Estimating Trends in the Proportion of Transmitted and Acquired HIV Drug Resistance in a Long Term Observational Cohort in Germany

Objective: We assessed trends in the proportion of transmitted (TDR) and acquired (ADR) HIV drug resistance and associated mutations between 2001 and 2011 in the German ClinSurv-HIV Drug Resistance Study. Method: The German ClinSurv-HIV Drug Resistance Study is a subset of the German ClinSurv-HIV Cohort. For the ClinSurv-HIV Drug Resistance Study all available sequences isolated from patients in five study centres of the long term observational ClinSurv-HIV Cohort were included. TDR was estimated using the first viral sequence of antiretroviral treatment (ART) naive patients. One HIV sequence/patient/year of ART experienced patients was considered to estimate the proportion of ADR. Trends in the proportion of HIV drug resistance were calculated by logistic regression. Results: 9,528 patients were included into the analysis. HIV-sequences of antiretroviral naive and treatment experienced patients were available from 34% (3,267/9,528) of patients. The proportion of TDR over time was stable at 10.4% (95% CI 9.1-11.8; p (for trend)=0.6; 2001-2011). The proportion of ADR among all treated patients was 16%, whereas it was high among those with available HIV genotypic resistance test (64%; 1,310/2,049 sequences; 95% CI 62-66) but declined significantly over time (OR 0.8; 95% CI 0.77-0.83; p (for trend)<0.001; 2001-2011). Viral load monitoring subsequent to resistance testing was performed in the majority of treated patients (96%) and most of them (67%) were treated successfully. Conclusions: The proportion of TDR was stable in this study population. ADR declined significantly over time. This decline might have been influenced by broader resistance testing, resistance test guided therapy and the availability of more therapeutic options and not by a decline in the proportion of TDR within the study population

Results of the first international HIV-1 coreceptor proficiency panel test

Background: Quality Assurance (QA) programs are essential to evaluate performance in diagnostics laboratories. Objectives: We present the results from the first QA for HIV-1 genotypic tropism testing, organized and coordinated by the Institute of Virology at the University of Cologne. Study design: 12 cell culture-derived viral strains of different HIV-1 clades from the NIH AIDS Reagent Program were sent to the participants to be processed with their standard diagnostic methods Fasta files containing the V3 region sequence were centrally analyzed at the Institute of Virology, Cologne. All samples were sent in parallel for phenotypic testing. Results: 36 laboratories from 16 countries reported genotypic results. The sequence-generation efficacy was 95.1%, while the phenotypic assays ESTA (R) and PhenXR only achieved results for 58.3% of the samples. All four X4 samples were identified by 31/36 laboratories, two laboratories amplified 3/4 x4 samples, and three detected 2/4 x4 samples. There was high concordance among the genotypic and phenotypic results, although differences in FPR values were detected. Most deficiencies in sequence editing did not result in wrong classification of X4 viruses as R5, with the exception of sample NRZ05 by laboratory 38, but in an overestimation of CXCR4 use. Conclusions: This demonstrates that genotypic tropism prediction is a safe procedure for clinical purposes. As we used homogeneous cell culture samples and all sequence fasta files were centrally analyzed, variations in FPR values can only be attributed to sample preparation, RT-PCR or sequence editing protocols

The SnoB study: frequency of baseline raltegravir resistance mutations prevalence in different non-B subtypes

The SnoB study analysed the variability of the integrase (IN) gene of non-B viruses from treatment-naive patients to determine whether non-B subtypes carry natural resistance mutations to raltegravir (RAL). Plasma viral RNA from 427 patients was gained, and IN sequences were subtyped and screened for subtype-specific highly-variable residues. Seven viruses of different subtypes were phenotypically tested for RAL susceptibility; 359/427 samples could be sequenced. One hundred and seventy samples (47%) were classified as non-B subtypes. No primary RAL resistance-associated mutations (RRAMs) were detected. Certain secondary mutations were found, mostly related to specific non-B subtypes. L74 M was significantly more prevalent in subtype 02_AG, T97A in A and 06_cpx, V151I in 06_cpx, and G163R in 12_BF. Various additional mutations were also detected and could be associated with the subtype too. While K156 N and S230 N were correlated with B subtype, V72I, L74I, T112I, T125A, V201I and T206S were more frequent in certain non-B subtypes. The resistance factors (RF) of 7 viral strains of different subtypes ranged from 1.0 to 1.9. No primary or secondary but subtype-associated additional RRAMs were present. No correlation between RF and additional RRAMs was found. The prevalence of RRAMs was higher in non-B samples. However, the RFs for the analysed non-B subtypes showed lower values to those reported relevant to clinical failure. As the role of baseline secondary and additional mutations on RAL therapy failure is actually not known, baseline IN screening is necessary

HIV prevalence and route of transmission in Turkish immigrants living in North-Rhine Westphalia, Germany

The high number of Turkish immigrants in the German state North-Rhine Westphalia (NRW) compelled us to look for HIV-infected patients with Turkish nationality. In the AREVIR database, we found 127 (107 men, 20 women) Turkish HIV patients living in NRW. In order to investigate transmission clusters and their correlation to gender, nationality and self-reported transmission mode, a phylogenetic analysis including pol gene sequences was performed. Subtype distribution and the number of HIV drug resistance mutations in the Turkish patient group were found to be similar to the proportion in the non-Turkish patients. Great differences were observed in self-reported mode of transmission in the heterosexual Turkish male subgroup. Neighbour-joining tree of pol gene sequences gave indication that 59% of these reported heterosexual transmissions cluster with those of men having sex with men in the database. This is the first study analysing HIV type distribution, drug resistance mutations and transmission mode in a Turkish immigrant population

Factors influencing the efficacy of rilpivirine in HIV-1 subtype C in low- and middle-income countries

The use of the NNRTI rilpivirine in low- and middle-income countries (LMICs) is under debate. The main objective of this study was to provide further clinical insights and biochemical evidence on the usefulness of rilpivirine in LMICs. Rilpivirine resistance was assessed in 5340 therapy-naive and 13aEuroS750 first-generation NNRTI-failed patients from Europe and therapy-naive HIV-1 subtype C (HIV-1C)-infected individuals from India (naEuroS=aEuroS617) and Ethiopia (naEuroS=aEuroS127). Rilpivirine inhibition and binding affinity assays were performed using patient-derived HIV-1C reverse transcriptases (RTs). Primary rilpivirine resistance was rare, but the proportion of patients with > 100aEuroS000 HIV-1 RNA copies/mL pre-ART was high in patients from India and Ethiopia, limiting the usefulness of rilpivirine as a first-line drug in LMICs. In patients failing first-line NNRTI treatments, cross-resistance patterns suggested that 73% of the patients could benefit from switching to rilpivirine-based therapy. In vitro inhibition assays showed similar to 2-fold higher rilpivirine IC50 for HIV-1C RT than HIV-1B RT. Pre-steady-state determination of rilpivirine-binding affinities revealed 3.7-fold lower rilpivirine binding to HIV-1C than HIV-1B RT. Structural analysis indicated that naturally occurring polymorphisms close to the NNRTI-binding pocket may reduce rilpivirine binding, leading to lower susceptibility of HIV-1C to rilpivirine. Our clinical and biochemical findings indicate that the usefulness of rilpivirine has limitations in HIV-1C-dominated epidemics in LMICs, but the drug could still be beneficial in patients failing first-line therapy if genotypic resistance testing is performed

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 2011(1). For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V-3)(2). This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL<1000 copies/mu l roughly increased in the last years (Fig. 3). The main steps for tropism testing (Fig. 4) demonstrated in this video: 1. Collection of a blood sample 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 3. Amplification of the env region 4. Amplification of the V3 region 5. Sequence reaction of the V3 amplicon 6. Purification of the sequencing samples 7. Sequencing the purified samples 8. Sequence editing 9. Sequencing data interpretation and tropism predictio

Novel tetra-peptide insertion in Gag-p6 ALIX-binding motif in HIV-1 subtype C associated with protease inhibitor failure in Indian patients

A novel tetra-peptide insertion was identified in Gag-p6 ALIX-binding region, which appeared in protease inhibitor failure Indian HIV-1C sequences (odds ratio=17.1, P<0.001) but was naturally present in half of untreated Ethiopian HIV-1C sequences. The insertion is predicted to restore ALIX-mediated virus release pathway, which is lacking in HIV-1C. The clinical importance of the insertion needs to be evaluated in HIV-1C dominating regions wherein the use of protease inhibitor drugs are being scaled up