Virological and immunological responses to efavirenz or boosted lopinavir as first-line therapy for patients with HIV.

BACKGROUND: Efavirenz and lopinavir boosted with ritonavir are both recommended as first-line therapies for patients with HIV when combined with two nucleoside reverse transcriptase inhibitors. It is uncertain which therapy is more effective for patients starting therapy with an advanced infection. METHODS: We estimated the relative effect of these two therapies on rates of virological and immunological failure within the Swiss HIV Cohort Study and considered whether estimates depended on the CD4(+) T-cell count when starting therapy. We defined virological failure as either an incomplete virological response or viral rebound after viral suppression and immunological failure as failure to achieve an expected CD4(+) T-cell increase calculated from EuroSIDA statistics. RESULTS: Patients starting efavirenz (n=660) and lopinavir (n=541) were followed for a median of 4.5 and 3.1 years, respectively. Virological failure was less likely for patients on efavirenz, with the adjusted hazard ratio (95% confidence interval) of 0.63 (0.50-0.78) then multiplied by a factor of 1.00 (0.90-1.12) for each 100 cells/mm(3) decrease in CD4(+) T-cell count below the mean when starting therapy. Immunological failure was also less likely for patients on efavirenz, with the adjusted hazard ratio of 0.68 (0.51-0.91) then multiplied by a factor of 1.29 (1.14-1.46) for each 100 cells/mm(3) decrease in CD4(+) T-cell count below the mean when starting therapy. CONCLUSIONS: Virological failure is less likely with efavirenz regardless of the CD4(+) T-cell count when starting therapy. Immunological failure is also less likely with efavirenz; however, this advantage disappears if patients start therapy with a low CD4(+) T-cell count.

Renal function in patients with HIV starting therapy with tenofovir and either efavirenz, lopinavir or atazanavir.

BACKGROUND: Tenofovir is associated with reduced renal function, but it is not clear whether there is a greater decline in renal function when tenofovir is co-administered with a boosted protease inhibitor rather than with a nonnucleoside reverse transcriptase inhibitor (NNRTI). METHODS: We calculated the estimated glomerular filtration rate (eGFR) for patients in the Swiss HIV Cohort Study. We estimated the difference in eGFR over time between first therapies containing tenofovir and either the NNRTI efavirenz or the protease inhibitors lopinavir (LPV/r) or atazanavir (ATV/r), both boosted with ritonavir. RESULTS: Patients on a first therapy of tenofovir co-administered with efavirenz (n  = 484), LPV/r (n = 269) and ATV/r (n =  187) were followed for a median of 1.7, 1.2 and 1.3 years, respectively. Relative to tenofovir and efavirenz, the estimated difference in eGFR for tenofovir and LPV/r was -2.6 ml/min per 1.73 m [95% confidence interval (CI) -7.3 to 2.2) during the first 6 months of therapy, then followed by a difference of 0.0 ml/min per 1.73 m (95% CI -1.1 to 1.1) for each additional 6 months of therapy. Relative to tenofovir and efavirenz, the estimated difference in eGFR for tenofovir and ATV/r was -7.6 ml/min per 1.73 m (95% CI -11.8 to -3.4) during the first 6 months of therapy, then followed by a difference of -0.5 ml/min per 1.73 m (95% CI -1.6 to 0.7) for each additional 6 months of therapy. CONCLUSION: Tenofovir with either boosted protease inhibitor leads to a greater initial decline in eGFR than tenofovir with efavirenz; this decline may be worse with ATV/r than with LPV/r.

Incidence and risk factors for chronic elevation of alanine aminotransferase levels in HIV-infected persons without hepatitis b or c virus co-infection.

BACKGROUND: Chronic liver disease in human immunodeficiency virus (HIV)-infected patients is mostly caused by hepatitis virus co-infection. Other reasons for chronic alanine aminotransferase (ALT) elevation are more difficult to diagnose. METHODS: We studied the incidence of and risk factors for chronic elevation of ALT levels (greater than the upper limit of normal at 2 consecutive semi-annual visits) in participants of the Swiss HIV Cohort Study without hepatitis B virus (HBV) or hepatitis C virus (HCV) infection who were seen during the period 2002-2008. Poisson regression analysis was used. RESULTS: A total of 2365 participants were followed up for 9972 person-years (median age, 38 years; male sex, 66%; median CD4+ cell count, 426/microL; receipt of antiretroviral therapy [ART], 56%). A total of 385 participants (16%) developed chronic elevated ALT levels, with an incidence of 3.9 cases per 100 person-years (95% confidence interval [CI], 3.5-4.3 cases per 100 person-years). In multivariable analysis, chronic elevated ALT levels were associated with HIV RNA level >100,000 copies/mL (incidence rate ratio [IRR], 2.23; 95% CI, 1.45-3.43), increased body mass index (BMI, defined as weight in kilograms divided by the square of height in meters) (BMI of 25-29.9 was associated with an IRR of 1.56 [95% CI, 1.24-1.96]; a BMI 30 was associated with an IRR of 1.70 [95% CI, 1.16-2.51]), severe alcohol use (1.83 [1.19-2.80]), exposure to stavudine (IRR per year exposure, 1.12 [95% CI, 1.07-1.17]) and zidovudine (IRR per years of exposure, 1.04 [95% CI, 1.00-1.08]). Associations with cumulative exposure to combination ART, nucleoside reverse-transcriptase inhibitors, and unboosted protease inhibitors did not remain statistically significant after adjustment for exposure to stavudine. Black ethnicity was inversely correlated (IRR, 0.52 [95% CI, 0.33-0.82]). Treatment outcome and mortality did not differ between groups with and groups without elevated ALT levels. CONCLUSIONS: Among patients without hepatitis virus co-infection, the incidence of chronic elevated ALT levels was 3.9 cases per 100 person-years, which was associated with high HIV RNA levels, increased BMI, severe alcohol use, and prolonged stavudine and zidovudine exposure. Long-term follow-up is needed to assess whether chronic elevation of ALT levels will result in increased morbidity or mortality.

The structure of human tRNALys3 anticodon bound to HIV genome is stabilized by modified nucleosides and adjacent mismatch base pairs

Replication of human immunodeficiency virus (HIV) requires base pairing of the reverse transcriptase primer, human tRNA(Lys3), to the viral RNA. Although the major complementary base pairing occurs between the HIV primer binding sequence (PBS) and the tRNA's 3'-terminus, an important discriminatory, secondary contact occurs between the viral A-rich Loop I, 5'-adjacent to the PBS, and the modified, U-rich anticodon domain of tRNA(Lys3). The importance of individual and combined anticodon modifications to the tRNA/HIV-1 Loop I RNA's interaction was determined. The thermal stabilities of variously modified tRNA anticodon region sequences bound to the Loop I of viral sub(sero)types G and B were analyzed and the structure of one duplex containing two modified nucleosides was determined using NMR spectroscopy and restrained molecular dynamics. The modifications 2-thiouridine, s(2)U(34), and pseudouridine, Psi(39), appreciably stabilized the interaction of the anticodon region with the viral subtype G and B RNAs. The structure of the duplex results in two coaxially stacked A-form RNA stems separated by two mismatched base pairs, U(162)*Psi(39) and G(163)*A(38), that maintained a reasonable A-form helix diameter. The tRNA's s(2)U(34) stabilized the interaction between the A-rich HIV Loop I sequence and the U-rich anticodon, whereas the tRNA's Psi(39) stabilized the adjacent mismatched pairs.

Étude de la résistance des sous-types non-B du VIH-1 aux antirétroviraux au Mali

Nous avons effectué ce travail afin d’évaluer l’impact d’une utilisation accrue des antirétroviraux (ARV) sur l’émergence de la résistance dans le cadre d’une cohorte de sujets infectés par le VIH-1, enrôlés au Mali pour recevoir la thérapie antirétrovirale. La première partie de ce travail a évalué la résistance primaire auprès de 101 sujets naïfs aux ARV. Cette étude a démontré que la majorité des sujets (71,3%) étaient infectés par le sous-type CRF02_AG. La prévalence de la résistance primaire était de 9,9%. Ce chiffre dépasse largement la moyenne de 5,5% observée dans les pays en développement et le seuil des 5% fixé par l’OMS dans le cadre de la surveillance de la résistance. Les mutations associées aux analogues de la thymidine ou « Thymidine-associated Mutations » (TAMs): M41L, D67N, L210W, T215A/Y, K219E liées à la résistance aux inhibiteurs nucléosidiques de la transcriptase inverse (INTI) ainsi que les mutations K103N, V108I, V179E et Y181C impliquées dans la résistance aux inhibiteurs non nucléosidiques de la transcriptase inverse (INNTI) étaient majoritairement observées. Ces mutations sont compatibles avec les régimes de traitement de première ligne utilisés au Mali, composés de stavudine/lamivudine/nevirapine. Nous n’avons pas trouvé de mutations majeures aux inhibiteurs de protéase (IP), probablement du fait que cette classe d’ARV est rarement utilisée au Mali. Cependant plusieurs polymorphismes au niveau du gène de la protéase, particulièrement L10I et L10V ont été observés à une fréquence très élevée (18,80%). Compte tenu de ces premiers résultats, une suite logique de ce travail était de savoir comment des souches de sous-type CRF02_AG évolueraient sous la pression de sélection des ARV. Nous avons abordé ces questions dans une étude de cohorte de 132 sujets infectés majoritairement avec le sous-type CRF02_AG débutant une thérapie de première ligne. Nos résultats suggèrent que la présence de mutation de résistance primaire pourrait avoir un effet sur l’efficacité du traitement. Par exemple, la présence d’une seule mutation INNTI avant traitement comme K103N ou V179E était suffisante pour mener à l’échec au traitement (charge virale supérieure à 400 copies/ml). Par ailleurs, nous avons effectué des expériences in vitro pour mieux évaluer l’impact du polymorphisme L10I/V chez le sous-type CRF02_AG. Il faut savoir que le rôle de ce polymorphisme reste incertain chez le sous-type CRF02_AG, car aucune étude in vitro n’avait été réalisée auparavant. Nos résultats indiquent chez le sous-type sauvage CRF02_AGwt_10L une légère augmentation de la concentration inhibitrice 50% (IC50) pour le darunavir, le lopinavir et le nelfinavir comparativement au sous-type de référence B HXB2_10L avec respectivement un « Fold Change » (FC) de 1,2, 1,3 et 1,5. Cette augmentation est plus importante pour le lopinavir avec un FC (1,3) très proche de son seuil biologique (1,6). Comparativement au type sauvage CRF02_AGwt_10L, nos deux mutants CRF02_AGL10I et CRF02_AGL10V ont démontré une légère augmentation d’IC50 pour l’indinavir (avec respectivement un FC de 1,3 et 1,2) et une diminution pour le lopinavir (avec respectivement un FC de 0,78 et 0,75). Toutes ces observations suggèrent que la mutation en position 10 pourrait avoir un impact chez le sous-type CRF02_AG. Toutefois, la signification clinique de ces observations doit être déterminée. En conclusion, nos résultats supportent d’une part la nécessité de renforcer la surveillance de la résistance aux ARV et d’autre part, il fournit des informations nécessaires à l’amélioration des stratégies thérapeutiques afin de prévenir les échecs aux traitements chez les sous-types non B, particulièrement le CRF02_AG.

Antiretroviral resistance mutations in human immunodeficiency virus type 1 infected patients enrolled in genotype testing at the Central Public Health Laboratory, São Paulo, Brazil: preliminary results

Antiretroviral resistance mutations (ARM) are one of the major obstacles for pharmacological human immunodeficiency virus (HIV) suppression. Plasma HIV-1 RNA from 306 patients on antiretroviral therapy with virological failure was analyzed, most of them (60%) exposed to three or more regimens, and 28% of them have started therapy before 1997. The most common regimens in use at the time of genotype testing were AZT/3TC/nelfinavir, 3TC/D4T/nelfinavir and AZT/3TC/efavirenz. The majority of ARM occurred at protease (PR) gene at residue L90 (41%) and V82 (25%); at reverse transcriptase (RT) gene, mutations at residue M184 (V/I) were observed in 64%. One or more thymidine analogue mutations were detected in 73%. The number of ARM at PR gene increased from a mean of four mutations per patient who showed virological failure at the first ARV regimens to six mutations per patient exposed to six or more regimens; similar trend in RT was also observed. No differences in ARM at principal codon to the three drug classes for HIV-1 clades B or F were observed, but some polymorphisms in secondary codons showed significant differences. Strategies to improve the cost effectiveness of drug therapy and to optimize the sequencing and the rescue therapy are the major health priorities.

Perfil metabólico de cálcio e ósseo de pacientes soropositivos para HIV em uso ou não da terapia antirretroviral

Pós-graduação em Alimentos e Nutrição - FCFAR

Microbiota bucal de pacientes HIV positivos: relação com o uso de drogas antirretrovirais e condições de saúde

Pós-graduação em Odontologia - FOA

Perfil de mutações e resistência do Vírus da Imunodeficiência Humana (HIV) em pacientes coinfectados com o Vírus da Hepatite B (VHB)

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

Background: Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. Methods/Results: We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in Sao Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naive individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion: The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3-5 x less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance.

Base-Paired and Base-Stacked Structures of the Anti-HIV Drug Lamivudine: A Nucleoside DNA-Mimicry with Unprecedented Topology

We have prepared a DNA-mimicry of nucleosides in which the anti-HIV drug lamivudine (beta-L-2',3'-dideoxy-3'-thiacytidine, 3TC) self-assembles into a base-paired and helically base-stacked hexagonal structure. Face-to-face and face-to-tail stacked 3TC=3TC dimers base-paired through two hydrogen bonds between neutral cytosines by either N-H center dot center dot center dot O or N-H center dot center dot center dot N atoms give rise to a right-handed DNA-mimicry of lamivudine with an unusual highly symmetric hexagonal lattice and topology. In addition, a base-paired and base-stacked supramolecular architecture of lamivudine hemihydrochloride hemihydrate was also obtained as a result of our crystal screenings. This structure is formed through partially face-to-face stacked lamivudine pairs held together by protonated and neutral fragments. However, no helical stacking occurs in this structure in which lamivudine also adopts unusual conformations as the C1'-endo and C1'-exo sugar puckers and cytosine orientations intermediate between the anti and syn conformations. As a conclusion drawn from the nucleoside duplex, the hexagonal DNA-mimicry of lamivudine reveals that such double-stranded helices can be assembled without counterions and organic solvents but with higher crystallographic symmetry instead, because only water crystallizes together with lamivudine in this structure.

Evaluation of inadequate anti-retroviral treatment in patients with HIV/AIDS

Introduction: Since the emergence of antiretroviral therapy, the survival of patients infected with human immunodeficiency virus has increased. Non-adherence to this therapy is directly related to treatment failure, which allows the emergence of resistant viral strains. Methods: A retrospective descriptive study of the antiretroviral dispensing records of 229 patients from the Center for Health Care, University Hospital, Federal University of Juiz de Fora, Brazil, was conducted between January and December 2009. Results: The study aimed to evaluate patient compliance and determine if there was an association between non-adherence and the therapy. Among these patients, 63.8% were men with an average age of 44.0 +/- 9.9 years. The most used treatment was a combination of 2 nucleoside reverse transcriptase inhibitors with 1 non-nucleoside reverse transcriptase inhibitor (55.5%) or with 2 protease inhibitors (28.8%). It was found that patients taking lopinavir/ritonavir with zidovudine and lamivudine had a greater frequency of inadequate treatment than those taking atazanavir with zidovudine and lamivudine (85% and 83.3%, respectively). Moreover, when the combination of zidovudine/lamivudine was used, the patients were less compliant (chi(2) = 4.468, 1 degree of freedom, p = 0.035). Conclusions: The majority of patients failed to correctly adhere to their treatment; therefore, it is necessary to implement strategies that lead to improved compliance, thus ensuring therapeutic efficacy and increased patient survival.

Treatment failure and mortality factors in patients receiving second-line HIV therapy in resource-limited countries

Context Long-term antiretroviral therapy (ART) use in resource-limited countries leads to increasing numbers of patients with HIV taking second-line therapy. Limited access to further therapeutic options makes essential the evaluation of second-line regimen efficacy in these settings. Objectives To investigate failure rates in patients receiving second-line therapy and factors associated with failure and death. Design, Setting, and Participants Multicohort study of 632 patients >14 years old receiving second-line therapy for more than 6 months in 27 ART programs in Africa and Asia between January 2001 and October 2008. Main Outcome Measures Clinical, immunological, virological, and immunovirological failure (first diagnosed episode of immunological or virological failure) rates, and mortality after 6 months of second-line therapy use. Sensitivity analyses were performed using alternative CD4 cell count thresholds for immunological and immunovirological definitions of failure and for cohort attrition instead of death. Results The 632 patients provided 740.7 person-years of follow-up; 119 (18.8%) met World Health Organization failure criteria after a median 11.9 months following the start of second-line therapy (interquartile range [IQR], 8.7-17.0 months), and 34 (5.4%) died after a median 15.1 months (IQR, 11.9-25.7 months). Failure rates were lower in those who changed 2 nucleoside reverse transcriptase inhibitors (NRTIs) instead of 1 (179.2 vs 251.6 per 1000 person-years; incidence rate ratio [IRR], 0.64; 95% confidence interval [CI], 0.42-0.96), and higher in those with lowest adherence index (383.5 vs 176.0 per 1000 person-years; IRR, 3.14; 95% CI, 1.67-5.90 for <80% vs ≥95% [percentage adherent, as represented by percentage of appointments attended with no delay]). Failure rates increased with lower CD4 cell counts when second-line therapy was started, from 156.3 vs 96.2 per 1000 person-years; IRR, 1.59 (95% CI, 0.78-3.25) for 100 to 199/μL to 336.8 per 1000 person-years; IRR, 3.32 (95% CI, 1.81-6.08) for less than 50/μL vs 200/μL or higher; and decreased with time using second-line therapy, from 250.0 vs 123.2 per 1000 person-years; IRR, 1.90 (95% CI, 1.19-3.02) for 6 to 11 months to 212.0 per 1000 person-years; 1.71 (95% CI, 1.01-2.88) for 12 to 17 months vs 18 or more months. Mortality for those taking second-line therapy was lower in women (32.4 vs 68.3 per 1000 person-years; hazard ratio [HR], 0.45; 95% CI, 0.23-0.91); and higher in patients with treatment failure of any type (91.9 vs 28.1 per 1000 person-years; HR, 2.83; 95% CI, 1.38-5.80). Sensitivity analyses showed similar results. Conclusions Among patients in Africa and Asia receiving second-line therapy for HIV, treatment failure was associated with low CD4 cell counts at second-line therapy start, use of suboptimal second-line regimens, and poor adherence. Mortality was associated with diagnosed treatment failure.

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.

Are plasma levels valid surrogates for cellular concentrations of antiretroviral drugs in HIV-infected patients?

Total plasma concentrations are currently measured for therapeutic drug monitoring of HIV protease inhibitors (PIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). However, the pharmacological target of antiretroviral drugs reside inside cells. To study the variability of their cellular accumulation, and to determine to which extent total plasma concentrations (TPC) correlate with cellular concentrations (CC), plasma and peripheral blood mononuclear cells (PBMCs) were simultaneously collected at single random times after drug intake from 133 HIV infected patients. TPC levels were analysed by high-performance liquid chromatography with ultraviolet detection and CC by LC-MS/MS from peripheral blood mononuclear cells. The best correlations between TPC and CC were observed for nelfinavir (NFV, slope=0.93, r=0.85), saquinavir (SQV, slope=0.76, r=0.80) and lopinavir (LPV, slope=0.87, r=0.63). By contrast, TPC of efavirenz (EFV) exhibited a moderate correlation with CC (slope=0.69, r=0.58), while no correlation was found for nevirapine (NVP, slope=-0.3, r=0.1). Interindividual variability in the CC/TPC ratio was lower for protease inhibitors (coefficients of variation 76%, 61%, and 80% for SQV, NFV and LPV, respectively) than for nonnucleoside reverse transcriptase inhibitors (coefficients of variation 101% and 318%, for EFV and NVP). As routine CC measurement raises practical difficulties, well-correlated plasma concentrations (ie, NFV, SQV and LPV) can probably be considered as appropriate surrogates for cellular drug exposure. For drugs such as EFV or NVP, there may be room for therapeutic drug monitoring improvement using either direct CC determination or other predictive factors such as genotyping of transporters or metabolizing enzyme genes.