Short Communication: In Vitro Accumulation of Drug Resistance Mutations in Chimeric Infectious Clones Containing Subtype B or C Reverse Transcriptase and Selected with Tenofovir or Didanosine.

Highly active antiretroviral therapy (HAART) contributed to the improvement in the life expectancy of HIV-infected patients. However, the emergence of drug-resistant mutations (DRM) is a major viral factor impacting therapeutic failure. Differences in DRM can occur among HIV-1 subtypes. We evaluate the kinetics of the selection of resistance mutations in vitro analyzing two chimeric clones that contain the reverse transcriptases of subtypes B or C (RTB' and RTC') in cells treated with increasing concentrations of tenofovir disoproxil fumarate (TDF) and didanosine (ddI). The mutation K65R is selected more quickly in RTC' than in RTB' viruses with TDF and ddI, and additional mutations (positions 45, 62, and 68) were selected after K65R fixation. Other primary mutations (M184V and Q151M) were selected with ddI treatment in conjunction with K65R only in RTC' viruses. Both patterns, M184V+K65R and Q151M+K65R, have a significant impact on NRTI resistance. Our data suggest that selection of TDF and ddI DRMs can occur earlier in subtype C HIV in patients when compared to subtype B.

Evaluation of HIV protease and nucleoside reverse transcriptase inhibitors on proliferation, necrosis, apoptosis in intestinal epithelial cells and electrolyte and water transport and epithelial barrier function in mice.

BACKGROUND: Protease inhibitors (PI's) and reverse transcriptase drugs are important components of highly active antiretroviral therapy (HAART) for treating human acquired immunodeficiency syndrome (AIDS). Long-term clinical therapeutic efficacy and treatment compliance of these agents have been limited by undesirable side-effects, such as diarrhea. This study aims to investigate the effects of selected antiretroviral agents on intestinal histopathology and function in vivo and on cell proliferation and death in vitro. METHODS: Selected antiretroviral drugs were given orally over 7 days, to Swiss mice, as follows: 100 mg/kg of nelfinavir (NFV), indinavir (IDV), didanosine (DDI) or 50 mg/kg of zidovudine (AZT). Intestinal permeability measured by lactulose and mannitol assays; net water and electrolyte transport, in perfused intestinal segments; and small intestinal morphology and cell apoptosis were assessed in treated and control mice. In vitro cell proliferation was evaluated using the WST-1 reagent and apoptosis and necrosis by flow cytometry analysis. RESULTS: NFV, IDV, AZT and DDI caused significant reductions in duodenal and in jejunal villus length (p < 0.05). IDV and AZT increased crypt depth in the duodenum and AZT increased crypt depth in the jejunum. NFV, AZT and DDI significantly decreased ileal crypt depth. All selected antiretroviral drugs significantly increased net water secretion and electrolyte secretion, except for DDI, which did not alter water or chloride secretion. Additionally, only NFV significantly increased mannitol and lactulose absorption. NFV and IDV caused a significant reduction in cell proliferation in vitro at both 24 h and 48 h. DDI and AZT did not alter cell proliferation. There was a significant increase in apoptosis rates in IEC-6 cells after 24 h with 70 ug/mL of NFV (control: 4.7% vs NFV: 22%) while IDV, AZT and DDI did not show any significant changes in apoptosis compared to the control group. In jejunal sections, IDV and NFV significantly increased the number of TUNEL positive cells. CONCLUSION: The PI's, NFV and IDV, increased cell apoptosis in vivo, water and electrolyte secretion and intestinal permeability and decreased villus length and cell proliferation. NFV was the only drug tested that increased cell apoptosis in vitro. The nucleoside reverse transcriptase inhibitors, AZT and DDI, did not affect cell apoptosis or proliferation. These findings may partly explain the intestinal side-effects associated with PI's.

Emergence of primary NNRTI resistance mutations without antiretroviral selective pressure in a HAART-treated child.

OBJECTIVE: The use of antiretrovirals (ARV) during pregnancy has drastically reduced the rate of the human immunodeficiency virus perinatal transmission (MTCT). As a consequence of widespread ARV use, transmission of drug resistant strains from mothers to their babies is increasing. Ultra-sensitive PCR techniques have permitted the quantification of minority viral populations, but little is known about the transmission of drug-resistant HIV-1 minority population in the setting of MTCT. METHODOLOGY/PRINCIPAL FINDINGS: We describe the case of a female child born to an HIV-infected mother, which had not taken any ARV during the pregnancy. The child's first genotype demonstrated a minor non-nucleoside reverse transcriptase inhibitor (K101E), and during her treatment with reverse transcriptase and protease inhibitors full resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) emerged (G190A). Phenotypic/genotypic analysis of variant quasispecies through yeast TyHRT assay was conducted to characterize minority resistant viral strains circulating in both mother and child. Maximum likelihood and Bayesian MCMC phylogenetic analyses were performed with samples from the pair to assess genetic relatedness among minor viral strains. The analysis showed that the child received a minor NNRTI resistant variant, containing the mutation K101E that was present in less than 1% of the mother's quasispecies. Phylogenetic analyses have suggested common ancestry between the mother's virus strain carrying K101E with the viral sequences from the child. CONCLUSION: This is the first documentation of MTCT of a minority resistant strain of HIV-1. The transmission of minor resistant variants carries the threat of emergence of multi-drug primary mutations without identified specific selective pressures.

Antirretrovirales. Realidades y limitaciones / Antiretrovirals. Realities and limitations

En el contexto de la investigación actual de la infección por VIH, los antirretrovirales ocupan un lugar preponderante. La síntesis de nuevos fármacos y el descubrimiento de resistencias virales ha provocado la creación de terapéuticas combinadas más agresivas contra el virus y, a su vez, más efectivas para limitar dichas resistencias. La valoración de la efectividad de los antivirales se realiza por dos métodos actualmente catalogados como indicadores de la progresión o limitación de la infección por el VIH: conteo de linfocitos CD4 y determinación de copias de RNA viral en plasma (carga viral). Gracias a éstos se puede determinar la necesidad de terapéuticas únicas o combinadas y limitar el daño provocado por el virus y evitar la progresión a SIDA

Effect of zidovudine and didanosine treatment on heart function in children infected with human immunodeficiency virus.

OBJECTIVES: Human immunodeficiency virus (HIV) infection in children can be complicated by the development of cardiac disease. Decreased left ventricular function has been temporally associated with the use of zidovudine (azidothymidine; AZT) in adults with HIV and has been associated with changes in cardiac muscle mitochondria in animal models. This study was done in an attempt to determine whether the cardiac disease is related to the antiretroviral therapy or to progressive HIV infection. METHODS: We retrospectively reviewed echocardiograms, clinical records, and laboratory data from 137 HIV-infected children who were being treated by the Pediatric Branch, National Cancer Institute, and who were receiving AZT or didanosine, both drugs, or no antiretroviral therapy. RESULTS: Despite correction of the echocardiographic results for HIV disease severity with markers such as CD4+ lymphocyte count, time since infection, mode of acquisition of HIV, and age, children who were treated with AZT had a lower average fractional shortening than those who were not treated with AZT (p < 0.00001). There was a nonlinear relation between days of AZT use and this There was a nonlinear relation between days of AZT use and this decrease in fractional shortening. The odds that a cardiomyopathy would develop was 8.4 times greater in children who had previously used AZT than in those who had never taken AZT (95% confidence interval, 1.7 to 42.0). Didanosine was not associated with the development of a cardiomyopathy. CONCLUSIONS: Treatment of HIV-infected children with AZT may be associated with the development of a cardiomyopathy; didanosine does not appear to increase the risk of cardiomyopathy. The continued use of AZT in a child in whom a cardiomyopathy develops should be carefully assessed, and all children receiving AZT should be followed by serial cardiac examination and echocardiograms.