Replicative fitness in vivo of HIV-1 variants with multiple drug resistance-associated mutations.

The relative fitness of HIV-1 viral variants containing a broad range of drug resistance-associated mutations has been little studied in vivo. Understanding the relative fitness associated with viruses containing mutations may aid future therapeutic management. The aim of this study was to investigate the relative fitness of mutant viruses by assessing a cohort of patients who had developed resistance to many drugs and subsequently stopped all therapy. Eleven patients were assessed for drug resistance associated mutations in the protease (PR) and reverse transcriptase (RT) genes before and, at multiple time points, after stopping therapy. Relative fitness was calculated as a function of the rate of disappearance of mutant viruses when therapy was stopped. The least fit viruses were associated with the RT mutation M184I/V (11.6% less fit) and the PR mutations D30N (12.4% less fit) and M46I/L (21% less fit). Mutations at these codons were associated with significant reductions in fitness levels compared to wild-type viruses. Mutations at codons 10, 20, 36, and 63 in the PR gene remained fairly constant when therapy was stopped and may not significantly reduce viral fitness. The rapid re-population of wild-type viruses may allow the recycling of antiretroviral drugs prescribed previously.

Relationships among the detection of p24 antigen, human immunodeficiency virus (HIV) RNA level, CD4 cell count, and disease progression in HIV-infected individuals with hemophilia.

The aim of this study was to assess the relationships among the detection of p24 antigen, human immunodeficiency virus (HIV) RNA level, CD4 cell count, and disease progression in 111 males with hemophilia who were infected with HIV for < or =20 years. Sixty-four individuals (58%) developed p24 antigenemia a median of 11.6 years after seroconversion. The time to first detection of p24 antigen was shorter among those who were older (P=.04) and those with a high initial HIV RNA level (P=.006). The median HIV RNA level and CD4 cell count at the time of the detection of p24 antigen were 4.95 log(10) copies/mL and 100 cells/mm(3), respectively. In univariate analyses, p24 antigenemia was associated with more-rapid progression to AIDS (relative hazard [RH], 5.50; P=.0001). The effect was reduced (RH, 1.85; P=.06) after adjusting for CD4 cell counts and HIV RNA levels during follow-up, age, and calendar year. A significant relationship between p24 antigenemia and death was nonsignificant after adjusting for CD4 cell count.

Gender differences in virologic response to treatment in an HIV-positive population: a cohort study.

OBJECTIVE: To establish whether a gender difference in virologic response to highly active antiretroviral treatment (HAART) exists. METHODS: A cohort of HIV-positive individuals was examined. OUTCOMES: Achievement of viral load <500 copies/ml and "failure" (failure to suppress viral load <500 copies/ml after 24 weeks or two consecutive measurements above this level after having suppressed below it). Hazard ratios (HRs) comparing the rate in women to that in men were derived using the Cox model. RESULTS: Of 366 male subjects, 79% were white and 82% were homosexual. Sixty-three percent of the 91 female subjects were African and 87% were heterosexual. The median follow-up after HAART was 94 weeks. The baseline CD4 count was higher in men (228 x 106 per liter) than in women (171 x 106 per liter) (p =.01), but the viral load was similar (p =.88). The median time to <500 copies/ml was 16 weeks. Women achieved a viral load of <500 copies/ml at a faster rate than men, with an adjusted HR of 1.46 (95% confidence interval [CI]: 0.99-2.16; p =.06). Some 261 patients failed treatment (58% of men and 53% of women) with an HR of 0.78 (95% CI: 0.51-1.21; p =.27). CONCLUSIONS: Women may achieve virologic suppression at a faster rate than men and have a more durable response. Further research should examine these responses in conjunction with clinical outcomes, because gender differences in virologic response may ultimately be of little relevance if clinical outcomes are similar.

The effects of the 32-bp CCR-5 deletion on HIV transmission and HIV disease progression in individuals with haemophilia.

The chemokine receptor CCR5 is an important co-receptor for cell fusion. A 32-bp deletion of the CCR5 gene, leading to complete absence of functional CCR5 expression, has been associated with resistance to human immunodeficiency virus (HIV) infection in homozygotes and slower HIV disease progression in heterozygotes. The objectives of this study were to assess the effects of this 32-bp deletion on transmission of HIV infection and on HIV disease progression in haemophilic individuals. Six HIV-negative patients from our centre, known to have been exposed to infectious factor VIII concentrates, have been analysed. Three of these patients possess the CCR5 32-bp deletion, two patients being homozygous. The presence of the CCR5 32-bp gene deletion has also been analysed in 71 HIV-positive patients. In this group of patients, there was a lower than expected incidence of the 32-bp deletion. Those who possess the 32-bp deletion progress to AIDS more slowly than those who do not (P = 0.05, log-rank test). Rates of CD4 loss were slower in those heterozygous for the gene deletion. We confirm that heterozygosity for the 32-bp gene deletion in CCR5 is partially protective against initial infection with HIV. In those heterozygous patients who became infected with HIV, disease progression was slower.

Immunological, virological and clinical response to highly active antiretroviral therapy treatment regimens in a complete clinic population. Royal Free Centre for HIV Medicine.

BACKGROUND: Highly active antiretroviral therapy (HAART) is now widely used in clinical practice and gives rise to a range of immunological, virological and clinical responses. OBJECTIVES: To describe the immunological, virological and clinical response to HAART and to examine the frequency of modification of the HAART regimen among patients from a single treatment centre. METHODS: Kaplan-Meier estimation and incidence rates were used to describe responses to HAART (a protease inhibitor or non-nucleoside drug in addition to at least two nucleoside analogues) among 421 patients from the Royal Free Hospital in London. RESULTS: The median CD4 cell count at starting HAART was 186 x 10(6) cells/l [interquartile range (IQR) 76-310] and viral load was 5.13 log10 copies/ml (IQR 4.66-5.56). At 6 months after starting HAART, 51.1% of patients were estimated to have experienced a 100 x 10(6) cells/l increase in CD4 cell count; the median time for viral load to fall below 400 copies/ml was 3.7 months (95% confidence interval 3.2-4.4). At 6 months after the first viral load was < 400 copies/ml, 16.4% of patients were estimated to have failed on the basis of a single viral load > 400 copies/ml and 12.4% were estimated to have failed if the more stringent definition of two viral loads above the limit of detection was used. Compared with the pre-HAART era, the incidence of death among patients on HAART was one sixth of that level; new AIDS-defining illnesses was one seventh; and hospital admissions was one fifth. In total, 141 patients (33.5%) stopped at least one of the antiretroviral agents included as part of their HAART regimen; the occurrence of side effects was the most common reason (n = 63; 44.7%). CONCLUSION: A good response occurred to an initial HAART regimen. There was a high rate of virological relapse, which varied considerably according to the definition of failure used. Even so, the rates of clinical progression and hospital admissions observed to date were low. Further follow-up of these patients is required to determine their long-term immunological, virological and clinical outcome.

Pre-AIDS mortality and its association with HIV disease progression in haemophilic men, injecting drug users and homosexual men.

OBJECTIVE: To study pre-AIDS mortality and its association with HIV disease progression in different exposure groups with known intervals of HIV seroconversion. DESIGN AND METHODS: The type and rate of pre-AIDS deaths were assessed in 111 HIV-infected haemophilic men followed in London, and 118 injecting drug users and 158 homosexual men followed in Amsterdam. In each group, the association between CD4+ T-cell count, HIV RNA and pre-AIDS mortality was studied using proportional hazards analysis. RESULTS: By 10 years after seroconversion 7.3% of the haemophilic men had died without AIDS and 38.2% had developed AIDS. These figures were 20.2 and 30.5% for injecting drug users, and 8.0 and 55.0% for homosexual men. The major causes of pre-AIDS mortality appear to differ in the three exposure groups. The risk of pre-AIDS death tended to increase with decreasing CD4 cell count and increasing HIV RNA levels in injecting drug users and homosexual men. In men with haemophilia the associations were less obvious, although the log-transformed CD4 cell count was predictive for pre-AIDS death. CONCLUSIONS: Pre-AIDS deaths occur and are at least partially related to HIV disease progression irrespective of how individuals became infected. Because of the longer life expectancy due to highly active antiretroviral therapy (HAART), pre-AIDS deaths are likely to show a further increase. Methods to incorporate these intermediate outcomes should be considered in the estimation of the size of the HIV epidemic and in the survival analysis of HIV-infected individuals. Prevention and treatment of non-AIDS infections, especially hepatitis C virus infection, and cancers will become increasingly important in HIV-infected individuals. The interaction between these therapies and HAART should be closely monitored.

Substantial correlation between HIV type 1 drug-associated resistance mutations in plasma and peripheral blood mononuclear cells in treatment-experienced patients.

The correlation of detectable HIV-1 drug resistance mutations in plasma and PBMCs in patients extensively treated with all antiretroviral drug classes has not been fully elucidated. The detection of mutations in PBMCs may reveal resistant HIV-1 associated with past therapies. In addition, these measures in PBMCs may have a practical value when plasma virus is at low levels that are difficult to detect with current assays. The reverse transcriptase (RT) and protease (P) genes were analyzed for drug resistance, using an in-house method carried out on 36 paired samples of plasma and PBMCs from 12 treatment-experienced patients in order to investigate resistance in the two compartments. When viruses in plasma and PBMCs were analyzed by patient, the mean of the Cohen kappa values was 0.728 (substantial agreement). When viruses were analyzed by codon the mean of the Cohen kappa values was 0.715 (substantial agreement). Baseline samples were concordant at 280 of 288 (97%) codons analyzed. This study shows that a minority of mutations associated with previous therapy can be detected in PBMCs and not in plasma. Overall, mutations in plasma and PBMCs showed a substantial correlation in extensively treated patients, suggesting that either compartment is suitable for the detection of mutations as a virological guide for clinical care.

Two decades of HIV infection in a cohort of haemophilic individuals: clinical outcomes and response to highly active antiretroviral therapy.

OBJECTIVES: Many haemophilic individuals infected with HIV died before receiving antiretroviral therapy (ART). Most who remain alive are chronically infected with hepatitis C virus (HCV), which has implications for their prognosis and choice of ART. The clinical status of a cohort of HIV-positive haemophilic men is reported together with their response to highly active antiretroviral therapy (HAART). DESIGN: Longitudinal cohort study. SETTING: A comprehensive care haemophilia centre. PATIENTS: A group of 111 haemophilic men who seroconverted to HIV in the period 1979 to 1985. RESULTS: The cohort has been followed since 1979. By 30 April 1999, 57 of the 111 men had developed AIDS and 65 had died: Kaplan-Meier rates of 57.0% [95% confidence interval (CI) 46.9-67.0) and 65.1% (95% CI 52.7-77.4) by 19.5 years, respectively. AIDS rates have declined since 1997 but death rates have remained high, largely owing to deaths from non-HIV-related causes. Thirty-five patients remain alive and under follow-up at the clinic. The 28 men who had received ART had lower CD4 cell counts than the seven patients who had not received ART, but the two groups were otherwise similar. In total, 21 patients are known to have started HAART while under care at the centre. By 10-12 months after starting HAART, viral loads dropped by 2.06 log10 copies/ml and CD4 cell counts increased by 60 x 10(6) cells/l. In 10 out of 18 patients with viral loads initially > 400 copies/ml, a viral load below this level was attained; four had changed therapy at the time. CONCLUSIONS: While the decision to initiate HAART in haemophilic men should be made carefully because of the possible adverse events, our results suggest that a good response rate was achieved in this group of men.

Cytomegalovirus seropositivity and human immunodeficiency virus type 1 RNA levels in individuals with hemophilia.

The effect of cytomegalovirus (CMV) seropositivity on the course of human immunodeficiency virus (HIV) type 1 RNA levels and HIV disease progression was assessed in a cohort of 109 hemophilic men infected with HIV-1 for a median of 12.7 years. There was no evidence of higher HIV RNA levels in the first year after HIV seroconversion (P=. 88) or faster rates of increase over infection (P=.20) in the 59 CMV-seropositive individuals than in the CMV-seronegative individuals. In univariate analyses, CMV seropositivity was associated with significantly faster progression to AIDS and death (relative hazards of 1.58 and 2.22, respectively). These effects were unchanged after adjusting for the RNA level, but they were reduced after adjusting for the CD4 cell count, age at seroconversion, and calendar year of follow-up. Thus, the effect of CMV seropositivity on clinical progression remains significant in this cohort but does not appear to be mediated through an increase in HIV RNA levels.

Reduction in human immunodeficiency virus type 1 mutations associated with drug resistance after initiating new therapeutic regimens in pretreated patients.

The objective of this study was to investigate the effect of initiating a salvage-therapy regimen on resistant viruses in heavily treated patients infected with human immunodeficiency virus type 1 (HIV-1). Nineteen patients receiving multiple antiretroviral drugs were tested for HIV-1 mutations associated with drug resistance by using an in-house method at baseline and at weeks 2, 4, and 8 of the salvage-therapy regimen. For the majority of mutations analyzed, the mean number of detectable mutations at baseline was significantly higher than the mean at weeks 2, 4, and 8 of salvage therapy. Introducing new and more potent therapy reduces the number of detectable drug resistance-associated mutations within 8 weeks, and no evidence was found that the new therapy promoted the emergence of novel mutations.

Course of viral load throughout HIV-1 infection.

HIV-1 RNA levels are routinely monitored as part of patient management. However, little is known about the course of HIV-1 RNA levels over the entire period of infection. The aim of this study was to investigate the course of HIV-1 RNA levels in a cohort of men with hemophilia who were observed for up to 17 years after HIV-1 seroconversion, and to assess the risk of HIV disease progression at any HIV-1 RNA level. Viral loads were measured on annual stored serum samples in 107 men with hemophilia A using the Roche Amplicor Monitor assay with non-B primers. On average, HIV-1 RNA levels increased significantly by 0.11 log10 per year over the course of HIV infection. This rate of increase was significantly faster in those who developed AIDS or died over the subsequent 12 to 17 year period, and in those who were older at HIV- 1 seroconversion. The risk of developing AIDS and death remained low when the HIV-1 RNA level was below 4 log10 copies/ml, but increased rapidly thereafter, supporting current guidelines for the initiation of antiretroviral therapy after the viral load has exceeded this level.

Changes in AIDS-defining illnesses in a London Clinic, 1987-1998.

OBJECTIVES: To describe the incidence of AIDS-defining illnesses within a single large clinic setting, to describe temporal changes over a 10-year period in the overall incidence and of individual AIDS-defining illnesses and to investigate the impact of HIV treatment regimen on the incidence of AIDS-defining illnesses. SUBJECTS AND METHODS: A person-years analysis was used to determine the incidence of AIDS-defining illnesses according to calendar year and stratification by CD4 lymphocyte count and treatment regimen in 1806 patients from the Royal Free Centre for HIV Medicine with at least one CD4 lymphocyte count and follow-up visit. RESULTS: Prior to 1992, the incidence of all AIDS-defining illnesses was 27.4/100 person-years of follow-up (PYFU; 95% confidence interval [CI], 22.8-32.0) and during 1997 this incidence had dropped to 6.9/100 PYFU (95% CI, 4.7-9.1; p < .0001, test for trend). The decline in incidence over time occurred in esophageal candidiasis, cytomegalovirus disease (including retinitis), Kaposi's sarcoma, lymphoma, wasting syndrome, and Pneumocystis carinii pneumonia (p < .05, test for trend), but there was no evidence of a decline in AIDS dementia or in Mycobacterium avium complex. In 1997, among patients with CD4 lymphocyte counts of < or =200 cells/mm3, the incidence rates for any AIDS-defining illness was 51.1/100 PYFU for patients taking no therapy (95% CI, 27.9-85.7), 34.5 among patients on monotherapy (95% CI, 4.2-124.6), 13.2 among patients taking dual combination therapy (95% CI, 3.6-33.8) and 6.1 among patients taking triple therapy or more complex regimens (95% CI, 0.7-22.0; p < .0001, test for trend). CONCLUSIONS: There was a considerable decline in AIDS-defining illnesses during 1996 and 1997, coinciding with the rapid development of new antiretroviral treatments and combinations of treatment. Further follow-up of large observational cohorts is essential to monitor the incidence of diagnoses less common than we were able to consider, such as tuberculosis, cryptosporidiosis, and cryptococcosis, and also to investigate whether the incidence of disease continues to fall, or whether it starts to rise again, as toxicities, compliance, drug resistance, and long-term side effects begin to appear.

In vivo HIV-1 replicative capacity in early and advanced infection.

OBJECTIVE: Previous studies on patients treated with potent antiretroviral therapy have shown that viral clearance rates do not tend to change between early and advanced HIV-1 infection. Our objective was to investigate whether the other major aspect of virus dynamics, viral replicative capacity, does change. In vitro work has indicated that the viral replicative, capacity increases but in vivo evidence has been lacking. METHODS: As an in vivo measure of the viral replicative capacity, we studied the rate of rebound of plasma HIV RNA level during a 1-week therapy interruption in previously untreated patients who had received 2 weeks of antiretroviral therapy. RESULTS: Such therapy in five previously drug-naive patients with high CD4 lymphocyte counts (mean, 611 x 10(6)/l) and five patients with low counts (mean, 49 x 10(6)/l) led to a mean 2.2 log10 copies/ml decrease in plasma HIV-1 levels (from 5-6 log10 copies/ml) in 2 weeks. This was similar in the two groups. Interruption of therapy for the ensuing week resulted in a stable HIV-1 level for approximately 2 days followed by a rebound towards pretherapy level, which was much more marked in the patients with low CD4 cell counts (estimated mean rise 2.22 log10 versus 1.06 log10 copies/ml; P < 0.02). After restarting therapy, HIV RNA levels returned to pre-interruption levels. CONCLUSIONS: These findings need confirmation, but the ability of HIV-1 to replicate in vivo appears to increase during HIV-1 infection. This increased replicative capacity, for which there are several potential explanations, may be the cause of gradual CD4 lymphocyte depletion.

Rapid decline in detectability of HIV-1 drug resistance mutations after stopping therapy.

OBJECTIVE: To investigate the rate of decline of drug resistant viruses after stopping therapy. DESIGN: Twenty-five patients receiving multiple combination therapies (mean five; range three to nine drugs) for more than 3 months were tested for HIV-1 resistance on therapy and off therapy. The sample off therapy was tested 6-175 days after stopping therapy. METHODS: Patients were tested for genotypic changes associated with drug resistance using an in-house automated DNA sequencing assay to detect resistance in the protease and reverse transcriptase genes. RESULTS: All samples tested when patients were on therapy showed evidence of drug resistance (range 1-9 mutations). The patients were divided into three groups: <2 weeks after stopping therapy, median 1.1 weeks (n = 8, group A); 2 weeks-2 months, median 6.4 weeks (n = 7, group B) and 2 months-6 months, median 12.9 weeks (n = 10, group C). Of primary mutations (protease: 30N, 461/L, 82A, 90M; reverse transcriptase: 70R, 184I/V, 215 Y/F) detected on therapy 100% remained after stopping therapy in group A; 68% remained in group B and 15% remained in group C. For secondary mutations 98% remained in group A; 99% remained in group B and 57% in group C. CONCLUSIONS: This study showed a rapid decline in detectability of the majority of primary mutations within 13 weeks of stopping combination therapy. From this data, HIV-1 resistance testing to direct patients' therapy should only be carried out when on existing therapy, or < 2 weeks off therapy, if reliable decisions are to be made relating to future combinations.

Automated DNA sequencing.

The polymerase chain reaction (PCR), first published by Mullis and Faloona in 1987 (1), has become an invaluable molecular biology tool in both research and routine applications. The combination of PCR with the chain-termination sequencing technique developed by Sanger et al. in 1977 (2) and the automation of both these techniques, has enabled DNA sequencing to also become widely used in a variety of research areas.

Influence of HIV-1 infection on GBV-C infection in multiply infected haemophilic patients.

Two hundred thirteen haemophilic patients were studied for the presence of GBV-C RNA and anti-E2 antibodies soon after their first treatment with unsterilised factor concentrates and in their most recent sample. An assessment was made to determine whether coinfection with HIV had any effect on the progression of GBV-C infection. All of the patients were infected with HCV and 81 patients (37%) were also infected with HIV. GBV-C RNA was detected using the Abbott LCx(TM) assay and by RT-PCR. Anti-E2 antibodies were detected using the microPLATE Anti-HGenv assay and by Abbott Laboratories. The HIV-negative patients were more likely than the HIV-positive patients to lose GBV-C RNA between the two time points. A proportion of the patients lost their anti-E2 antibodies over the time period, however, the majority of these were HIV-positive. This study shows that infection with HIV does affect the progression of GBV-C infection, however, this effect is little understood as yet.