary receptor on CD4+ T lymphocytes for certain strains of human immunodeficiency virus-type 1 (HIV-1). The CKR5 structural gene was mapped to human chromosome 3p21, and a 32-base pair deletion allele (CKR5Δ32) was identified that is present at a frequency of ∼0.10 in the Caucasian population of the United States. An examination of 1955 patients included among six well-characterized acquired immunodeficiency syndrome (AIDS) cohort studies revealed that 17 deletion homozygotes occurred exclusively among 612 exposed HIV-1 antibody-negative individuals (2.8 percent) and not at all in 1343 HIV-1-infected individuals. The frequency of CKR5 deletion heterozygotes was significantly elevated in groups of individuals that had survived HIV-1 infection for more than 10 years, and, in some risk groups, twice as frequent as their occurrence in rapid progressors to AIDS. Survival analysis clearly shows that disease progression is slower in CKR5 deletion heterozygotes than in individuals homozygous for the normal CKR5 gene. The CKR5Δ32 deletion may act as a recessive restriction gene against HIV-1 infection and may exert a dominant phenotype of delaying progression to AIDS among infected individuals.

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

The Risk of Pneumocystis carinii Pneumonia among Men Infected with Human Immunodeficiency Virus Type 1

We assessed the risk of pneumonia due to Pneumocystis carinii in 1665 participants in the Multicenter AIDS Cohort Study who were seropositive for human immunodeficiency virus type 1 (HIV-1) but did not have the acquired immunodeficiency syndrome (AIDS) and were not receiving prophylaxis against P. carinii. During 48 months of follow-up, 168 participants (10.1 percent) had a first episode of P. carinii pneumonia. The risk was greatly increased in participants with CD4+ cell counts at base line of 200 per cubic millimeter or less (relative risk, 4.9; 95 percent confidence interval, 3.1 to 8.0). Although most participants (60.7 percent) described no HIV-1-related symptoms at the clinic visit at which a CD4+ cell count of 200 per cubic millimeter or less was first noted, this finding during follow-up was also associated with an increased risk of P. carinii pneumonia. The development of thrush or fever significantly and independently increased the risk of P. carinii pneumonia in these patients (adjusted relative risks, 1.86 and 2.15 for thrush and fever, respectively). Most participants with CD4+ cell counts above 200 per cubic millimeter who had P. carinii pneumonia within six months were symptomatic. We conclude that P. carinii pneumonia is unlikely to develop in HIV-1-infected patients unless their CD4+ cells are depleted to 200 per cubic millimeter or below or the patients are symptomatic, and therefore that prophylaxis should be reserved for such patients.

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.

Natural History of Human Immunodeficiency Virus Type 1 Viremia after Seroconversion and Proximal to AIDS in a Large Cohort of Homosexual Men

The natural history of human immunodeficiency virus type 1 (HIV-1) viremia and its association with clinical outcomes after seroconversion was characterized in a cohort of homosexual men. HIV-1 RNA was measured by reverse-transcription polymerase chain reaction (RT-PCR) in stored longitudinal plasma samples from 269 seroconverters. Subjects were generally antiretroviral drug naive for the first 3 years after seroconversion. The decline in CD4 lymphocyte counts was strongly associated with initial HIV RNA measurements. Both initial HIV RNA levels and slopes were associated with AIDS-free times. Median slopes were +0.18, +0.09, and -0.01 log10 copies/mL, respectively, for subjects developing AIDS <3, 3-7, and>7 years after seroconversion. In contrast, HIV RNA slopes in the 3 years preceding AIDS and HIV RNA levels at AIDS diagnosis showed little variation according to total AIDS-free time. HIV RNA load at the first HIV-seropositive visit ( approximately 3 months after seroconversion) was highly predictive of AIDS, and subsequent HIV RNA measurements showed even better prognostic discrimination.

Escape from the Dominant HLA-B27-Restricted Cytotoxic T-Lymphocyte Response in Gag Is Associated with a Dramatic Reduction in Human Immunodeficiency Virus Type 1 Replication

ABSTRACT Human leukocyte antigen (HLA)-B27-positive subjects are uncommon in their ability to control infection with human immunodeficiency virus type 1 (HIV-1). However, late viral escape from a narrowly directed immunodominant Gag-specific CD8+ T-lymphocyte (CTL) response has been linked to AIDS progression in these individuals. Identifying the mechanism of the immune-mediated control may provide critical insights into HIV-1 vaccine development. Here, we illustrate that the CTL escape mutation R264K in the HLA-B27-restricted KK10 epitope in the capsid resulted in a significant defect in viral replication in vitro. The R264K variant was impaired in generating late reverse transcription products, indicating that replication was blocked at a postentry step. Notably, the R264K mutation was associated in vivo with the development of a rare secondary mutation, S173A, which restored viral replication in vitro. Furthermore, infectivity of the R264K variant was rescued by the addition of cyclosporine A or infection of a cyclophilin A-deficient cell line. These data demonstrate a severe functional defect imposed by the R264K mutation during an early step in viral replication that is likely due to the inability of this variant to replicate efficiently in the presence of normal levels of cyclophilin A. We conclude that the impact of the R264K substitution on capsid structure constrains viral escape and enables long-term maintenance of the dominant CTL response against B27-KK10, providing an explanation for the protective effect of HLA-B27 during HIV infection.

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.

Low sensitivity of rapid diagnostic test for influenza

The QuickVue Influenza A+B Test (Quidel) was used to test nasal swab specimens obtained from persons with influenza-like illness in 3 different populations. Compared with reverse-transcriptase polymerase chain reaction, the test sensitivity was low for all populations (median, 27%; range, 19%-32%), whereas the specificity was high (median, 97%; range, 96%-99.6%).

The Effects on Survival of Early Treatment of Human Immunodeficiency Virus Infection

BACKGROUND Zidovudine has been shown to prolong survival in patients with the acquired immunodeficiency syndrome (AIDS) and, in persons with human immunodeficiency virus (HIV) infection but not AIDS, to delay the progression to AIDS. However, it is still uncertain whether treatment before the development of AIDS prolongs survival. METHODS We analyzed data from a cohort of 2162 high-risk men who were already seropositive for HIV type 1 (HIV-1) and 406 men who seroconverted from October 1986 through April 1991. There were 306 deaths. The probabilities of death were compared among men at similar stages of disease who began zidovudine therapy before the diagnosis of AIDS and among those who did not. Relative risks of death were calculated for each of five initial disease states on the basis of CD4+ cell counts and clinical symptoms and signs appearing over follow-up periods of 6, 12, 18, and 24 months. Adjustments were also made for the use of prophylaxis against Pneumocystis carinii pneumonia (PCP). RESULTS After we controlled for CD4+ cell count and symptoms, the use of zidovudine with or without PCP prophylaxis before the development of AIDS significantly reduced mortality in all follow-up periods. The relative risks of death were 0.43 (95 percent confidence interval, 0.23 to 0.78) at 6 months, 0.54 (95 percent confidence interval, 0.38 to 0.78) at 12 months, 0.59 (95 percent confidence interval, 0.44 to 0.79) at 18 months, and 0.67 (95 percent confidence interval, 0.52 to 0.86) at 24 months. After we adjusted for the effects of PCP prophylaxis, zidovudine alone significantly reduced mortality at 6, 12, and 18 months (relative risks, 0.45, 0.59, and 0.70, respectively), but not at 24 months (relative risk, 0.81). Among zidovudine users, those who also used PCP prophylaxis before the development of AIDS had significantly lower mortality at 18 and 24 months than those who did not (relative risks, 0.62 and 0.60, respectively). CONCLUSIONS The results of this study support the hypothesis that in HIV-1 infection, early treatment with zidovudine and PCP prophylaxis improves survival in addition to slowing the progression to AIDS.