A.M.J. Wensing

The Calculated Genetic Barrier for Antiretroviral Drug Resistance Substitutions Is Largely Similar for Different HIV-1 Subtypes

Background: The genetic barrier, defined as the number of mutations required to overcome drug-selective pressure, is an important factor for the development of HIV drug resistance. Because of high variability between subtypes, particular HIV-1 subtypes could have different genetic barriers for drug resistance substitutions. This study compared the genetic barrier between subtypes using some 2000 HIV-1 sequences (>600 of non-B subtype) isolated from anti-retroviral-naive patients in Europe. Methods: The genetic barrier was calculated as the sum of transitions (scored as 1) and/or transversions (2.5) required for evolution to any major drug resistance substitution. In addition, the number of minor protease substitutions was determined for every subtype. Results: Few dissimilarities were found. An increased genetic barrier was calculated for I82A (subtypes C and G), V108I (subtype G), V118I (subtype G), Q151M (subtypes D and F), L210W (subtypes C, F, G, and CRF02_AG), and P225H (subtype A) (P < 0.001 compared with subtype B). A decreased genetic barrier was found for I82T (subtypes C and G) and V106M (subtype C) (P < 0.001 vs subtype B). Conversely, minor protease substitutions differed extensively between subtypes. Conclusions: Based on the calculated genetic barrier, the rate of drug resistance development may be similar for different HIV-1 subtypes. Because of differences in minor protease substitutions, protease inhibitor resistance could be enhanced in particular subtypes once the relevant major substitutions are selected.

Long-term complications in patients with poor immunological recovery despite virological successful HAART in Dutch ATHENA cohort.

Objective:We investigated the risk of AIDS and serious non-AIDS-defining diseases (non-ADDs) according to the degree of immunological recovery after 2 years of virological successful antiretroviral therapy (HAART). Design:Retrospective observational cohort study including HIV-infected patients treated with HAART resulting in viral suppression (<500 copies/ml). Methods:Patients were grouped according to their CD4 cell count after 2 years of HAART: CD4 cell count less than 200 cells/&mgr;l (group A), 200–350 cells/&mgr;l (group B), 351–500 cells/&mgr;l (group C) or more than 500 cells/&mgr;l (group D). Analysis was done to assess predictors for poor immunological recovery and the occurrence of a composite endpoint [death, AIDS, malignancies, liver cirrhosis and cardiovascular events (CVEs)], non-ADDs, CVEs and non-AIDS-defining malignancies (non-ADMs). Results:Three thousand and sixty-eight patients were included. Older age, lower CD4 cell nadir and lower plasma HIV-RNA at the start of HAART were independent predictors for a poor immunological recovery. The composite endpoint, non-ADDs and CVE were observed most frequently in group A (overall log rank, P < 0.0001, P = 0.002 and P = 0.01). In adjusted analyses, age was a strong independent predictor for all endpoints. Compared with group A, patients in group D had a lower risk for the composite endpoint [hazard ratio 0.54 (95% confidence interval [CI] 0.33–0.87]; patients in group B had a lower risk for CVEs [hazard ratio 0.34 (95% CI 0.14–0.86)]. Conclusion:Poor immunological recovery despite virological successful HAART is associated with a higher risk for overall morbidity and mortality and CVEs in particular. This study underlines the importance of starting HAART at higher CD4 cell counts, particularly in older patients.

Update on World Health Organization HIV drug resistance prevention and assessment strategy: 2004-2011

The HIV drug resistance (HIVDR) prevention and assessment strategy, developed by the World Health Organization (WHO) in partnership with HIVResNet, includes monitoring of HIVDR early warning indicators, surveys to assess acquired and transmitted HIVDR, and development of an accredited HIVDR genotyping laboratory network to support survey implementation in resource-limited settings. As of June 2011, 52 countries had implemented at least 1 element of the strategy, and 27 laboratories had been accredited. As access to antiretrovirals expands under the WHO/Joint United Nations Programme on HIV/AIDS Treatment 2.0 initiative, it is essential to strengthen HIVDR surveillance efforts in the face of increasing concern about HIVDR emergence and transmission.

Prediction of Response to Antiretroviral Therapy by Human Experts and by the EuResist Data-Driven Expert System (the EVE study)

The EuResist expert system is a novel data‐driven online system for computing the probability of 8‐week success for any given pair of HIV‐1 genotype and combination antiretroviral therapy regimen plus optional patient information. The objective of this study was to compare the EuResist system vs. human experts (EVE) for the ability to predict response to treatment.

Impact of triplicate testing on HIV genotypic tropism prediction in routine clinical practice

Guidelines state that the CCR5-inhibitor Maraviroc should be prescribed to patients infected with R5-tropic HIV-1 only. Therefore, viral tropism needs to be assessed phenotypically or genotypically. Preliminary clinical trial data suggest that genotypic analysis in triplicate is associated with improved prediction of virological response by increasing the detection of X4-tropic variants. Our objective was to evaluate the impact of triplicate genotypic analysis on prediction of co-receptor usage in routine clinical practice. Samples from therapy-naive and therapy-experienced patients were collected for routine tropism testing at three European clinical centres. Viral RNA was isolated from plasma and proviral DNA from peripheral blood mononuclear cells. Gp120-V3 was amplified in a triplicate nested RT-PCR procedure and sequenced. Co-receptor usage was predicted using the Geno2Pheno([coreceptor]) algorithm and analysed with a false-positive rate (FPR) of 5.75%, 10%, or an FPR of 20% and according to the current European guidelines on the clinical management of HIV-1 tropism testing. A total of 266 sequences were obtained from 101 patient samples. Discordance in tropism prediction for the triplicates was observed in ten samples using an FPR of 10%. Triplicate testing resulted in a 16.7% increase in X4-predicted samples and to reclassification from R5 to X4 tropism for four cases rendering these patients ineligible for Maraviroc treatment. In conclusion, triplicate genotypic tropism testing increases X4 tropism detection in individual cases, which may prove to be pivotal when CCR5-inhibitor therapy is applied.

Primary resistance to integrase strand-transfer inhibitors in Europe

OBJECTIVES The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

Therapy Failure following Selection of Enfuvirtide-Resistant HIV-1 in Cerebrospinal Fluid

We report the selection of enfuvirtide-resistant human immunodeficiency virus type 1 in cerebrospinal fluid, resulting in subsequent loss of viral suppression in the plasma. This case report emphasizes the potential danger of low-level penetration of entry inhibitors into the central nervous system.

Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir.

HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01 ± 0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA.

Consensus statement of the European guidelines on clinical management of HIV-1 tropism testing.

7‐11 November 2010, Tenth International Congress on Drug Therapy in HIV Infection, Glasgow, UK

Low HIV-1 transmitted drug resistance in Bulgaria against a background of high clade diversity

OBJECTIVES To determine transmitted drug resistance (TDR) and HIV-1 genetic diversity in Bulgaria. METHODS The prevalence of TDR and HIV-1 subtypes was determined in 305/1446 (21.1%) persons newly diagnosed with HIV/AIDS from 1988 to 2011. TDR mutations (TDRMs) in protease and reverse transcriptase were defined using the WHO HIV drug mutation list. Phylogenetic analysis was used to infer polymerase (pol) genotype. RESULTS TDRMs were found in 16/305 (5.2%) persons, 11 (3.6%) with resistance to NRTIs, 5 (1.6%) with resistance to NNRTIs and 3 (0.9%) with resistance to PIs. Dual-class TDRMs were found in three (1.0%) patients and one statistically supported cluster of TDRMs comprising two individuals with subtype B infection. TDRMs were found in 10 heterosexuals, 4 MSM and two intravenous drug users. Phylogenetic analyses identified high HIV-1 diversity consisting of mostly subtype B (44.6%), subtype C (3.3%), sub-subtype A1 (2.6%), sub-subtype F1 (2.3%), sub-subtype A-like (3.6%), subtype G (0.3%), CRF14_BG (1.6%), CRF05_DF (1.3%), CRF03_AB (0.3%) and unique recombinant forms (1.3%). CONCLUSIONS We found a low prevalence of TDR against a background of high HIV-1 genetic diversity among antiretroviral-naive patients in Bulgaria. Our results provide baseline data on TDR and support continued surveillance of high-risk populations in Bulgaria to better target treatment and prevention efforts.