John W. Mellors

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.

Treatment with Indinavir, Zidovudine, and Lamivudine in Adults with Human Immunodeficiency Virus Infection and Prior Antiretroviral Therapy

BACKGROUND The new protease inhibitors are potent inhibitors of the human immunodeficiency virus (HIV), and in combination with other antiretroviral drugs they may be able to cause profound and sustained suppression of HIV replication. METHODS In this double-blind study, 97 HIV-infected patients who had received zidovudine treatment for at least 6 months and had 50 to 400 CD4 cells per cubic millimeter and at least 20,000 copies of HIV RNA per milliliter were randomly assigned to one of three treatments for up to 52 weeks: 800 mg of indinavir every eight hours; 200 mg of zidovudine every eight hours combined with 150 mg of lamivudine twice daily; or all three drugs. The patients were followed to monitor the occurrence of adverse events and changes in viral load and CD4 cell counts. RESULTS The decrease in HIV RNA over the first 24 weeks was greater in the three-drug group than in the other groups (P<0.001 for each comparison). RNA levels decreased to less than 500 copies per milliliter at week 24 in 28 of 31 patients in the three-drug group (90 percent), 12 of 28 patients in the indinavir group (43 percent), and none of 30 patients in the zidovudine-lamivudine group. The increase in CD4 cell counts over the first 24 weeks was greater in the two groups receiving indinavir than in the zidovudine-lamivudine group (P< or =0.01 for each comparison). The changes in the viral load and the CD4 cell count persisted for up to 52 weeks. All the regimens were generally well tolerated. CONCLUSIONS In most HIV-infected patients with prior antiretroviral therapy, the combination of indinavir, zidovudine, and lamivudine reduces levels of HIV RNA to less than 500 copies per milliliter for as long as one year.

Antiretroviral Drug Resistance Testing in Adult HIV-1 Infection: 2008 Recommendations of an International AIDS Society-USA Panel

Resistance to antiretroviral drugs remains an important limitation to successful human immunodeficiency virus type 1 (HIV-1) therapy. Resistance testing can improve treatment outcomes for infected individuals. The availability of new drugs from various classes, standardization of resistance assays, and the development of viral tropism tests necessitate new guidelines for resistance testing. The International AIDS Society-USA convened a panel of physicians and scientists with expertise in drug-resistant HIV-1, drug management, and patient care to review recently published data and presentations at scientific conferences and to provide updated recommendations. Whenever possible, resistance testing is recommended at the time of HIV infection diagnosis as part of the initial comprehensive patient assessment, as well as in all cases of virologic failure. Tropism testing is recommended whenever the use of chemokine receptor 5 antagonists is contemplated. As the roll out of antiretroviral therapy continues in developing countries, drug resistance monitoring for both subtype B and non-subtype B strains of HIV will become increasingly important.

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

A computer protocol to predict myocardial infarction in emergency department patients with chest pain.

To achieve more appropriate triage to the coronary care unit of patients presenting with acute chest pain, we used clinical data on 1379 patients at two hospitals to construct a simple computer protocol to predict the presence of myocardial infarction. When we tested this protocol prospectively in 4770 patients at two university hospitals and four community hospitals, the computer-derived protocol had a significantly higher specificity (74 vs. 71 percent) in predicting the absence of infarction than physicians deciding whether to admit patients to the coronary care unit, and it had a similar sensitivity in detecting the presence of infarction (88.0 vs. 87.8 percent). Decisions based solely on the computer protocol would have reduced the admission of patients without infarction to the coronary care unit by 11.5 percent without adversely affecting the admission of patients in whom emergent complications developed that required intensive care. Although this protocol should not be used to override careful clinical judgment in individual cases, the computer protocol for the most part yields accurate estimates of the probability of myocardial infarction. Decisions about admission to the coronary care unit based on the protocol would have been as effective as those actually made by the unaided physicians who cared for the patients, and less costly. Whether physicians who are aided by the protocol perform better than unaided physicians cannot be determined without further study.

Class-sparing regimens for initial treatment of HIV-1 infection.

BACKGROUND The use of either efavirenz or lopinavir-ritonavir plus two nucleoside reverse-transcriptase inhibitors (NRTIs) is recommended for initial therapy for patients with human immunodeficiency virus type 1 (HIV-1) infection, but which of the two regimens has greater efficacy is not known. The alternative regimen of lopinavir-ritonavir plus efavirenz may prevent toxic effects associated with NRTIs. METHODS In an open-label study, we compared three regimens for initial therapy: efavirenz plus two NRTIs (efavirenz group), lopinavir-ritonavir plus two NRTIs (lopinavir-ritonavir group), and lopinavir-ritonavir plus efavirenz (NRTI-sparing group). We randomly assigned 757 patients with a median CD4 count of 191 cells per cubic millimeter and a median HIV-1 RNA level of 4.8 log10 copies per milliliter to the three groups. RESULTS At a median follow-up of 112 weeks, the time to virologic failure was longer in the efavirenz group than in the lopinavir-ritonavir group (P=0.006) but was not significantly different in the NRTI-sparing group from the time in either of the other two groups. At week 96, the proportion of patients with fewer than 50 copies of plasma HIV-1 RNA per milliliter was 89% in the efavirenz group, 77% in the lopinavir-ritonavir group, and 83% in the NRTI-sparing group (P=0.003 for the comparison between the efavirenz group and the lopinavir-ritonavir group). The groups did not differ significantly in the time to discontinuation because of toxic effects. At virologic failure, antiretroviral resistance mutations were more frequent in the NRTI-sparing group than in the other two groups. CONCLUSIONS Virologic failure was less likely in the efavirenz group than in the lopinavir-ritonavir group. The virologic efficacy of the NRTI-sparing regimen was similar to that of the efavirenz regimen but was more likely to be associated with drug resistance. (ClinicalTrials.gov number, NCT00050895 [ClinicalTrials.gov].).

Antiretroviral Drug Resistance Testing in Adults Infected with Human Immunodeficiency Virus Type 1: 2003 Recommendations of an International AIDS Society-USA Panel

New information about the benefits and limitations of testing for resistance to human immunodeficiency virus (HIV) type 1 (HIV-1) drugs has emerged. The International AIDS Society-USA convened a panel of physicians and scientists with expertise in antiretroviral drug management, HIV-1 drug resistance, and patient care to provide updated recommendations for HIV-1 resistance testing. Published data and presentations at scientific conferences, as well as strength of the evidence, were considered. Properly used resistance testing can improve virological outcome among HIV-infected individuals. Resistance testing is recommended in cases of acute or recent HIV infection, for certain patients who have been infected as long as 2 years or more prior to initiating therapy, in cases of antiretroviral failure, and during pregnancy. Limitations of resistance testing remain, and more study is needed to refine optimal use and interpretation.

Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy

Residual viremia can be detected in most HIV-1-infected patients on antiretroviral therapy despite suppression of plasma RNA to <50 copies per ml, but the source and duration of this viremia is currently unknown. Therefore, we analyzed longitudinal plasma samples from 40 patients enrolled in the Abbott M97-720 trial at baseline (pretherapy) and weeks 60 to 384 by using an HIV-1 RNA assay with single-copy sensitivity. All patients were on therapy (lopinavir/ritonavir, stavudine, and lamivudine) with plasma HIV RNA <50 copies per ml by week 96 of the study and thereafter. Single-copy assay results revealed that 77% of the patient samples had detectable low-level viremia (≥1 copy per ml), and all patients had at least one sample with detectable viremia. A nonlinear mixed effects model revealed a biphasic decline in plasma RNA levels occurring over weeks 60 to 384: an initial phase of decay with a half-life of 39 weeks and a subsequent phase with no perceptible decay. The level of pretherapy viremia extrapolated for each phase of decay was significantly correlated with total baseline viremia for each patient (R2 = 0.27, P = 0.001 and R2 = 0.19, P < 0.005, respectively), supporting a biological link between the extent of overall baseline viral infection and the infection of long-lived reservoirs. These data suggest that low-level persistent viremia appears to arise from at least two cell compartments, one in which viral production decays over time and a second in which viral production remains stable for at least 7 years.

New Real-Time Reverse Transcriptase-Initiated PCR Assay with Single-Copy Sensitivity for Human Immunodeficiency Virus Type 1 RNA in Plasma

ABSTRACT More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 106 per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

Depletion of latent HIV-1 infection in vivo: a proof-of-concept study

BACKGROUND Persistent infection in resting CD4+ T cells prevents eradication of HIV-1. Since the chromatin remodeling enzyme histone deacetylase 1 (HDAC1) maintains latency of integrated HIV, we tested the ability of the HDAC inhibitor valproic acid to deplete persistent, latent infection in resting CD4+ T cells. PROCEDURES We did a proof-of-concept study in four volunteers infected with HIV and on highly-active antiretroviral therapy (HAART). After intensifying the effect of HAART with subcutaneous enfuvirtide 90 mug twice daily for 4-6 weeks to prevent the spread of HIV, we added oral valproic acid 500-750 mg twice daily to their treatment regimen for 3 months. We quantified latent infection of resting CD4+ T cells before and after augmented treatment by limiting-dilution culture of resting CD4+ T cells after ex-vivo activation. FINDINGS The frequency of resting cell infection was stable before addition of enfuvirtide and valproic acid, but declined thereafter. This decline was significant in three of four patients (mean reduction 75%, range 68% to >84%). Patients had slight reactions to enfuvirtide at the injection site, but otherwise tolerated treatment well. INTERPRETATION Combination therapy with an HDAC inhibitor and intensified HAART safely accelerates clearance of HIV from resting CD4+ T cells in vivo, suggesting a new and practical approach to eliminate HIV infection in this persistent reservoir. This finding, though not definitive, suggests that new approaches will allow the cure of HIV in the future.