Viral Load Group Research Articles

Comparison of fluorescence quantitative PCR and direct PCR sequencing in the detection of HBV polymerase RT region

To explore the feasibility of Direct PCR sequencing in clinic and the significance of Direct PCR sequencing for retrieval treatment plan. To address this issue, a cross-sectional study on the drug resistance in the HBV polymerase RT region was performed using Fluorescence quantitative PCR and Direct PCR sequencing in 60 chronic hepatitis B patients, who responded failure to long-term LAM and/or ADV therapy. Compared with Fluorescence quantitative PCR, Direct PCR sequencing expressed a higher positive rate (43.2% vs 38.6%; 66.7% vs 54.2%, respectively) and a lower missing rate (9.5% vs 19.0%; 5.9% vs 23.5%, respectively) in the detection of drug resistance in the patients treated with LAM or combination therapy of LAM with ADV. The sensitivity of experiment using Direct PCR sequencing seemed better than Fluorescence quantitative PCR, although the difference was not significant. Further analysis on sensitivity and specificity of detection between high and low viral loads groups indicated that the consensus of two experiments in high viral load group is better than that in low viral load group (Chi-square test = 5.18, probability value = 0.023). In low viral load group, Direct PCR sequencing expressed higher sensitivity and higher specificity than Fluorescence quantitative PCR, although the difference did not approach significant. Direct PCR sequencing is better than Fluorescence quantitative PCR in the detection of drug resistance in clinic, not only with higher sensitivity and specificity, but also with comprehensive information of HBV polymerase RT region.

Comparison and evaluation of 3 diagnostic kits for quantification (PCR-Fluorescence Probing) of hepatitis B virus DNA

Objective The goal of this clinic study is to evaluate the application performance for 2 new HBV DNA Quantitative Fluorescence Diagnostic Kits, which are recently emerged in the market.Methods Serial diluted HBV serum samples and 1001 clinical serum samples with random virus load were tested quantitatively with the 3 diagnostic kits A, B and C. By studying their linear range, specificity,precision and sensitivity, the two new reagents (A and B ) were used to test these samples and also to compare them with the quantitative results from the boiling method kit (C) which is of better quality and reliability than similar diagnostic kits in current market. Furthermore, the immunoassy results of these samples were evalued and compared with their quantitative results. Results The quantitative results of 767 samples showed that their average values of the 3 kits have no significant difference. However, in the low viral load group, the results of kit A showed the best sensitivity(1.00E + 01 IU/ml)and had much better sensitivity than kit B (1.00E + 02 IU/ml), while kit C kit ( 5.00E + 02 IU/ml ) failed to test positive for most of the low concentration samples. Conclusion The nucleic acid extraction-free method ( kit B) showed much better accuracy and much larger linear range than the conventional method. In this method, the nucleic acid templates extracted by lysis buffer all went into the PCR reaction, resulting in high extraction efficiency and minimum nucleic acid loss. With a simple procedure, great accuracy and good sensitivity, this new test kit is suitable for routine clinical lab usage. Key words: Hepatitis B; DNA; Polymerase chain reaction; Sensitivity and specificity

Red blood cell deformability in patients with human immunodeficiency virus infection

Red blood cell (RBC) deformability is a major determinant of the ability of the RBC to pass repeatedly through the microcirculation. A decrease in RBC deformability leads to tissue perfusion and organ dysfunction. The purpose of this study was to measure the rigidity of RBCs from human immunodeficiency virus (HIV) seropositive individuals and investigate its relation to immune status and viral load. A filtration method based on the initial flow rate principle was used to determine the index of rigidity (IR) of 53 samples from HIV patients and 53 healthy individuals. The mean IR was significantly increased in patients with HIV compared to healthy individuals (P < 0.01). IR was inversely correlated with current CD4+ T-lymphocyte counts (P < 0.0001). High CD4 cell counts (>200 cells/microl) are related to low IR values, independently of the viral load (VL). No differences in rigidity were noted between the VL groups, although there was a trend towards an increased IR in patients with high VL within the group of CD4<200. RBC deformability is decreased in HIV disease, in a degree mainly related to CD4 depletion. Further studies are needed to elucidate the underlying mechanisms and the role of VL in highly immunocompromised HIV patients.

Epidemiologie der primären Medikamentenresistenz bei chronisch HIV-Infizierten in Nordrhein-Westfalen 2001-2005

Primary HIV drug resistance, characterized by mutant virus strains in untreated HIV-infected persons, is of significant epidemiological significance. Primary resistance is associated with reduced efficacy of antiretroviral therapy (ART). We determined the prevalence of primary resistance in Nordrhein-Westfalen, Germany. Genotypic resistance testing was performed in a prospective multicenter study in chronically infected previously untreated HIV-positive patients before administration of first-line ART. Mutations were classified according to the International AIDS Society USA guidelines and the geno2pheno interpretation tool. Between January 2001 and December 2005, resistance testing was performed in 831 patients. 77.4% were males, the mean age was 39 years (SD: 10.5). The mean duration of diagnosis of HIV infection was 1.6 years (SD: 3.4). 32.4% of patients were at CDC stage C, mean CD4 cell count was 236 /microl (SD: 205), and mean viral load was 206,855 copies/ml (SD: 450,610). In total, resistance-associated mutations were detected in 75 patients (9.0%; 95%CI, 7.1-11.0). After inclusion of mutations E44D and V118I, resistance was identified in 99 patients (11.9%; 95%CI, 9.7-14.1). 5.4% had mutations indicating nucleoside reverse transcriptase inhibitor (NRTI) resistance (95%CI, 3.9-7.0), 3.0% had non-NRTI resistance (95%CI, 1.8-4.2), and 2.4% had protease inhibitor resistance (95%CI, 1.4-3.4), respectively. Two-class resistance was detected in 0.8% (95%CI, 0.2-1.5), three-class resistance in 0.5% (95%CI, 0.01-1.0). Mutations indicating revertant variants of resistant strains were found in 3.9% (95%CI, 2.5-5.2). Considering the variables age, gender, time since diagnosis, CDC stage, CD4 cell count, viral load, HIV subtype, ethnic origin, and HIV transmission group, no significant risk factor for the presence of primary resistance was demonstrated in univariate and mutlivariate analyses. The prevalence of primary resistant virus strains was about 10% in chronically infected ART-naive HIV-patients in the largest federal state of Germany. The majority of these patients had NRTI-associated resistance. No risk factor for the presence of primary drug resistance was identified. Because of the high prevalence and the possible impact on efficacy of drug treatment, routine genotypic resistance testing should be performed in untreated HIV-positive patients before administration of first-line ART.

Quality of life in HIV subtype C infection among asymptomatic subjects and its association with CD4 counts and viral loads – a study from South India

To study the association between quality of life (QOL) domains and biological markers of disease progression of HIV infection, i.e. viral load (VL) and CD4 counts among asymptomatic subjects with HIV subtype C infection in South India. Quality of life was measured using the locally validated version of the WHOQOL HIV-BREF. The subjects were neurologically asymptomatic, non psychiatrically ill HIV infected men and women participating in a cohort study. The results indicated mixed findings, with some QOL dimensions being associated with high VLs and low CD4 counts while several others did not show any associations. Significant associations were seen between low CD4 counts and the psychological and social relationships domain, with lower mean scores in these domains being reported by subjects having CD4 counts <200/mm. However, there were no significant differences between the CD4 subgroups for the domains related to physical health, level of independence, environment, and spirituality domains. Significant lower mean QOL scores were found in the highest VL subgroup compared to other groups for the following WHOQOL HIV-BREF domains: physical, psychological, level of independence, and environmental. In this sample of HAART naïve asymptomatic HIV infected subjects, some QOL dimensions were associated with the biological markers of disease progression i.e. VL and CD4 counts, while several were not. The associations were significant only in the high VL and low CD4 groups.

Past HBV Viral Load as Predictor of Mortality and Morbidity from HCC and Chronic Liver Disease in a Prospective Study

In a prospective cohort study with 11 yr of follow-up, we assessed the relationship between past hepatitis B virus (HBV) viral load and mortality. Surviving cohort members were evaluated for current liver disease. We measured HBV viral load by real-time polymerase chain reaction on stored samples from cohort entry (1992-1993) in 2,763 hepatitis B surface antigen (HBsAg)-positive adults. Major end points were death from hepatocellular carcinoma (HCC) or chronic liver disease (CLD). There were 447 deaths. In the 1,683 survivors, we assessed severity of liver disease on a return visit in 2003. Viral load was divided into three categories: undetected (<1.6 x 10(3) copies/mL); low titer (<10(5) copies/mL); and high titer (> or =10(5) copies/mL). For HCC, there was a significant increase in mortality across viral load categories (p(trend) < 0.001). Compared to the HBV undetected category, the relative risk (RR) for HCC mortality in the low viral load group was 1.7 (95% confidence interval [CI] 0.5-5.7) and 11.2 (3.6-35.0) in the high viral load group. For CLD mortality, the RRs were 1.5 (0.2-12.1) and 15.2 (2.1-109.8), respectively (p(trend) < 0.001). The RR associated with high viral load did not change with increased follow-up time. In surviving cohort members evaluated for liver disease in 2003, there was also a significant association of viral load with disease severity. In this prospective study, viral load is associated with increased mortality from HCC and CLD in HBV-infected subjects. Viral load may be a useful prognostic tool in HBV infection, and interventions aimed at its reduction should be explored.

Pilot study of interferon‐α–ribavirin–interleukin‐2 for treatment of nonresponder patients with severe liver disease infected by hepatitis C virus genotype 1

The aim of this randomized prospective study was to assess the efficacy and safety of a triple therapy with interferon-alpha (IFN-alpha)-ribavirin-interleukin-2 (IL-2) for the treatment of patients with genotype 1 infection and high viral load nonresponsive to primary IFN-ribavirin therapy. Twenty hepatitis C virus (HCV) genotype 1 patients with high viral load and Metavir fibrosis score >or=2 without HIV co-infection who were previously nonviral responders to standard treatment with IFN plus ribavirin were intensively re-treated with IFN-alpha2a (3 millions (M) IU every 2 days) combined with ribavirin (1000-1200 mg/day) for a 24-week period. Patients were randomized to receive four cycles of subcutaneous injection of IL-2 (3 MIU/day, 5 days a week every 3 weeks) during either the first 12 weeks (group 1, n = 10) or the last 12 weeks (group 2, n = 10) of combination therapy. At the end of triple therapy, six patients (30%) achieved a biochemical response and 4 (20%) a viral response followed by a relapse after triple therapy withdrawal. After 12 weeks of therapy, no difference in viral load was observed between the groups. The decrease in viral load in group 2 was not raised after the addition of IL-2 to IFN plus ribavirin combination therapy. No serious adverse effects were observed. In conclusion, in patients with poor predictive factors of response, the addition of IL-2 to IFN ribavirin combination therapy does not exert a favourable impact on HCV treatment.

Assessment of drug resistance mutations in plasma and peripheral blood mononuclear cells at different plasma viral loads in patients receiving HAART

HIV drug resistance mutations both in peripheral blood mononuclear cells (PBMCs) and plasma have the ability to influence the outcome of highly active antiretroviral therapy for HIV patients. PBMCs harbor archival proviral DNA, are a major source of HIV and also underdo latent infection during suppressive HAART. The main objectives of this study were to assess whether specific viral load groups are better predictors of drug resistance and to examine the utility of PBMCs for drug resistance testing during HAART. Patients were grouped into a plasma panel comprising of 100 patients and a PBMC/plasma panel of 45 patients. These two groups were further divided according to plasma viral load (low, medium and high). Therapy naive patients were also included. Resistance to protease and reverse transcriptase inhibitors was assessed in each group over different viral load categories. Our data indicated that in addition to plasma, PBMCs also are a reliable predictor of drug resistance. Drug resistance mutations analyzed from each panel demonstrated that intermediate and high viral loads were strong indicators of drug resistance in both the plasma and PBMC compartments. Despite this, a significant portion of patients with high viral loads showed reduced levels of drug resistance indicating that factors including poor compliance, drug pharmacokinetics and host genetic factors are also likely to contribute to therapy failure. A significant degree of resistance to NRTI and PI resistance was found in treatment-naive individuals, demonstrating the transmission of circulating drug resistant HIV-1 variants. Our data emphasize the need for stronger pharmacokinetic evaluation during HAART, especially for patients with intermediate or high plasma viremia. The utility of PBMCs as an alternative source of resistance profiling was also demonstrated, and this approach may benefit the assessment of future drug regimens for HIV-infected patients.

The relationship between plasma viral load and neuropsychological functioning in HIV-1 infection

Plasma viral load is helpful in monitoring systemic HIV infection but the relationship between plasma viral load and CNS functioning remains unclear. Equivocal results have been reported on the relationship between plasma viral load and cognitive functioning. The present study tested cognitive functions with a standardized neuropsychological battery consisting of 28 test scores. Participants ( N=140) were grouped into undetectable (<400 copies), low (401–10,000 copies), or moderate (10,001–100,000 copies) viral load groups. Statistical analyses (MANOVA and MANCOVA) revealed no differences in neuropsychological test performance between the viral load groups. Fairly healthy patients with moderate plasma viral loads do not appear to show increased neuropsychological dysfunction. CSF viral load may be more helpful in evaluating cognitive correlates of HIV encephalitis.

Plasma RNA viral load is not associated with intrapatient quasispecies heterogeneity in HIV-1 infection

The human immunodeficiency virus type 1 (HIV-1) viral set point has been associated with the rate of, disease progression and with the level of HIV-specific immune response. The analysis of the possible association between viral set point and quasispecies heterogeneity has important consequences in the understanding of HIV-1 in vivo evolution. In this study, we analyzed the association between intrapatient viral diversity and RNA viral load in 16 antiretroviral therapy-naive HIV-1-infected patients at a single time point, during the disease free period. Patients were separated into low and high viral load groups according to plasma RNA values. HIV-1 quasispecies complexity was assessed in the C2-V5 env region. The average intrapatient quasispecies heterogeneity in both groups was not significantly different (t-test, P > 0.05). However, while within the low viral load group both synonymous and non-synonymous mutations contribute to the variation observed, in the heterogeneity observed in the high viral load group there was an increase in the contribution of the non-synonymous mutations. Thus, this study show that although intrapatient quasispecies heterogeneity is not associated with viral set point in HIV-1 infection, some differences exist between the two groups in the pattern of mutation accumulation.

Short-term risk of AIDS according to current CD4 cell count and viral load in antiretroviral drug-naive individuals and those treated in the monotherapy era.

One key piece of information required when deciding whether to initiate antiretroviral therapy is the risk of AIDS before the next clinic visit. Information on the short-term (6-month) risk of AIDS according to the current viral load and CD4 cell count in untreated individuals and those treated in the zidovudine monotherapy era (i.e., pre-September 1995), especially in those with CD4 cell count > 200 x 10 cells/l, is lacking. Risk of AIDS was assessed in 3226 subjects with viral load and CD4 cell count known before initiation of antiretroviral therapy or during the zidovudine monotherapy era. These were from CASCADE Collaboration in which data from 20 cohorts of individuals with known dates of seroconversion to HIV, based in clinics in Europe and Australia, have been combined. During a total 5126.0 person-years of follow-up, 219 individuals developed AIDS. In those with current CD4 cell count < 200 x 10 cells/l, 6-month risks were 4.9, 12.7, 17.7 and 22.4% for viral load groups < 10 000, 10 000-29 999, 30 000- 99 999 and > or = 100 000 copies/ml, respectively. For CD4 cell counts 200-349 x 10 cells/l risks were 0.5, 1.6, 3.2 and 4.7%, respectively, for the four viral load groups while the corresponding values for group with CD4 cell count > or = 350 x 10 cells/l were 0.2%, 0.5%, 0.9% and 2.2%, respectively. Results were similar when analysis was restricted to those with no antiretroviral drug experience. Older people had a higher risk of AIDS for a given CD4 cell count and viral load than younger people. Combined with consideration of other issues, these estimates should prove useful information when deciding whether to initiate antiretroviral therapy in HIV-infected individuals.

Strategies to Decrease Viral Load Rebound, and Prevent Loss of Cd4 and Onset of Resistance during Structured Treatment Interruptions

Toxicity and other drug adherence-related factors have contributed to decreased compliance to antiretroviral regimens amongst HIV-infected patients. Irregular therapy disruption causes loss of CD4 T cells, onset of drug resistance and rapid rebound of plasma viral load (VL). However, an appropriate choice of drugs and properly scheduled structured treatment interruptions (STIs) may limit VL rebound, maintain CD4 counts and minimize resistance. We conducted a clinical study of STIs, RIGHT 901, involving 60 drug-naive patients with chronic HIV infection (CD4 >300, VL >10,000) randomized to receive didanosine-stavudine-indinavir (IDV group) or didanosine-stavudine-hydroxyurea (HU group), for 12 weeks. Subsequently, all patients were randomized again to start STI (short induction) or to continue the therapy for an additional 24 weeks before starting STI (long induction). Both groups underwent four STI cycles and then stopped therapy as long as viraemia remained below 10,000 copies/ml before reinitiating another four cycles of STI. During continuous therapy VLs were suppressed at similar rates in both the HU and IDV groups, while a blunted CD4 count was documented in the HU group. Following the first stop median VL rebounded close to baseline values in both groups, however, during the following STI median VL rebound decreased in the HU group, while in the IDV group VL continued to rebound to values close to baseline, and the difference between the two groups was statistically significant. Moreover, patients treated with HU had a constant and stable CD4 increase during STI, whereas CD4 counts fluctuated in the IDV group, with sharp falls during treatment interruptions and partial CD4 recovery following treatment restart. Even in the presence of IDV resistance predisposing mutations at baseline, no genotypic change in the protease sequence was observed during STI. A relevant mutation in the reverse transcriptase sequence (K70R) emerged in one patient interrupting treatment after 36 weeks of continuous therapy and in one patient after four STI cycles. Side effects (no major events) were similar among groups. An appropriate choice of STI schedule and regimens containing drugs less prone to resistance and/or able to prevent CD4 fluctuation may contribute to optimizing STI for chronically infected patients with respect to limiting viral rebound, improving CD4 counts and maintaining a resistance profile comparable to continuous highly active antiretroviral therapy.

Experimental herpes simplex virus encephalitis: a combination therapy of acyclovir and glucocorticoids reduces long-term magnetic resonance imaging abnormalities.

Despite early antiviral treatment, herpes simples virus encephalitis (HSVE) still remains a life-threatening sporadic disease with high mortality and morbidity. In patients and in experimental disease, chronic progressive magnetic resonance imaging (MRI) abnormalities have been found even after antiviral therapy. Secondary autoimmune-mediated and not directly virus-mediated mechanisms might play a key role for the outcome of disease. This study aimed to evaluate a possible beneficial effect of a therapy of acyclovir and corticosteroids versus acyclovir only. In a mouse model of HSVE (intranasal inoculation with 10(5) pfu [plaque-forming units] of HSV-1 strain F), a long-term MRI study was realized. Cranial MRI was performed serially at days 2, 7, 14, 21, 60, and 180 in different therapy groups: 1, saline; 2, acyclovir; 3, acyclovir, subsequently methylprednisolone; 4, sham-infected with saline. Brain viral load peaked at day 7 to decline thereafter to a low baseline value. Viral load in group 1 was significantly higher than in animals with antiviral therapy. In group 4, no viral DNA was detectable. Viral load did not differ significantly between acyclovir and acyclovir/corticosteroid-treated groups, suggesting that the use of corticosteroids in addition to acyclovir does not increase viral burden. MRI findings in untreated and acyclovir-treated animals revealed chronic progressive changes. In contrast, there was a significant reduction of the severity of long-term MRI abnormalities in acyclovir/corticosteroid-treated animals. With respect to abnormal MRI findings, this study demonstrates a clear beneficial effect of an acyclovir and corticosteroid therapy without influencing brain viral load.

Efficacy of Nevirapine-Based HAART in HIV-1-Infected, Treatment-Naive Persons with High and Low Baseline Viral Loads

Purpose: The objective of this analysis was to assess the efficacy of nonnucleoside reverse transcriptase inhibitor (NNRTI)-containing, protease inhibitor (PI)-sparing, three-drug highly active antiretroviral therapy (HAART) in HIV- 1-infected, treatment-naive patients with high and low baseline viral loads. Method: A composite analysis was performed of clinical studies including the NNRTI nevirapine that were presented at international conferences between 1998 and October 2000. In all of the studies, nevirapine was administered in combination with two nucleoside reverse transcriptase inhibitors (NRTIs). For a study to be included in the analysis, more than 25% of participants must have had baseline viral loads >100,000 copies/mL, and more than 25% of participants must have had viral loads <100,000 copies/mL. Results: After 6 months, 139 of 156 (89%) and 82 of 99 (83% ) patients in the low and high baseline viral load groups, respectively, had on-treatment viral loads <200 to 500 copies/mL (depending on assay used). After 12 months, 95 of 124 patients (77%) with lower baseline viral loads and 63 of 76 patients (83%) with high baseline viral loads had on-treatment viral loads below the limit of quantification. Conclusion: Baseline viral load does not affect virologic outcome in HIV-1-infected, antiretroviral-naive participants treated with nevirapine-containing, PI-sparing HAART.

Clinical usefulness of the branched DNA assay version 2 in predicting the efficacy of Interferon treatment in a group of chronic HCV patients based on serotype

Interferon (IFN) response depends upon pretreatment viral loads and viral genotype/serotype. This study investigated the difference in response to IFN in different viral load groups of 96 chronic hepatitis C patients and compared version 1 (bDNA1.0) and version 2 (bDNA2.0) of the branched DNA assay, according to serotype. On univariate analysis, viral load ( P=0.0001, by bDNA 1.0; P=0.0020, by bDNA 2.0,), serotype ( P=0.0053) and age ( P=0.0073) were significant predictors of IFN response. On multivariate analysis, serotype (odds ratio, 5.44; 95% confidence interval, 1.94–15.24; P<0.01) was a stronger predictor of IFN response than age or viral load (by bDNA2.0). In very high (>6.7 log eq/ml), high (6.0∼6.69 log eq/ml) and low (<6 log eq/ml) viral load groups (by bDNA2.0), complete response was 25, 55 and 92.6% in serotype 2 (sero-2), and 10, 20 and 71.4% in serotype 1 (sero-1), respectively. In sero-2, bDNA2.0 can detect high viral loads underestimated by bDNA1.0. In a low viral load group (by bDNA2.0), IFN response is dependent upon serotype; sero-2 responded better than sero-1. However, in high and very high viral load groups, sensitivities of bDNA1.0 and bDNA2.0 are not effective in clinically distinguishing CR from non-response, and aid in patient selection for IFN therapy.

HIV-Associated Multinucleated Giant Cells in Lymphoid Tissue of the Waldeyer's Ring: A Detailed Study

Hyperplastic lymphoid tissues of the Waldeyer's ring in human immunodeficiency virus (HIV)–infected patients may occasionally contain multinucleated giant cells (MGCs). These cells, which are unrelated to any opportunistic infection, previously have been demonstrated to harbor significant amounts of HIV. Studies undertaken to characterize these MGCs have generated conflicting results: some reports suggested a macrophage origin, whereas others supported a dendritic cell lineage. This study was performed to determine the occurrence of MGCs in a series of adenoid/tonsil specimens from HIV-seropositive patients showing no histological evidence of opportunistic infection in order to further characterize the phenotype of these cells and to investigate the role of a viral infection in their pathogenesis. Adenoid/tonsil tissue specimens from 21 HIV-seropositive patients with no documented opportunistic infection were scrutinized for the presence of MGCs and evaluated immunohistochemically on paraffin sections by antibodies directed against various macrophage and DC antigens. These antigens included CD68, the macrophage marker 3A5, major histocompatibility complex Class II, S-100 protein, CD1a, and CD83. Additional immunostainings directed at CD21 and CD35 as well as at the HIV-associated p24 antigen were also performed. Finally, the presence of Epstein-Barr virus and human herpesvirus 8 viral sequences was investigated by in situ hybridization and by polymerase chain reaction analysis, respectively. MGCs were found in 14 patients (66.7%), regardless of gender, age, method of viral transmission, CD4 cell count, viral load, or ethnic group. These cells were mostly localized at the lymphoepithelium layer of the tonsillar crypts and, to a lesser extent, in the interfollicular areas of the underlying lymphoid tissue, which consistently exhibited features of follicular hyperplasia. Phenotypically, MGCs were found to be CD68+, 3A5+, major histocompatibility complex Class II+, S-100 protein+/−, CD1a−, CD21−, CD35−, and CD83−. Although the HIV-associated p24 protein was consistently present in the cytoplasm of these cells, no sign of Epstein-Barr virus or human herpesvirus 8 infection could be demonstrated. Consequently, our study didn't show any conclusive evidence to support that MGCs in hyperplastic lymphoid tissues of the Waldeyer's ring from HIV-seropositive patients originated from dendritic cells. The definite nature of these cells has yet to be elucidated, but it is plausible that they simply represent activated macrophages that are infected with HIV present in the oropharyngeal secretions during the circulation of their precursor through the lymphoepithelium area of adenoids and tonsils.

Regulatory dysfunction of the interleukin-2 receptor during HIV infection and the impact of triple combination therapy.

The interleukin-2 (IL-2)/IL-2 receptor (IL-2R) system is the main regulatory determinant of T cell reactivity. Although it is well known that IL-2 secretion is impaired during HIV infection, up to now IL-2R expression has not been extensively studied in HIV-infected patients despite the use of IL-2 in clinical therapy trials. We show here that IL-2R expression in HIV patients with high viral load (group 1 in the study) is greatly enhanced on B lymphocytes, CD8 T lymphocytes, and monocytes, but not on CD4 T lymphocytes, compared with noninfected individuals. Paradoxically, this modified IL-2R expression does not lead to increased IL-2 responsiveness, except for B lymphocytes. In patients receiving triple combination therapy (TCT, two reverse transcriptase inhibitors and one protease inhibitor) that has triggered a drastic reduction in plasma viral load and an increase in CD4 counts (group 2 patients), IL-2R expression is significantly lower than in group 1 patients. Moreover, cells involved in cellular immunity and CD4 T lymphocytes have the capacity to respond to IL-2 after TCT. These results allow us to anticipate a beneficial role of IL-2 immunotherapy in combination with TCT.

Lack of evidence of a stable viral load set-point in early stage asymptomatic patients with chronic HIV-1 infection.

To address the question of whether individuals with chronic HIV-1 infection have a stable viral load set-point and to assess the influence of host and viral factors on the evolution of viral load in a subset of stable asymptomatic patients with a baseline viral load below 5000 copies/ml and CD4+ T-cell count above 500 x 10(6)/l. Medical visits were performed at least every 6 months including routine blood analysis, viral load and CD4+ T-cell count. HIV-1 RNA was measured in frozen (-70 degrees C) plasma samples using PCR. Patients were classified into three groups according to baseline viral load: group A, < 200 copies/ml (undetectable); group B, 201-2000 copies/ml; group C, 2001-5000 copies/ml. A survival analysis and a Cox regression model were performed to assess the influence of viral and host factors in the increase of baseline viral load. The endpoint was the time to increase viral load to a stable level > 0.5 log10 copies/ml above baseline viral load in groups B and C and to a stable detectable viral load (> 200 copies/ml) in group A. A cohort of 114 patients with viral load below 5000 copies/ml was followed for a median of 12 months (6-42 months). Overall, 22 (19%) out of 114 patients had an increase > 0.5 log10 copies/ml of baseline viral load. Baseline viral load increased in two (5%) out of 37 patients in group A, four (12%) out of 33 patients in group B, and 16 (36%) out of 44 patients in group C (survival analysis, P<0.002). Patients of group C had a eightfold higher risk of increasing baseline viral load than patients in the other two groups pooled together (hazards ratio, 8.28; 95% confidence interval, 1.78-38; P = 0.006). Patients with an increase of viral load to the virological endpoint had a threefold higher risk of decreasing baseline CD4+ T-cell counts > 100 x 10(6)/I than patients with stable viral load (hazards ratio, 2.78; 95% confidence interval, 1.12-14; P = 0.03). In our cohort of chronically HIV-1-infected asymptomatic patients with a baseline viral load < 5000 copies/ml and CD4+ cell count > 500 x 10(6)/l, a true viral load set-point did not seem to exist. Patients with baseline viral load of 2000-5000 copies/ml had an eightfold higher risk of increasing the level of viral load than patients with a baseline viral load below 2000 copies/ml.

Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype.

Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV-1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor-alpha stimulated and unstimulated ACH-2 cells demonstrated the ability to detect < 5% HIV RNA positive cells with a sensitivity of < 30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV-seropositive patients on no, single, combination, or triple drug therapy and 8 HIV-seronegative patients were examined. The majority of HIV-positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV-infected cell populations in the peripheral blood of HIV-positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease.

Viral Load and Neuropsychological Functioning in HIV Seropositive Individuals:

We have theorized a direct relationship between viral burden and deleterious effects on the central nervous systems. It was hypothesized that HIV+ individuals would manifest poorer neuropsychological functioning after increased viral load. To address this, we compared viral burden to neuropsychological performance in subjects who participated in ACTG 116-118. Plasma samples and neuropsychological assessments completed at the same time were available for 64 observations on 21 subjects. Subjects who had a viral peak of over 1000 TCID per ml were classified as high viral load, those who did not as low viral load. Mean performances of the high viral load subjects were poorer, even though baseline performances were slightly poorer in the low viral load group. Mean post-viral peak performances were poorer than pre-viral peak performances. Declines in neuropsychological performance were found significantly more often in the high viral load group. These findings support the viral load hypothesis.