Angélica Corral

Plasma Levels of Atazanavir and the Risk of Hyperbilirubinemia Are Predicted by the 3435C→T Polymorphism at the Multidrug Resistance Gene 1

The 3435C-->T polymorphism at the multidrug resistance gene 1 (MDR1) was examined in 74 patients with human immunodeficiency virus who initiated atazanavir therapy. The MDR1 genotype distribution at position 3435 was 28% CC, 45% CT, and 27% TT. Plasma levels of atazanavir were significantly higher in patients with genotype CC than in those with CT or TT, and bilirubin levels correlated with atazanavir concentrations.

High concordance between HIV-1 drug resistance genotypes generated from plasma and dried blood spots in antiretroviral-experienced patients.

Objective:Dried blood spots (DBS) are a convenient alternative to plasma for drug resistance testing in resource-limited settings. We investigated the correlation between resistance genotypes generated from DBS and plasma. Design:Sixty DBS specimens from HIV-1 subtype B-infected antiretroviral-experienced (n = 58) and naive patients (n = 2) were tested. DBS were prepared using 50 μl blood and were stored with desiccant at −20°C. Methods:Resistance genotypes from DBS were obtained using the ViroSeq HIV-1 assay and were compared with genotypes derived from plasma. The frequency of amplification of proviral DNA from DBS was evaluated using an in-house nested polymerase chain reaction assay. Results:Fifty of the 60 DBS specimens were successfully genotyped including all 38 specimens collected from patients with plasma viral loads greater than 2000 copies/ml and 12 of 22 DBS (54.5%) from patients with viral loads less than 2000 copies/ml. HIV-1 DNA was detected in 44.4% of the DBS. Despite the presence of DNA, genotypes from DBS and plasma were highly concordant. Of the 316 mutations found in plasma sequences, 306 (96.8%) were also found in DBS. Discrepancies were mostly caused by mixtures at minor protease positions or unusual amino acid changes, and in only two cases were caused by major protease (M46L) or reverse transcriptase (K103N) mutations absent in DBS sequences. Conclusion:We demonstrated a high concordance between resistance genotypes from plasma and DBS, and that resistance testing from DBS can achieve sensitive levels similar to those seen using plasma. Our results indicate that DBS may represent a feasible alternative to plasma for drug resistance testing in treated individuals.

Correlation between Human Immunodeficiency Virus Type 1 (HIV-1) RNA Measurements Obtained with Dried Blood Spots and Those Obtained with Plasma by Use of Nuclisens EasyQ HIV-1 and Abbott RealTime HIV Load Tests

ABSTRACT The plasma human immunodeficiency virus (HIV) RNA load is used in the clinical routine for the monitoring of HIV infection and the patients response to antiretroviral therapy. Other body fluids or dried blood spots (DBS) can be used, however, to assess the level of viremia. The use of DBS may be especially helpful for the monitoring of HIV-infected patients in resource-poor settings, where access to adequate laboratory facilities is often difficult. However, the correlation between the HIV RNA levels in plasma and those in DBSs has not been well established. Paired plasma and DBS samples obtained from HIV type 1 (HIV-1)-infected patients were tested for HIV RNA copy numbers by using two different commercial assays, the Nuclisens EasyQ HIV-1 (version 1.1) test (the Nuclisens test; Biomerieux) and the m2000rt RealTime HIV test (the m2000rt test; Abbott). Nucleic acid extraction was performed manually by using either the Nuclisens isolation kit (which uses the Boom methodology) or the m2000rt sample preparation kit (an iron particle-based method). A total of 103 paired plasma and DBS samples were tested. Viral load results were obtained for 97 (94.2%) samples with the Nuclisens isolation kit and 81 (78.6%) samples with the m2000rt kit. The overall correlation between the RNA loads in plasma and DBS was good, although better results were obtained by the Nuclisens test (R2 = 0.87, P < 0.001) than by the m2000rt test (R2 = 0.70, P < 0.001). While the specificities were excellent and similar for both the Nuclisens and the m2000rt tests (97.1% and 100%, respectively), the sensitivity was greater by the Nuclisens test than by the m2000rt test (75.8% and 56.6%, respectively). Overall, the viral loads in DBS tended to be lower than those in plasma, with mean differences of 0.3 log unit (standard deviation, 0.5 log unit) and 0.76 log unit (standard deviation, 0.8 log unit) for the Nuclisens and the m2000rt tests, respectively. The levels of agreement between the measurements in plasma and DBS were assessed by using the Bland-Altman plot for each assay. The Nuclisens test gave results within its defined limits (−0.65 to 1.26) for 95.9% of the samples, while the m2000rt test gave results within its limits (−0.83 to 2.33) for 100% of the samples. In summary, the HIV-1 load can accurately be quantified by testing DBS by either the Nuclisens or the m2000rt test, although the Nuclisens test may outperform the m2000rt test when nucleic acids are extracted manually.

Resistance to Nonnucleoside Reverse-Transcriptase Inhibitors and Prevalence of HIV Type 1 Non-B Subtypes Are Increasing among Persons with Recent Infection in Spain

The prevalence of drug resistance mutations was 12.1% among 198 persons who experienced human immunodeficiency virus (HIV) seroconversion identified in Spain during 1997-2004. There was a significant increase of K103N and of non-B subtypes over time. Transmission of HIV infection around the time of seroconversion was shown in 8 couples and in 2 clusters of 3 individuals.

Risk of selecting K65R in antiretroviral-naive HIV-infected individuals with chronic hepatitis B treated with adefovir

Seven antiretroviral-naive HIV-infected individuals with chronic hepatitis B treated with adefovir for longer than 6 months were assessed. Using bulk population sequencing and a sensitive limiting dilution analysis, the selection of K65R or other resistance mutations did not occur in HIV, suggesting that adefovir can be confidently used as hepatitis B virus (HBV) therapy in HIV/HBV-co-infected patients who do not require antiretroviral therapy.

Evidence for different susceptibility to tipranavir and darunavir in patients infected with distinct Hiv-1 subtypes

Background:Tipranavir (TPV) and darunavir (DRV) are potent against protease inhibitor (PI)-resistant viruses. Efficacy of these compounds when confronting distinct HIV subtypes is not known. Methods:All clinical specimens from HIV-positive patients sent to our institution for drug resistance testing between 1999 and 2006 were analysed. The prevalence of TPV and DRV resistance mutations was assessed based on the latest International AIDS Society-USA panel list. Phenotypic susceptibility to DRV and TPV was examined in a subset of these samples using the PhenoSense assay. Results:A total of 1364 genotypes were analysed, including 1178 from individuals infected with clade B (285 drug naive) and 186 with non-B subtypes (137 drug naive). The mean number (±SD) of DRV resistance-associated mutations was higher in clade B than non-B (0.4 ± 0.9 versus 0.06 ± 0.3; P < 0.001), and more frequent among PI-experienced than drug-naive patients (0.6 ± 1.02 versus 0.02 ± 0.21; P < 0.001). In contrast, the mean number of TPV resistance-associated mutations was higher in non-B than B subtypes (2.7 ± 1 versus 1.2 ± 1.6; P < 0.001), regardless of PI experience. Susceptibility to TPV and DRV was examined in 29 drug-naive patients infected with non-B subtypes (1A, 3C, 2CRF01_AE, 9CRF02_AG, 1CRF12_BF, 3CRF14_BG, 3F and 7G). All showed susceptibility to DRV and 93% to TPV. Interestingly, two subtype F specimens showed reduced TPV susceptibility, with fold-changes of 2.7 and 2.1, respectively. Conclusions:Non-B subtypes show a greater number of TPV resistance-associated mutations than B viruses, regardless of PI exposure. While HIV clade has no influence on DRV susceptibility, some F subtypes may show reduced TPV susceptibility.

Predictors of selection of K65R: tenofovir use and lack of thymidine analogue mutations

Over the past 5 years, 1846 HIV-infected patients underwent drug resistance testing at our institution. None out of 216 drug-naive subjects showed K65R. However, it was recognized in 53 out of 1630 antiretroviral-experienced patients (3.3%), of whom 10 had never been exposed to tenofovir. The rate of K65R increased from 0.6% in 1999 to 11.5% in 2004. The recognition of K65R correlated negatively with the presence of thymidine analogue mutations but positively with Q151M.

Changes in the human immunodeficiency virus p7-p1-p6 gag gene in drug-naive and pretreated patients.

ABSTRACT Resistance to antiretroviral agents often results from mutations within the human immunodeficiency virus (HIV) pol gene. Moreover, insertions within the p6 gag-pol region have recently been found to be involved with resistance to nucleoside analogs. Overall, we found that 21% of 156 specimens collected from HIV-infected individuals (17.6% from 74 drug-naive patients and 24.4% from 82 pretreated patients) harbored these insertions. Insertions around the KQE (Lys-Gln-Glu) motif were found in 12.2% of the pretreated patients but in none of the drug-naive subjects (P = 0.002). In contrast, insertions around the PTAP (Prol-Thre-Ala-Prol) motif were seen at similar rates (∼15%) among drug-naive and pretreated patients, which supports the idea that they may be natural polymorphisms.

Prevalence of the HIV-1 protease mutation I47A in clinical practice and association with lopinavir resistance.

Mutation proI47A has recently been associated with lopinavir/ritonavir (LPV/r) resistance. Only four out of 1859 specimens (0.2%) sent for drug resistance testing (219 drug-naive and 1650 antiretroviral-experienced) showed I47A. All belonged to patients failing LPV/r. The prevalence among protease inhibitor-experienced patients was 0.6%. Phenotypic testing showed that proI47A caused high-level lopinavir resistance (> 100-fold) and cross-resistance to amprenavir, whereas it caused hypersusceptibility to saquinavir. ProI47A should thus be considered the primary lopinavir resistance mutation.

Decline in the rate of genotypic resistance to antiretroviral drugs in recent HIV seroconverters in Madrid.

Genotypic resistance to antiretroviral drugs was analysed in plasma from 57 acute or recent HIV seroconverters in Madrid. The overall prevalence of drug-associated primary resistance mutations was 25.8% in 1997-1999, but declined to 3.8% in 2000-2001. The lower rate in recent years suggested that most new HIV infections derive from viraemic individuals unaware of their HIV- positive status, rather than from those failing antiretroviral treatment. Drug-resistance testing is thus not required before beginning antiretroviral therapy.