Ana Garcia-Diaz

Comparative Evaluation of the ExaVir Load Version 3 Reverse Transcriptase Assay for Measurement of Human Immunodeficiency Virus Type 1 Plasma Load

ABSTRACT In resource-limited settings, the virological monitoring of antiretroviral therapy is limited by high cost and the lack of infrastructure. The Cavidi ExaVir Load assay employs a simple and inexpensive enzyme-linked immunosorbent assay format to measure human immunodeficiency virus (HIV) reverse transcriptase activity, which correlates with plasma RNA load. The version 3 assay has been described as having improved precision and sensitivity. There are limited data on its performance relative to those of current real-time assays. Our objective was to compare HIV type 1 (HIV-1) RNA load measurement in plasma by ExaVir Load version 3 (designated ExaVir), Abbott M2000sp/M2000rt RealTime HIV-1 assay (designated RealTime), and Roche COBAS Ampliprep/COBAS TaqMan HIV-1 version 1 assay (designated TaqMan). Plasma from 119 patients (34 with subtype B infection, 85 with non-subtype B infection [A-H, CRF01, CRF02, CRF06, CRF12, CRF14, and complex]; 48 subjects were treatment experienced, 71 were naive) and serial dilutions of the second international standard (IS) were tested. Assay relationship and agreement were determined by linear regression, correlation analysis, and the Bland-Altman method. The ExaVir assay quantified 77/83 (92.8%) samples with viral loads of >2.3 log10 copies/ml by the molecular assays. Results were linearly associated and strongly correlated with RealTime and TaqMan measurements (R of 0.94 and 0.92, respectively) for both subtype B (R of 0.97 and 0.95, respectively) and non-subtype B (R of 0.93 and 0.91, respectively) samples. Mean differences were 0.28 and 0.18 log10 copies/ml in favor of the two molecular assays; 7/119 (5.9%) and 5/119 (4.2%) samples were outside the 95% level of agreement. ExaVir underquantified the IS by a mean of 0.2 (range, 0.0 to 0.5) log10 copies/ml. The ExaVir assay showed excellent concordance with real-time molecular assays, offering a suitable option for virological monitoring in settings with limited infrastructure.

Comparative Evaluation of the Performance of the Abbott Real-Time Human Immunodeficiency Virus Type 1 (HIV-1) Assay for Measurement of HIV-1 Plasma Viral Load following Automated Specimen Preparation

ABSTRACT The performance of the new Abbott real-time human immunodeficiency virus type 1 (HIV-1) assay for HIV-1 RNA load determination in plasma was compared to that of the Abbott LCx HIV-1 RNA quantitative assay following automated RNA isolation by the Abbott m1000 extractor. The measured viral loads of 89 clinical specimens differed by mean 0.19 log10 copies/ml (95% confidence interval, 0.12 to 0.26 log10 copies/ml). Although the difference in viral load determinations was positively skewed in favor of the LCx assay, it did not reach statistical significance (P = 0.42). Results were linearly associated (R2 = 0.94) and strongly correlated (R = 0.96). Good performance was observed with HIV-1 subtypes other than B and circulating recombinant forms, although results obtained with two subtype G specimens and one H specimen showed a more substantial difference.

Exploring the bidirectional interactions between human cytomegalovirus and hepatitis C virus replication after liver transplantation

Recurrence of Hepatitis C (HCV) post‐liver transplantation (LT) is universal and its course is more aggressive than in immunocompetent individuals. Human cytomegalovirus (CMV) infection is a common post‐LT infection and has immunomodulatory effects that could adversely affect the outcome of HCV. To date, the effect of HCV replication on the dynamics of CMV have not been investigated. From 2000 to 2004, a cohort of 69 HCV‐infected liver transplant recipients and 188 HCV‐negative liver transplant recipients (NON‐HCV cohort) were monitored for CMV infection twice weekly by CMV polymerase chain reaction (PCR) with preemptive therapy initiated after 2 consecutive positive results. None of the patients received CMV prophylaxis. A subset of 18 HCV‐infected patients had their HCV viral load monitored regularly post‐LT by quantitative PCR. CMV DNAemia (>200 genomes/mL blood) did not influence the level of HCV replication within 150 days posttransplantation or the stage of liver fibrosis in liver biopsies at 1 yr post‐LT. There were no differences in the incidence of CMV DNAemia or replication dynamics in the HCV cohort compared to the NON‐HCV cohort. In conclusion, short term CMV viremia does not enhance the replication of HCV after LT, while HCV replication does not alter the replication dynamics of CMV. Liver Transpl 13:130–135, 2007.

Analysis of transmitted HIV-1 drug resistance using 454 ultra-deep-sequencing and the DeepChek®-HIV system

Next‐generation sequencing (NGS) is capable of detecting resistance‐associated mutations (RAMs) present at frequencies of 1% or below. Several studies have found that baseline low‐frequency RAMs are associated with failure to first‐line HAART. One major limitation to the expansion of this technology in routine diagnostics is the complexity and laboriousness integral to bioinformatics analysis. DeepChek (ABL, TherapyEdge) is a CE‐marked software that allows automated analysis and resistance interpretation of NGS data.

Prevalence of baseline polymorphisms for potential resistance to NS5A inhibitors in drug-naive individuals infected with hepatitis C genotypes 1–4

BACKGROUND The non-structural 5A (NS5A) protein of HCV is a multifunctional phosphoprotein involved in regulation of viral replication and virion assembly. NS5A inhibitors targeting domain I of NS5A protein have demonstrated high potency and pan-genotypic antiviral activity, however they possess a low genetic barrier to resistance. At present, only genotype 1, the most prevalent HCV genotype has been studied in detail for resistant variants. METHODS Utilizing a panel of genotypic-specific resistance assays, population sequencing was performed on plasma-derived viral RNA isolated from 138 patients infected with HCV genotypes 1-4 and not treated with direct-acting antiviral agents. Amino acid changes in HCV NS5A domain I at codon positions 28, 30, 31, 32 and 93, reported to confer reduced susceptibility to certain NS5A inhibitors were examined. Additionally, genotypic outcome based on NS5A sequences were compared with VERSANT HCV Genotype Assay (LiPA) 1.0 (Siemens Healthcare Diagnostics, Surrey, UK) and Abbott m2000 RealTime HCV genotype II assay (Abbott Molecular, Maidenhead, Berkshire, UK). RESULTS Amino acid substitutions associated with moderate to high level resistance to NS5A inhibitors were detected in 2/42 (4.76%) HCV-1a, 3/23 (13.04%) HCV-1b, 4/26 (15.38%) HCV-2, 1/24 (4.17%) HCV-3 and 1/23 (4.35%) HCV-4 infected patients who had not been treated with NS5A inhibitors. Genotype prediction based on NS5A sequences were concordant with LiPA and/or Abbott RealTime for 97.10% of cases. CONCLUSIONS Primary resistance mutations associated with resistance to first-generation NS5A inhibitors such as daclatasvir were observed in all genotypes, albeit at low frequencies. An excellent correlation based on NS5A genotyping and LiPA or Abbott RealTime was achieved.

Telaprevir or boceprevir based therapy for chronic hepatitis C infection: Development of resistance-associated variants in treatment failure

The use of triple-therapy, pegylated-interferon, ribavirin and either of the first generation hepatitis C virus (HCV) protease inhibitors telaprevir or boceprevir, is the new standard of care for treating genotype 1 chronic HCV. Clinical trials have shown response rates of around 70-80%, but there is limited data from the use of this combination outside this setting. Through an expanded access programme, we treated 59 patients, treatment naïve and experienced, with triple therapy. Baseline factors predicting treatment response or failure during triple therapy phase were identified in 58 patients. Thirty seven (63.8%) of 58 patients had undetectable HCV RNA 12weeks after the end of treatment. Genotype 1a (p=0.053), null-response to previous treatment (p=0.034), the rate of viral load decline after 12weeks of previous interferon-based treatment (p=0.033) were all associated with triple-therapy failure. The most common cause of on-treatment failure for telaprevir-based regimens was the development of resistance-associated variants (RAVs) at amino acids 36 and/or 155 of HCV protease (p=0.027) whereas in boceprevir-based regimens mutations at amino acid 54 were significant (p=0.015). SVR12 rates approaching 64% were achieved using triple therapy outside the clinical trial setting, in a patient cohort that included cirrhotics.

Predicting antiretroviral drug resistance from the latest or the cumulative genotype.

BACKGROUND This study evaluates the added benefit when estimating antiretroviral drug resistance of combining all available resistance test results in a cumulative genotype relative to using the latest genotype alone. METHODS The prevalence of resistance and genotypic sensitivity scores (GSS) predicted by the latest and the cumulative genotype, together with virological outcomes after the latest genotype, were measured in treatment-experienced patients who underwent ≥2 resistance tests in 1999-2008. RESULTS Comparing the latest with the cumulative genotype in 227 patients, 4 (1.7%) versus 0 (0.0%) showed no major resistance mutations, whereas 74 (32.6%) versus 46 (20.3%), 88 (38.8%) versus 76 (33.5%) and 61 (26.9%) versus 105 (46.3%) showed single-class, dual-class and triple-class resistance mutations, respectively. The median (IQR) number of fully or partially active drugs was 6 (5-6) versus 5 (4-6) for the nucleoside/nucleotide reverse transcriptase inhibitors, 3 (1-3) versus 1 (1-3) for the non-nucleoside reverse transcriptase inhibitors and 7 (7-7) versus 7 (7-7) for the protease inhibitors, respectively. Among 163 patients who started a new regimen after the latest genotype, both the latest and the cumulative GSS were predictive of early (≤24 weeks) virological responses. The GSS decreased by median 1 unit (IQR 0.5-1.0) in the cumulative genotype and larger differences relative to the latest genotype corresponded to smaller decreases in viral load. CONCLUSIONS The cumulative genotype offers a more comprehensive evaluation of the burden of resistance. This approach can guide small but appreciable improvements in the selection of antiretroviral regimens for treatment-experienced patients.

The utility of different bioinformatics algorithms for genotypic HIV-1 tropism testing in a large clinical cohort with multiple subtypes

Objectives:HIV-1 tropism needs to be determined before the use of CCR5 antagonist drugs such as maraviroc (MVC), which are ineffective against CXCR4-using HIV-1. This study assessed how different computational methods for predicting tropism from HIV sequence data performed in a large clinical cohort. The value of adding clinical data to these algorithms was also investigated. Design and methods:PCR amplification and sequence analysis of the HIV-1 gp120 V3 loop region was performed on triple replicates of plasma viral RNA or proviral DNA extracted from peripheral blood monocytes (PBMCs) in 242 patients. Coreceptor usage was predicted from V3 sequences using seven bioinformatics interpretation algorithms, combined with clinical data where appropriate. An intention-to-treat approach was employed for exploring outcomes and performance for different viral subtypes was examined. Results:The frequency of R5 predictions varied by 22.6%, with all seven algorithms agreeing for only 75.3% of tests. The identification of individuals likely to fail was poor for all algorithms. The addition of clinical data improved this, but at the expense of their ability to predict success. The clinical algorithms varied across subtypes, whereas other algorithms were more consistent. Furthermore, individuals with discordant clonal and clinical predictions were more likely to fail MVC treatment. Conclusion:Eligibility for MVC varied depending on the algorithm method used. The addition of clinical parameters alongside sequence data may help predict X4 emergence during treatment. It could be that V3 loop analysis in isolation may not be the best method for selecting individuals for MVC.

Detection of low-frequency K103N mutants after unstructured discontinuation of efavirenz in the presence of the CYP2B6 516 TT polymorphism

OBJECTIVES To measure antiretroviral drug plasma levels in newly diagnosed HIV-1 seropositive persons who presented with an undetectable plasma HIV-1 RNA load but gave no history of antiretroviral drug exposure and to determine the impact of interrupting undisclosed or unknown antiretroviral therapy on the emergence of drug resistance. PATIENTS AND METHODS Five newly diagnosed, reportedly drug-naive HIV-1 seropositive persons were included in the study. Drug resistance was determined by population and clonal sequencing of reverse transcriptase and protease. CYP2B6 polymorphisms were assayed by real-time PCR allelic discrimination on pre-amplified exons. RESULTS Efavirenz was detected in the plasma of one of the five persons coinciding with a viral load <40 copies/mL by two different assays. When efavirenz became undetectable, the viral load rebounded. The patient was CYP2B6-516T homozygous. Population sequencing showed wild-type subtype D virus, whereas clonal sequencing detected low-frequency (2%) K103N. The patient firmly denied antiretroviral exposure but described the use of Ugandan remedies. CONCLUSIONS In migrating populations seeking HIV testing, careful and compassionate counselling is required to facilitate the disclosure of previous diagnosis and therapy. The use of remedies of dubious content should also be discussed and investigated.

Baseline drug resistance mutations are detectable in HCV genes NS3 and NS5A but not NS5B in acute and chronic HIV-coinfected patients.

In 2012, Plaza et al. [1] reported the prevalence of natural polymorphisms in the HCV NS5A gene associated with resistance to daclatasvir (DCV) in 78 HIV– HCV-coinfected patients and 635 HCV-monoinfected derived NS5A sequences deposited in Los Alamos HCV database. They did not observe NS5A resistanceassociated variants (RAVs) in HCV-1a and HCV-3 NS5A sequences, whereas, major RAVs (Y93H) were detected in 7% and 13% in NS5A sequences from coinfected patients infected with HCV-1b and HCV-4, respectively, with a similar frequency of NS5A RAVs observed in HCV-monoinfected patients for these HCV genotypes (gts) [1]. Additionally, the L31M NS5A variant was observed in 7% of HCV gt-1b patients, irrespective of coinfection status and occurred in >93% of HCV gt-4 monoinfected and coinfected patients [1]. Interestingly, the presence of naturally occurring drug resistance variants in acutely HCV-infected, treatmentnaive HIV patients have been detected by population and deep sequencing in HCV NS3 in a large proportion of subjects [2]. The significance of the threshold at which these RAVs are detectable and whether these will impact on response to therapy with NS3 and NS5A inhibitors in clinical practice is not fully clear. In acute HCV amongst those who are HIV-infected, the role for new HCV direct-acting antivirals (DAAs) has not been established. Treatment with pegylated interferon and ribavirin (PEG-IFN/RBV) early in HCV infection is often successful for most gts [3]. Telaprevir, a firstgeneration protease inhibitor (PI), has been used in a small study of acute HCV infection in HIV, and results suggest sustained virological response (SVR) may be improved using ‘triple therapy’ with shortened treatment durations [4]. It is likely that in the near future DAA-based therapy will be standard of care for both acute and chronic HCV infections, with PIs forming part of this armamentarium [5,6] alongside other DAAs, including NS5B polymerase inhibitors [7] and H-CV NS5A inhibitors [8]. Ultimately, an interferonfree future is heralded, where drug regimens will consist of combinational DAAs, targeting different HCV gene products [9]. It is therefore important to establish the frequency of RAVs in all patients infected with HCV. In this study, we investigated the prevalence of RAVs by population sequencing from three groups of HIV– HCV-coinfected patients: acute HCV infections (n=25), chronic treatment-naive patients (n=20) and chronic treatment-experienced (PEG-IFN/RBV) patients who did not achieve an SVR (n=34) and compared with the prevalence of RAVs in 85 chronic HCV-monoinfected patients. Genomic regions (sites of known RAVs) were amplified from HCV RNA using reverse transcriptase PCR followed by a nested PCR. Typically, amino acids 1–181 of HCV NS3 protease (gt1 only), amino acids 1–213 of domain I of NS5A (for gt-1a, 1b, 2, 3 and 4) and amino acids 219–347 of NS5B (pan-genotypic) were included. Purified PCR amplicons were sequenced using ABI PRISM 3730 genetic analyser (Applied Biosystems, Life Technologies Ltd, Paisley, UK) and consensus sequences aligned against HCV reference sequences. Baseline RAVs were detected in all three cohorts of coinfected patients in NS3 and NS5A but baseline S282T, associated with resistance to sofosbuvir, was not detected in any of our cohorts (Table 1), possibly attributable to the low fitness of this mutation [10]. The Q80K polymorphism was the predominant NS3 variant for gt-1a conferring resistance to firstand secondgeneration PIs [11], and increased in frequency in acute Letter