James W. Bremer

Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response

The duration of disease-free survival after infection with human immunodeficiency virus type 1 (HIV-1) varies considerably during antiretroviral therapy. Patients with similar CD4+ lymphocyte counts progress at different rates when they are given the same antiretroviral therapy. Better prediction of risk for progression and its association with viral suppression may help improve antiretroviral management for individual patients and speed the development of new drugs. Higher plasma HIV-1 RNA levels are associated with poorer clinical status and lower CD4+ lymphocyte counts [1-3] and predict subsequent outcome [4-11]. The biological variability of plasma HIV-1 RNA levels in patients receiving stable therapeutic regimens must be quantified to define the magnitude of an antiviral effect that can be reliably detected after antiretroviral treatment is initiated. Determination of infectious HIV-1 titers in mononuclear cells of peripheral blood by quantitative microculture [12, 13] or syncytium-inducing phenotype of an HIV-1 isolate may provide information that is different from or complementary to the information gleaned from measuring plasma HIV-1 RNA levels [14-16]. However, studies have not yet conclusively determined whether measurements of CD4+ lymphocytes in conjunction with any or all of these virological variables should be recommended to optimize prediction or guide antiretroviral treatment more effectively. In this report, we quantify the relative roles of CD4+ lymphocyte counts, plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and the syncytium-inducing viral phenotype as predictors of disease progression during a clinical trial of combination therapy [17]. Our approach was to assess the value of plasma HIV-1 RNA levels and CD4+ lymphocyte count, both of which are readily available to clinicians, and then to assess the additional value of the infectious HIV-1 titer in mononuclear cells of peripheral blood and the syncytium-inducing viral phenotype. We also quantify the variability of plasma HIV-1 RNA levels. Our results suggest guidelines for using these measures in clinical practice for predicting the effectiveness of antiretroviral therapy over 1 year. Methods Study Design We prospectively evaluated virological, immunologic, and clinical data from patients who participated in the intensive virology substudy of ACTG (AIDS Clinical Trials Group) Protocol 241; ACTG Protocol 241 was a multicenter, randomized, double-blind, placebo-controlled trial of 398 patients receiving nevirapine, zidovudine, and didanosine compared with zidovudine and didanosine [17]. All patients at 8 of the 16 AIDS Clinical Trials Units who participated in the main study were enrolled in the substudy (n = 198). For 48 weeks, all 198 patients received open-label zidovudine (600 mg/d) and didanosine (400 mg/d for patients weighing 60 kg and 250 mg/d for patients weighing <60 kg). One hundred of the substudy patients were randomly assigned to receive nevirapine (200 mg/d for the first 2 weeks and 400 mg/d thereafter), and 98 were assigned to receive matching placebo. Participants gave written informed consent, and the protocol was approved by the institutional review board at each participating AIDS Clinical Trials Unit. The study was funded by the ACTG of the National Institute of Allergy and Infectious Diseases; supplemental funding for virology was provided by Boehringer Ingelheim Pharmaceuticals (Ridgefield, Connecticut). Study drugs were provided by Glaxo Wellcome (Research Triangle Park, North Carolina), Bristol-Myers Squibb (Princeton, New Jersey), and Boehringer Ingelheim Pharmaceuticals. However, all data were gathered by members of the ACTG and were analyzed and interpreted by the authors, who had sole responsibility for the decision to submit the manuscript for publication. Evaluation of Patients Stable therapy at baseline was defined as the absence of reported change in antiretroviral therapy from 30 days before the preentry visit until the entry visit. All patients were followed prospectively for progression of HIV-related disease. Progression was defined as the development of a new acquired immunodeficiency syndrome (AIDS)-defining event [18]; a newly diagnosed, deep-seated bacterial infection or bacteremia that was not related to injection drug use or an intravascular catheter; pulmonary or extrapulmonary tuberculosis; recurrent Pneumocystis carinii pneumonia; recurrent toxoplasmosis of the central nervous system; or death. Reports of disease progression were reviewed by the study chair; only events that could be confirmed were used in the analysis. We measured CD4+ lymphocyte counts, plasma HIV-1 RNA levels, and infectious HIV-1 titers in mononuclear cells of peripheral blood at the preentry visit (within 14 days of starting study treatment), at the entry visit (before starting study treatment and at least 72 hours after the preentry visit), and at the visits 8 and 48 weeks after the start of study treatment. Specimens could be obtained at any time of day. We used the geometric mean of preentry and entry measurements as the baseline value for each variable. The presence of the syncytium-inducing viral phenotype was determined at the entry visit. Standardized assays were used to determine CD4+ lymphocyte counts [19, 20], infectious HIV-1 titer in mononuclear cells of peripheral blood (in infectious units per million cells) using real-time testing [13, 21], and syncytium-inducing viral phenotype of a virus isolated from mononuclear cells of peripheral blood using MT-2 cells [22]. Plasma samples were frozen at 70C; HIV-1 RNA levels were measured by quantitative reverse transcription polymerase chain reaction assay (Roche Molecular Systems, Alameda, California, and Branchburg, New Jersey) [23]. The lower limit of detection for this assay was 200 HIV-1 RNA copies/mL. Levels of HIV-1 RNA in plasma samples collected from the same patient at the preentry, entry, week 8, and week 48 visits were determined in a single laboratory assay. Statistical Analysis Analysis of plasma HIV-1 RNA levels and infectious HIV-1 titers in mononuclear cells of peripheral blood was done after log10 transformation. Plasma levels of HIV-1 RNA that were below the detectable limit were assigned the value of 200 copies/mL. Infectious HIV-1 titers in mononuclear cells of peripheral blood outside the measurable range (0.22 to 7493 infectious units per million cells) were assigned the value of 0.22 infectious units per million cells if they were below the range and 7493 infectious units per million cells if they were above the range. Linear regression analysis [24] was used to compare the mean plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and CD4+ lymphocyte counts according to patient characteristics at baseline and to assess factors associated with the long-term change (from baseline to week 48) in CD4+ lymphocyte counts. Logistic regression analysis [25] was used to assess the association at baseline of the percentage of patients who had AIDS with virological measures and CD4+ lymphocyte counts. The intrapatient SD of plasma HIV-1 RNA levels was estimated using the method of moments for variance components [26]. Spearman correlation coefficients were used to assess the association between preentry and entry measurements. Proportional hazards models [27] were used to assess the association between the risk for disease progression or death and baseline levels and early changes (from baseline to week 8) in plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and log-transformed CD4+ lymphocyte counts as well as baseline syncytium-inducing viral phenotype. These models were stratified by study treatment to control for any differential effects of the two study regimens. Results Patient Characteristics at Study Entry The mean CD4+ lymphocyte count of the 198 patients before treatment was 145 cells/mm3 (range, 1 to 443 cells/mm3). Patients were a median of 39 years of age, predominantly male (81%), predominantly white (76%), and predominantly free of a previous AIDS-defining diagnosis (86%). All but 3 patients had taken zidovudine before study entry, 44% had taken didanosine, and 35% had taken zalcitabine. The median duration of cumulative previous nucleoside therapy was 25 months, and 34% of patients had received therapy for longer than 36 months. Virological Measures at Baseline by Patient Characteristics Table 1 shows the mean plasma HIV-1 RNA levels, infectious HIV-1 titers in mononuclear cells of peripheral blood, and CD4+ lymphocyte counts at baseline for patients stratified by characteristics that were significantly associated with viral load. We also assessed the associations with age, sex, racial or ethnic group, self-reported homosexuality, and duration of previous nucleoside therapy, but these associations were not significant. Table 1. Plasma HIV-1 RNA Level, Infectious HIV-1 Titer in Mononuclear Cells of Peripheral Blood, and CD4+ Lymphocyte Count at Baseline* Patients with a history of AIDS had a significantly higher mean baseline level of HIV-1 RNA in plasma and a significantly lower mean CD4+ lymphocyte count than did those without such a history (Table 1). More patients with a history of AIDS than those without had baseline HIV-1 isolates with the syncytium-inducing viral phenotype (58% compared with 36%; P = 0.015). However, in a multivariate analysis, only the CD4+ lymphocyte count at baseline was significantly associated with a history of AIDS. Thus, disease status at baseline was explained by CD4+ lymphocyte counts and not by any of the virological measures that were considered. Variability of Virological Measures in Patients Receiving Stable Treatment Variation in plasma HIV-1 RNA levels was evaluated by comparing the preentry and entry measures from the 167 patients who reported no changes in treatment from 30 days

Female Genital-Tract HIV Load Correlates Inversely with Lactobacillus Species but Positively with Bacterial Vaginosis and Mycoplasma hominis

BACKGROUND Bacterial vaginosis (BV) is associated with human immunodeficiency virus (HIV) acquisition. We examined the association between BV and BV-associated bacteria and expression of HIV in the female genital tract. METHODS HIV RNA, lactobacilli, Gardnerella vaginalis, and Mycoplasma hominis in cervicovaginal lavage (CVL) samples were quantified by polymerase chain reaction (PCR). Gynecologic evaluation included Nugent score assessment, Amsel criteria assessment, detection of other genital-tract infections, and dysplasia grading. CD4 cell count, plasma HIV RNA level, and antiretroviral history were obtained. RESULTS A total of 203 CVL samples from women with Nugent scores of 7-10 (BV group) and 203 samples from women with Nugent scores of 0-3 (no-BV group) were matched by plasma HIV RNA level and analyzed. After controlling for plasma HIV RNA level and Nugent score in univariate analyses, we found that G. vaginalis and M. hominis bacterial counts, Candida vaginitis, and herpes simplex virus (HSV) were positively associated with CVL HIV RNA levels. In multivariate analysis, only lactobacilli bacterial counts (P=.006; inverse association), M. hominis bacterial counts (P=.0001; positive association), Candida vaginitis (P=.007), and HSV (P=.03) were significantly associated with CVL HIV RNA levels. CONCLUSION Bacteria associated with BV increase genital-tract HIV RNA levels. Quantitative bacterial counts for lactobacilli and M. hominis are better correlates of CVL HIV RNA than are Nugent score or Amsel criteria. Since plasma virus and CD4 cell levels did not differ between the BV and no-BV groups, these data suggest that the bacterial flora associated with BV influence genital-tract HIV shedding.

Multicenter Evaluation of Use of Dried Blood and Plasma Spot Specimens in Quantitative Assays for Human Immunodeficiency Virus RNA: Measurement, Precision, and RNA Stability

ABSTRACT Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at −70°C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy.

HIV-1 in genital tract and plasma of women: Compartmentalization of viral sequences, coreceptor usage, and glycosylation

Worldwide, 90% of HIV-1 infections are transmitted heterosexually. Because the genital mucosa are the sites of initial contact with HIV-1 for most exposed individuals, study of the virus from the genital tract is critical for the development of vaccines and therapeutics. Previous analyses of HIV-1 in various tissues have documented compartmentalization of viral genomes. Whether compartmentalization was associated with viral phenotypic differences or immune status, however, was not well understood. We compared HIV-1 gp120 env sequences from the genital tract and plasma of 12 women. Eight women displayed compartmentalized HIV-1 RNA genomes, with viral sequences from each site that were clearly discrete, yet phylogenetically related. The remaining four exhibited env sequences that were intermingled between the two sites. Women with compartmentalized HIV-1 genomes had higher CD4+ cell counts than those displaying intermingled strains (P = 0.02). Intrapatient HIV-1 recombinants comprising sequences that were characteristic of both sites were identified. We next compared viral phenotypes in each compartment. HIV-1 coreceptor usage was often compartmentalized (P ≤ 0.01). The number of N-linked glycosylation sites, associated with neutralization resistance, also differed between compartments (P < 0.01). Furthermore, disparities between the density of gp120 glycosylations in each compartment correlated with higher CD4+ counts (P = 0.03). These data demonstrate that the genital tract and plasma can harbor populations of replicating HIV-1 with different phenotypes. The association of higher CD4+ cell counts with compartmentalization of viral genomes and density of gp120 glycosylations suggests that the immune response influences the development of viral genotypes in each compartment. These findings are relevant to the prevention and control of HIV-1 infection.

Blinded, Multicenter Comparison of Methods To Detect a Drug-Resistant Mutant of Human Immunodeficiency Virus Type 1 at Low Frequency

ABSTRACT We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.1 to 0.4%. These technologies should help define the impact of low-frequency drug-resistant mutants on response to antiretroviral therapy.

Analysis and Characterization of Antiviral Drug–Resistant Cytomegalovirus Isolates from Solid Organ Transplant Recipients

The development of cytomegalovirus (CMV) disease and subsequent emergence of drug-resistant strains was examined in a large group of solid organ transplant recipients; drug-resistant CMV was detected in a total of 30 transplant recipients (20 lung, 5 kidney, 4 heart, and 1 liver). Drug resistance was confirmed both phenotypically and genotypically. The sequences of drug-resistant CMV strains from the same patient differed from drug-susceptible baseline sequences only at single sites previously confirmed to confer drug resistance. At least 1 isolate from each patient had a mutation in the UL97 phosphotransferase coding sequence. Mutations in the DNA polymerase gene were found in 6 of 38 sequenced strains. Lung transplant recipients had the highest incidence of drug-resistant virus: of the 30 patients, 28 were CMV-seronegative transplant recipients of CMV-seropositive organs, which strongly supports the premise that drug resistance is most prevalent in that transplant population.

Underestimation of HIV Type 1 Drug Resistance Mutations: Results from the ENVA-2 Genotyping Proficiency Program

Current recommendations indicate the use of HIV-1 drug resistance genotyping in the treatment of HIV-1 infection, primarily on treatment failure, and in specific instances also before the initiation of therapy. As such, HIV-1 genotyping is becoming a standard of care parameter in HIV-1 treatment monitoring and a rapidly increasing number of laboratories now use this technology routinely. A study of proficiency, using the ENVA-2 panel, was performed to evaluate the current HIV-1 resistance genotyping quality in 34 laboratories from different parts of the world. The results demonstrated extensive interlaboratory variation in the quality of genotyping and a significant underestimation of resistance mutations, even in samples expressing pure mutant genotype. The level of variation could not be attributed to the sequencing technology used and was therefore considered to be laboratory associated. The direct clinical consequences of this may be inadequate treatment of HIV-1-infected individuals and a more rapid exhaustion of therapeutic options for the patients. Drug resistance mutations are frequently missed. Therefore, quality control programs are urgently needed. Until these are widely implemented, clinicians must consider this issue and interpret the reported genotyping results with caution.

Evaluation of Dried Blood Spots for Human Immunodeficiency Virus Type 1 Drug Resistance Testing

ABSTRACT Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at −20°C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at −20°C or at −70°C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that −20°C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.

Cell-associated genital tract virus and vertical transmission of human immunodeficiency virus type 1 in antiretroviral-experienced women.

To determine the association between genital tract shedding of human immunodeficiency virus (HIV) type 1 and vertical transmission, a case-control substudy was conducted within the Women and Infants Transmission Study. Antenatal cervicovaginal lavage specimens were assessed for HIV-1 RNA in the supernatant and HIV-1 RNA and DNA in cell pellets. Multivariate analyses compared 26 women who transmitted HIV to their infants with 52 women who did not; 33% received combination antiretroviral therapy, and 65% received monotherapy. Adjusted odds ratios (ORs) for the presence (OR, 3.42; 95% confidence interval [CI], 0.76-15.4; P=.11) and titer (OR, 1.66; 95% CI, 0.93-2.99; P=.09) of HIV-1 DNA suggested that there is an independent association with vertical transmission. When analyses were restricted to vaginal and nonelective cesarean deliveries, each one-log increase in mean titer of HIV-1 DNA was associated with a significantly higher risk of transmission (OR, 2.28; 95% CI, 1.09-4.78; P=.03). The cell-associated genital tract compartment is important in the pathophysiology and prevention of vertical HIV-1 transmission.

The contribution of assay variation and biological variation to the total variability of plasma HIV-1 RNA measurements

OBJECTIVES To assess the specific contributions of assay variation and biological variation to the total variation of plasma HIV-1 RNA measured by the Roche Monitor assay and the extent to which batch assays reduced both assay variability and total variability compared with real-time determinations. DESIGN A retrospective analysis of data obtained from three trials conducted by the Adult and Pediatric AIDS Clinical Trials Groups (ATCG), the Women and Infants Transmission Study (WITS) and the NIAID-sponsored Virology Quality Assurance Program. METHODS Within-subject variation was assessed from stored, serially collected plasma samples from 663 subjects enrolled in the ACTG and WITS studies. Interassay and intra-assay variation were estimated from two of the clinical trials and 22 laboratories that participated in a quality assurance program and were used to estimate the effect of real-time testing on total variation. RESULTS The total variation (standard deviation) from a random effects model was 0.26 log10 RNA copies/ml. The estimated interassay variation was 0.08 log10 and intra-assay variation was 0.12 log10 RNA copies/ml. Biological variation accounted for 56-80% of total variation. The effect of real-time testing compared with batch testing was minimal. CONCLUSION Our estimates of total within-subject HIV-1 RNA variation support the current recommendation to obtain at least two specimens, preferably obtained less than 2 weeks apart, for viral RNA measurement before starting therapy. The major contribution of biological variation to the total variation supports the use of real-time HIV-1 RNA assays, provided that consistent specimen collection procedures are followed and acceptable assay proficiency is maintained.