Antiviral Therapy

Significant advances in the treatment of the morbidity and mortality associated with AIDS are also associated with undesirable side-effects in fat redistribution (lipodystrophy), insulin resistance and cardiovascular risk, which is directly linked to protease inhibitor (PI) treatment.

The effects of four different PIs on triglyceride (TG) storage and adipokine production (leptin, adiponectin, and acylation stimulating protein [ASP]) in omental (OM) and subcutaneous (SC) adipose tissues were examined.

Initial results demonstrated that saquinivir (SQV) and ritonivir (RTV) had little observed effect on de novo TG synthesis ([³H]glucose incorporation into TG) or fatty acid re-esterification ([¹⁴C]oleate incorporation into TG), whereas amprenivir (APV) and indinivir (IDV) reduced TG synthesis, especially in SC tissue up to 30 ±5.8% P<0.05 and 46 ±7.8% P<0.001, at 20 μM, respectively. There was no observed effect on phospholipid synthesis, tissue protein or toxicity. Only APV and IDV decreased leptin and adiponectin secretion in SC tissue, in a time- and concentration-dependent manner: at 18 h, leptin was inhibited by 54 ±3.1% (P<0.001) and 44 ±6.4% (P<0.001) by APV and IDV (40 μM), respectively, and adiponectin was inhibited by 35 ±5.6%(P<0.001) and 25 ±12.3% (P<0.05) by APV and IDV (40 μM), respectively. By contrast, both IDV and APV decreased ASP secretion in OM tissues by a maximum of 53 ±7.8% (P<0.001) and 59 ±5.9% (P<0.001), respectively, and by a maximum of 86 ±1.6% (P<0.001) and 72 ±4% (P<0.001), respectively, in SC tissues.

PI have a direct effect on human adipose tissue which are site, PI and adipokine specific; these effects may contribute to the overall adipose imbalance and development of lipodystrophy, and metabolic syndrome in HIV-positive individuals.

To determine the potential impact of reverse transcriptase (RT) mutations, other than those currently known to confer nucleoside reverse transcriptase inhibitors (NRTIs) resistance, on the virological response to didanosine (ddI).

In the placebo-controlled Jaguar trial, 168 patients were randomly assigned to receive ddI (n=111) or placebo (n=57) in addition to their currently failing regimen for 4 weeks.

The virological response was a reduction of HIV-1 RNA from baseline to week 4. In an univariate analysis, we investigated the impact on the virological response to ddI of all the mutations in the RT gene (codons 21–236), except those known to confer NRTI resistance. Using the removing procedure, with a test for trend (Jonckheere's test), a new potential score was calculated incorporating all potential mutations associated to the week 4 virological response.

Two RT polymorphisms were associated with a reduced virological response to ddI, R211A/D/G/K/S and L228H/M/R, and one with a better virological response: F214L. A mutation score (M41L+D67N+T69D-K70R +L74V-M184V/I+T215Y/F+K219Q/E+R211A/D/G/K/S+ L228H/M/R), including two RT polymorphisms not previously described to be associated with ddI resistance (211 and 228) and RT mutations previously described, was associated with a continuum of virological response and increased the predictability of virological response to ddI.

RT polymorphisms should be taken into account to define algorithms able to correctly define resistance to NRTIs and more specifically ddI.

Previous studies have described the presence of the L74V reverse transcriptase (RT) mutation in highly experienced individuals as a risk factor for reduced response to tenofovir and selection of the K65R mutation. This study examined whether, in the context of a first-line abacavir/lamivudine highly active anti-retroviral therapy (HAART) regimen, K65R might occur as a minority population where L74V was detected at virological failure.

Four previously ART-naive individuals undergoing virological failure were selected, all receiving abacavir and lamivudine with either protease inhibitors or efavirenz and with virus displaying L74V and M184V or M184V alone following transient L74V emergence. Clonal analyses from plasma viral RNA were performed on samples taken at baseline and after virological failure. Clones were screened for the presence of K65R by real time K65R-selective PCR (K65R-sPCR). RT genes from clones displaying K65R were sequenced.

At baseline, all clones were wild type at codon 65 by K65R-sPCR, except in one patient in which one of 720 clones exhibited K65R. At failure with M184V and L74V present by bulk sequencing, this K65R was retained in one of 855 clones assessed. One additional patient (M184V alone by bulk sequencing) displayed K65R in one of 466 failure clones. Virological failure samples from the remaining two patients were wild type at codon 65 in all of 524 and 270 clones, respectively.

Minority species harbouring K65R are uncommon and occur at very low population densities in patients experiencing virological failure on first-line abacavir/lamivudine-containing HAART.

Combination antiretroviral therapy (cART) may vary in ability to suppress viral load and increase CD4⁺ T-cell count in people infected with different HIV-1 subtypes, possibly due to differences in resistance development. Antiretroviral drugs have predominantly been developed in Western Europe/North America on the basis of the most prevalent subtype, B. However, non-B subtypes are increasingly spreading worldwide.

To compare virological and immunological response to cART between patients infected with B and non-B subtypes across Europe.

EuroSIDA prospective, observational cohort with 11,928 HIV-1-infected patients.

Response to cART was analysed in patients with subtypes determined pre-cART, via multivariable logistic regression on the first measurements 6–12 months after starting cART. A virological response was defined as a viral load <500 copies/ml and immunological response as a CD4⁺ T-cell count increase of ≥100 cells/mm³.

Forty-five percent of patients were antiretroviral naive at initiation of cART. Virological suppression was achieved by 58% of 689 subtype-B-infected patients and 66% of 102 non-B-infected patients (P=0.159). After adjustment for potential confounders, there was no significant difference in odds of achieving virological suppression (non-B compared with B; odds ratio [OR]: 1.05, 95% confidence interval [CI]: 0.58–1.93, P=0.866). An immunological response was achieved by 43% of 753 B-infected patients and 48% of 114 non-B-infected patients (P=0.334). After adjustment, there was no significant difference in odds of an immunological response (OR: 1.17, 95% CI: 0.73–1.87, P=0.524).

There was no evidence of significant differences in virological or immunological response to cART between patients infected with HIV-1 B and non-B subtypes.

The aetiopathogenic bases of HIV-1-/highly active antiretroviral treatment (HAART)-associated lipodystrophy (HALS) are poorly known, but this syndrome indicates that adipose tissue is highly sensitive to either HIV-1 infection, antiretroviral drugs or their combination.

To assess the relative contribution of infection and drugs, we compared the expression of marker genes corresponding to mitochondrial function, adipocyte differentiation and metabolism, and adipokines in subcutaneous adipose tissue from healthy controls, untreated HIV-1-infected patients, and HIV-1-infected patients treated with HAART with or without HALS.

Subcutaneous adipose tissue from HIV-1-infected patients contained lower concentrations of the mRNA of the mitochondrial DNA-encoded cytochrome c oxidase subunit II than that of controls. These concentrations decreased further in association with HAART. The expression of nuclear genes coding for mitochondrial proteins, peroxisome proliferator-activated receptor-γ, and adipocyte-specific markers was reduced in HIV-1-infected patients, treated or not, with respect to the controls. In contrast, the mRNA concentrations of uncoupling protein-3 and preadipocyte factor-1 increased in lipodystrophic HAART-treated patients. The genes coding for adipokines were strongly affected: tumour necrosis factor-α was upregulated, whereas adiponectin and leptin were downregulated in HIV-1-infected patients, treated or not. Thus, substantial alterations of gene expression were already present when naive patients were compared with controls. Further changes were associated with HAART and with the diagnosis of HALS.

Disturbances in adipose tissue gene expression are already present in untreated HIV-1-infected patients, thus indicating a role of HIV-1 infection itself in eliciting adipose tissue alterations that are worsened by HAART, which ultimately leads to HALS.

To describe the prevalence, incidence and predictors of severe anaemia in previously untreated symptomatic HIV-infected adults with CD4⁺ T-cells <200 cells/mm³ initiating zidovudine-containing regimens in Africa.

DART is a randomized trial comparing two strategies for HIV/AIDS management in Uganda and Zimbabwe.

We analysed the occurrence of anaemia at weeks 4 and 12, and then every 12 weeks. We also evaluated sex, age, WHO stage, body mass index (BMI), baseline laboratory measurements and first regimen as predictors of developing grade 4 anaemia (<6.5 mg/dl) by week 48 using logistic regression.

To May 2005, 3,314 participants (65% women, 23% at WHO stage 4, median age=37 years, baseline CD4⁺ T-cell=86 cells/mm³ and median baseline haemo-globin=11.4 g/dl) had a median 72 weeks follow-up. Prevalence of grade 4 anaemia was 0.7%, 2.0%, 0.5% and <0.5% at weeks 4, 12, 24 and ≥36, respectively. Overall, 219 (6.6%) participants developed grade 4 anaemia by week 48; women and those with lower haemoglobin, CD4⁺ T-cell count and BMI at baseline were at significantly higher risk (P<0.05), but not those with lower neutrophils or receiving cotrimoxazole at baseline.

We observed a higher incidence of grade 4 anaemia than in studies from industrialized countries, which is likely to be due in part to population characteristics and in part to a higher rate of concurrent HIV-related clinical events. Clinical vigilance and haemoglobin measurements 4, 8 and 12 weeks after starting zidovudine could help to manage serious anaemia.

Lamivudine (3TC) therapy can cause the emergence of M184I/V. Previous studies suggest a higher fidelity of the mutant reverse transcriptase and lower replication capacity of the mutant virus. No data exist from clinical comparative studies evaluating the benefit of M184I/V in patients receiving combination antiretroviral therapy (cART).

HIV-1-infected adults failing a 3TC-containing regimen were randomized to continue (On-3TC) or discontinue 3TC (Off-3TC) whilst receiving cART. The primary efficacy measure was the log₁₀ average-area-under-the-curve-minus-baseline reduction in HIV RNA over 48 weeks. Cryopreserved plasma samples from patients with baseline and ≥1 follow-up sample with HIV RNA >500 copies/ml were sequenced for a nucleotide distances substudy. Evolutionary distances were compared between treatment arms and between viruses with and without M184I/V

The overall 48-week log₁₀ HIV RNA change was -1.4 (95% CI: -1.6, -1.1) for On-3TC (n=65) and -1.5 (95% CI: -1.7, -1.2) for Off-3TC (n=66; P=0.51). No difference was seen in the magnitude of the CD4⁺ T-cell count increases (median increase: 87 vs 76 cells/ml for 3TC vs Off-3TC, respectively). Thirty-seven patients had baseline and follow-up sequencing. Overall, there were 1.2 (95% CI: -2.2, 4.6) more nucleotide substitutions from baseline for Off-3TC patients (P=0.50), and 10.7 (95% CI: 7.5, 14.0) fewer nucleotide changes in viruses containing M184I/V (P<0.0001).

This study found no added virological or immunological benefit of continuing 3TC in patients on cART harbouring M184I/V Evolutionary distances from baseline were larger in viruses that did not contain M184I/V. More discernable benefits may be seen in patients with fewer drug options as potent cART may eclipse a benefit of M184I/V in COLATE.

The incidence of adefovir dipivoxil (ADV) resistance in patients with lamivudine (3TC)-resistant mutants who received ADV therapy remains unclear. The aims of this study were to determine the virological response to ADV, the incidence and the risk factors of ADV resistance, and the associated factors of initial virological response (IVR) in lamivudine-resistant patients.

Forty-six consecutive lamivudine-resistant chronic hepatitis B patients treated with ADV for more than 12 months with or without 3TC overlapping were prospectively examined for virological response and adefovir resistance.

IVR was documented in 24 (52.2%) of patients. Of the 46 patients, 11 had ADV resistance (5 rtN236T, 5 rtA181T, 1 rtA181T and rtN236T). The cumulative incidence of ADV resistance at month 6, 12, 18 and 24 was 0%, 6.5%, 24.6% and 38.3% respectively. Compared with those without ADV resistance, patients with ADV resistance had a significantly higher rate of liver cirrhosis. Based on Cox regression analysis, the significant risk factor of ADV resistance was younger age (OR=0.92, 95% CI=0.86–0.99, P=0.023) and liver cirrhosis (OR=5.3, 95% CI=1.12–25.09, P=0.036). In addition, patients with ADV resistance were associated with higher HBV DNA levels and lower HBV DNA reduction in first 6 months of ADV treatment than those without ADV resistance.

Only half of our patients achieved IVR on ADV treatment. The incidence of ADV resistance was high in 3TC-resistant patients treated with ADV.

Comprehensive study on viral factors predicting treatment responsiveness to lamivudine is lacking.

To define the significance of various viral factors and changes of viral population with lamivudine treatment.

Hepatitis B virus (HBV) DNA levels at baseline, week 24, 52 and year 3 were measured in 80 patients on continuous lamivudine therapy for 3 years. Genotypes, core promoter/precore mutations, YMDD mutations, polymorphic sequence of polymerase gene (rt91I/L, rt256S/C) were determined at baseline, week 12, 24 and 52. YMDD mutations were also determined at year 3.

High alanine aminotransferase levels and presence of core promoter/ precore mutations at baseline were associated with higher chance of achieving HBV DNA <1,000 copies/ml (good response) and higher rate of hepatitis Be antigen (HBeAg) seroconversion at week 52. Achieving HBV DNA levels <1,000 copies/ml at week 24 as well as baseline core promoter/precore mutations were associated with higher chance of achieving good response, higher rate of HBeAg seroconversion and lower rate of YMDD mutations at year 3. Lamivudine reversed core promoter mutations to wild type in 25% of patients. All 5 patients with rt256C had poor HBV DNA response, persistent HBeAg and YMDD mutations by year 3. There was no difference in treatment response between patients with genotype B and C.

Achieving HBV DNA levels <1,000 copies/ml at 24 week is the best target for short- and long-term treatment efficacy. Core promoter and precore mutations were associated with better treatment outcome, and rt256C polymorphism in the polymerase gene with poor response.

Antiretroviral therapy (ART) is a risk factor for cardiovascular disease (CVD) and smoking the most important modifiable cardiovascular risk factor.

We prospectively evaluated a smoking cessation programme (SCP) in HIV-infected individuals (intervention: counselling and nicotine replacement therapy). Primary endpoint was the smoking cessation rate at 12 months; secondary endpoints were CVD morbidity and mortality. Controls were a not randomized control group of smokers not participating in the SCP.

Four-hundred and seventeen of 680 (61%) patients were smokers, and 34 of these participated in the SCP. Of these 34 individuals, 82% were male, the median age was 43 years, prior AIDS was recorded in 29%, and depressive disorder was recorded in 18%. Twenty-five (74%) patients were receiving ART. Additional risk factors were dyslipidaemia (68%), a prior cardiovascular event (24%), hypertension (15%), and a family history of CVD in 2/34 (6%) individuals. According to the Framingham equation, the 10-year risk of CVD was higher in SCP participants than in controls (11.2% versus 8.5%, P=0.06). At termination of the SCP, 17/34 (50%) individuals had stopped smoking compared with 57/383 (15%) controls. Self-reported smoking abstinence for ≥12 months was 13/34 (38%) in the intervention group and 27/383 (7%) in the control group (odds ration 6.2, 95% confidence interval 2.8–14.3). During the follow-up, two SCP participants and 4 controls experienced a myocardial infarction. One patient in the control group died of CVD.

SCP in HIV-infected individuals is feasible and should be encouraged. The long-term impact of smoking cessation on CVD morbidity and mortality should be evaluated in comparative trials.

To study the antiviral efficacy and the mutations selected by a triple therapy with zidovudine (AZT), lamivudine (3TC) and tenofovir disoproxil fumarate (TDF).

Antiretroviral-naive patients received 300 mg AZT/150 mg 3TC twice a day plus 300 mg TDF once a day in an open pilot study. Follow-up was assessed at baseline therapy (M0) and at months 1, 3, 6, 9 and 12. Reverse transcriptase (RT) genotypic resistance analysis and in selected cases, a recombinant drug susceptibility and replication capacity assay were performed from plasma RNA at baseline and in case of virological failure (VF); that is, rebound of viral load >50 copies/ml on therapy.

Twenty-four patients were included. At baseline, the median CD4⁺ T-cell count was 443 cells/μl and the median plasma viral load (VL) was 4.38 log₁₀ copies/ml. RT resistance mutations were observed at M0 in 4 patients. At M12, the proportion of patients with a VL <50 copies/ml reached 88% using an on-treatment analysis and 67% with an intent-to-treat analysis. The median increase in CD4⁺ T cells at M12 was 94 cells/μl. Four patients had a VF on therapy: two with wild-type viruses, one with selection of M184V and thymidine analogue mutations (TAMs) on a background of TAMs, and one with selection of K65R and M184V, with a replication capacity at 2.4%.

The virological response in our study demonstrates the antiviral efficacy of the AZT/3TC/TDF combination therapy, which needs further evaluation. The moderate frequency of selection of K65R could be due to the presence of AZT in the regimen.