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

Tenth International Congress on Drug Therapy in HIV Infection 7-11 November 2010 Glasgow, UKIntroduction: Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. To date, in most European countries HIV tropism is determined using a phenotypic test. Recently, new data have emerged supporting the use of a genotypic HIV V3-loop sequence analysis as the basis for tropism determination. The European guidelines group on clinical management of HIV-1 tropism testing was established to make recommendations to clinicians and virologists. Methods: We searched online databases for articles from Jan 2006 until March 2010 with the terms: tropism or CCR5-antagonist or CCR5 antagonist or maraviroc or vicriviroc. Additional articles and/or conference abstracts were identified by hand searching. This strategy identified 712 potential articles and 1240 abstracts. All were reviewed and finally 57 papers and 42 abstracts were included and used by the panel to reach a consensus statement. Results: The panel recommends HIV-tropism testing for the following indications: i) drug-naïve patients in whom toxicity or limited therapeutic options are foreseen; ii) patients experiencing therapy failure whenever a treatment change is considered. Both the phenotypic Enhanced Trofile assay (ESTA) and genotypic population sequencing of the V3-loop are recommended for use in clinical practice. Although the panel does not recommend one methodology over another it is anticipated that genotypic testing will be used more frequently because of its greater accessibility, lower cost and shorter turnaround time. The panel also provides guidance on technical aspects and interpretation issues. If using genotypic methods, triplicate PCR amplification and sequencing testing is advised using the G2P interpretation tool (clonal model) with an FPR of 10%. If the viral load is below the level of reliable amplification, proviral DNA can be used, and the panel recommends performing triplicate testing and use of an FPR of 10%. If genotypic DNA testing is not performed in triplicate the FPR should be increased to 20%. Conclusions: The European guidelines on clinical management of HIV-1 tropism testing provide an overview of current literature, evidence-based recommendations for the clinical use of tropism testing and expert guidance on unresolved issues and current developments. Current data support both the use of genotypic population sequencing and ESTA for co-receptor tropism determination. For practical reasons genotypic population sequencing is the preferred method in Europe.Ye

A21 HIV-1 sub-subtype F1 outbreak among MSM in Belgium

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Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies

<p>Abstract</p> <p>Background</p> <p>Antibodies are the main effectors against malaria blood-stage parasites. Evaluation of functional activities in immune sera from Phase 2a/b vaccine trials may provide invaluable information in the search for immune correlates of protection. However, the presence of anti-malarial-drugs, improper collection/storage conditions or concomitant immune responses against other pathogens can contribute to non-specific anti-parasite activities when the sera/plasma are tested <it>in vitro</it>. Purification of immunoglobulin is a standard approach for reducing such non-specific background activities, but the purification method itself can alter the quality and yield of recovered Ag-specific antibodies.</p> <p>Methods</p> <p>To address this concern, various immunoglobulin (Ig) purification methods (protein G Sepharose, protein A/G Sepharose, polyethylene glycol and caprylic acid-ammonium sulphate precipitation) were evaluated for their impact on the quality, quantity and functional activity of purified rabbit and human Igs. The recovered Igs were analysed for yield and purity by SDS-PAGE, for quality by Ag-specific ELISAs (determining changes in titer, avidity and isotype distribution) and for functional activity by <it>in vitro </it>parasite growth inhibition assay (GIA).</p> <p>Results</p> <p>This comparison demonstrated that overall polyethylene glycol purification of human serum/plasma samples and protein G Sepharose purification of rabbit sera are optimal for recovering functional Ag-specific antibodies.</p> <p>Conclusion</p> <p>Consequently, critical consideration of the purification method is required to avoid selecting non-representative populations of recovered Ig, which could influence interpretations of vaccine efficacy, or affect the search for immune correlates of protection.</p

Epidemiological study of phylogenetic transmission clusters in a local HIV-1 epidemic reveals distinct differences between subtype B and non-B infections

<p>Abstract</p> <p>Background</p> <p>The number of HIV-1 infected individuals in the Western world continues to rise. More in-depth understanding of regional HIV-1 epidemics is necessary for the optimal design and adequate use of future prevention strategies. The use of a combination of phylogenetic analysis of HIV sequences, with data on patients' demographics, infection route, clinical information and laboratory results, will allow a better characterization of individuals responsible for local transmission.</p> <p>Methods</p> <p>Baseline HIV-1 <it>pol </it>sequences, obtained through routine drug-resistance testing, from 506 patients, newly diagnosed between 2001 and 2009, were used to construct phylogenetic trees and identify transmission-clusters. Patients' demographics, laboratory and clinical data, were retrieved anonymously. Statistical analysis was performed to identify subtype-specific and transmission-cluster-specific characteristics.</p> <p>Results</p> <p>Multivariate analysis showed significant differences between the 59.7% of individuals with subtype B infection and the 40.3% non-B infected individuals, with regard to route of transmission, origin, infection with <it>Chlamydia </it>(p = 0.01) and infection with Hepatitis C virus (p = 0.017). More and larger transmission-clusters were identified among the subtype B infections (p < 0.001). Overall, in multivariate analysis, clustering was significantly associated with Caucasian origin, infection through homosexual contact and younger age (all p < 0.001). Bivariate analysis additionally showed a correlation between clustering and syphilis (p < 0.001), higher CD4 counts (p = 0.002), <it>Chlamydia </it>infection (p = 0.013) and primary HIV (p = 0.017).</p> <p>Conclusions</p> <p>Combination of phylogenetics with demographic information, laboratory and clinical data, revealed that HIV-1 subtype B infected Caucasian men-who-have-sex-with-men with high prevalence of sexually transmitted diseases, account for the majority of local HIV-transmissions. This finding elucidates observed epidemiological trends through molecular analysis, and justifies sustained focus in prevention on this high risk group.</p

Generation of representative primary virus isolates from blood plasma after isolation of HIV-1 with CD44 MicroBeads

Infection of cell cultures with cell-free virus isolated from HIV-infected patients is notoriously difficult and results in a loss of viral variation. Here, we describe viral sequences from PBMC, U87.CD4.CCR5 and U87.CD4.CXCR4 cell cultures and compare them to those from blood plasma from 12 patients from whom virus particles were isolated using CD44 MicroBeads. In both PBMC and U87.CD4.CCR5 cultures, 66% of the plasma viral strains were retrieved after culturing. In addition, coreceptor use was predicted based on the env-V3 sequence and tested in U87.CD4 cells expressing either CCR5 or CXCR4. Recovery was lower for the CXCR4-using viruses. Only 50% of the virus clusters predicted to use CXCR4 could be retrieved from cell cultures, while 71% of CCR5-using strains were found in U87.CCR5 cultures. Therefore, isolation of primary viruses with CD44 MicroBeads results in a good representation in cell culture of the in vivo divergence

Comparison of phenotypic and genotypic tropism determination in triple-class-experienced HIV patients eligible for maraviroc treatment

BACKGROUND: Determination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care. METHODS: Forty-nine consecutive plasma samples for which an original Trofile(TM) assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call. RESULTS: According to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with Trofile(TM) population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value. CONCLUSIONS: In line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc

Transmitted drug resistance, selection of resistance mutations and moderate antiretroviral efficacy in HIV-2: Analysis of the HIV-2 Belgium and Luxembourg database

BACKGROUND: Guidelines established for the treatment of HIV-1 infection and genotype interpretation do not apply for HIV-2. Data about antiretroviral (ARV) drug efficacy and resistance mutations is scarce. METHODS: Clinical data about HIV-2 infected patients in Belgium and Luxembourg were collected and the effect of ARV therapy on plasma viral load and CD4 counts were analysed. Viral RNA encoding for protease (PR) and reverse transcriptase (RT) from ARV-naive and treated patients were sequenced. RESULTS: Sixty-five HIV-2 infected patients were included in this cohort. Twenty patients were treated with 25 different ARV combinations in a total of 34 regimens and six months after the start of ARV therapy, only one third achieved viral load suppression. All of these successful regimens bar one contained protease inhibitors (PIs). Mean CD4 gains in the group of viral load suppressors and the group of patients treated with PI-containing regimens were respectively significantly higher than in the group of non-suppressors and the group of PI-sparing regimens. The most frequent mutations selected under therapy (compared to HIV-2 ROD) were V71I, L90M and I89V within PR. Within RT, they were M184V, Q151M, V111I and K65R. All of these mutations, except K65R and M184V, were also found in variable proportions in ARV-naive patients. CONCLUSION: Despite a high rate of ARV treatment failure, better virological and immunological results were achieved with PI-containing regimens. The analysis of polymorphic positions and HIV-2 specific mutations selected during therapy showed for the first time that transmission of drug resistant viruses has occurred in Belgium and Luxembourg. The high heterogeneity in ARV combinations reflects a lack of guidelines for the treatment of HIV-2 infection

Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases