Tropism Testing Research Articles

Use of Cellular HIV DNA to Predict Virologic Response to Maraviroc: Performance of Population-Based and Deep Sequencing

A tropism test is required before administration of the antiretroviral drug maraviroc. However, plasma RNA testing is not possible in patients with undetectable plasma viral loads. Here we assess genotypic testing of cellular human immunodeficiency virus (HIV) DNA from peripheral blood mononuclear cells (PBMCs) to predict virologic responses in treatment-experienced patients beginning maraviroc-containing regimens. PBMC samples from 181 maraviroc recipients at study entry in MOTIVATE or A4001029 (51% R5 by original Trofile). The V3 loop was amplified in triplicate from cellular HIV DNA, and matching plasma RNA (n = 156). Sequencing was performed using standard population-based methods and next-generation deep sequencing, with tropism assessment as previously defined. Genotypic DNA-based tropism testing from the cellular compartment had 78%-81% sensitivity relative to RNA-based Trofile at the same time point. Cell-based genotypic tropism methods and plasma-based phenotypic and genotypic methods were predictive of virologic response. However, when classifications were discordant, the outcomes favored the plasma predictions over the DNA ones. Genotypic determination of HIV tropism can be performed using cell-derived viral DNA, and is a predictor of virologic success on maraviroc in therapy-experienced patients. However, the PBMC compartment appears to be a suboptimal predictor compared to plasma.

High prevalence of CXCR4 usage among treatment-naive CRF01_AE and CRF51_01B-infected HIV-1 subjects in Singapore

BackgroundRecent studies suggest HIV-1 inter-subtype differences in co-receptor usage. We examined the correlation between HIV-1 subtype and co-receptor usage among treatment-naïve HIV-1 subjects in Singapore. Additionally, we investigated whether the subtype co-receptor association was influenced by stage of infection.MethodsV3 sequences of HIV-1 envelope protein gp120 were obtained from 110 HIV treatment-naïve patients and genotypic co-receptor tropism determination was performed using Geno2pheno. Two false-positive rate (FPR) cut-offs, 10% and 5.75% were selected for tropism testing.ResultsSubtype assignment of viral strains from 110 HIV-infected individuals based on partial sequencing of HIV-1 pol, gp120 and gp41 were as follows: 27 subtype B, 64 CRF01_AE, 10 CRF51_01B, and 9 other subtypes. At FPR=10%, 10 (100%) CRF51_01B-infected subjects and 26 (40.6%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 7 (25.9%) subtype B subjects and 1 (11.1%) CRF33_01B-infected subject (P < 0.001). At FPR=5.75%, 10 (100%) CRF51_01B-infected subjects and 20 (31.3%) CRF01_AE-infected subjects had CXCR4-using virus, compared to 4 (14.8%) subtype B and 1 (11.1%) CRF33_01B-infected subjects (P < 0.001). Among those with evidence of seroconversion within 2 years prior to study enrolment, 100% of CRF51_01B-infected subjects had CXCR4-using virus, independent of Geno2pheno FPR.ConclusionCRF51_01B and CRF01_AE-infected individuals have higher prevalence of CXCR4-usage compared to subtype B infected individuals. Further studies examining these differences could help optimise the use of CCR5-antagonist in populations with these subtypes, and increase our understanding of HIV-1 biology.

Switching the third drug of antiretroviral therapy to maraviroc in aviraemic subjects: a pilot, prospective, randomized clinical trial

To evaluate the safety and efficacy of switching the third drug of antiretroviral treatment to maraviroc in aviraemic subjects infected with R5 HIV. This is a pilot, prospective, randomized clinical trial (ClinicalTrials ID: NCT00966329). Eighty HIV-1-infected aviraemic adults on stable antiretroviral treatment for ≥1 year and no antiretroviral drug resistance were screened for the presence of non-R5 HIV by triplicate proviral V3 population sequencing. From them, 30 subjects with R5 HIV-1 were randomized 1 : 1 to switch the non-nucleoside reverse transcriptase inhibitor or ritonavir-boosted protease inhibitor to maraviroc (n = 15) or to continue the same antiretroviral treatment (controls, n = 15). The principal endpoint was the proportion of subjects with HIV-1 RNA <50 copies/mL at week 48. Ultrasensitive proviral HIV-1 tropism testing (454 sequencing) was performed retrospectively at weeks 0, 4, 12, 24, 36 and 48. One subject in the maraviroc arm and one control had non-R5 HIV in proviral DNA by retrospective 454 sequencing. The subject receiving maraviroc was the only individual to develop virological failure. However, plasma HIV at failure was R5. Switching to maraviroc was well tolerated and associated with small, but statistically significant, declines in total, high-density lipoprotein and low-density lipoprotein cholesterol. Median (IQR) triglyceride [1 (0.67-1.22) versus 1.6 (1.4-3.1) mmol/L, P = 0.003] and total cholesterol [4.3 (4.1-4.72) versus 5.4 (4-5.7) mmol/L, P = 0.059] values were lower in the maraviroc arm than in controls at week 48. In this pilot, prospective, randomized clinical trial, switching the third drug to maraviroc was safe, efficacious and improved lipid parameters.

Concordance between HIV-2 genotypic coreceptor tropism predictions based on plasma RNA and proviral DNA

In this study, assessing HIV-2 tropism among 43 paired plasma/peripheral blood mononuclear cell specimens, the concordance between proviral DNA and plasma RNA genotypic tropism prediction was 74%. All the discordances were attributable to the prediction of R5 in RNA and X4/dual-mixed in DNA. HIV-2 genotypic tropism test based on proviral DNA is a suitable tool for tropism determination in HIV-2-infected patients with low or undetectable viral load.

Detecting and understanding genetic and structural features in HIV-1 B subtype V3 underlying HIV-1 co-receptor usage

To define V3 genetic elements and structural features underlying different HIV-1 co-receptor usage in vivo. By probabilistically modeling mutations in the viruses isolated from HIV-1 B subtype patients, we present a unique statistical procedure that would first identify V3 determinants associated with the usage of different co-receptors cooperatively or independently, and then delineate the complicated interactions among mutations functioning cooperatively. We built a model based on dual usage of CXCR4 and CCR5 co-receptors. The molecular basis of our statistical predictions is further confirmed by phenotypic and molecular modeling analyses. Our results provide new insights on molecular basis of different HIV-1 co-receptor usage. This is critical to optimize the use of genotypic tropism testing in clinical practice and to obtain molecular-implication for design of vaccine and new entry-inhibitors.

HIV‐1 Tropism Testing and Clinical Management of CCR5 Antagonists: Quebec Review and Recommendations

HIV-1 tropism assays play a crucial role in determining the response to CCR5 receptor antagonists. Initially, phenotypic tests were used, but limited access to these tests prompted the development of alternative strategies. Recently, genotyping tropism has been validated using a Canadian technology in clinical trials investigating the use of maraviroc in both experienced and treatment-naive patients. The present guidelines review the evidence supporting the use of genotypic assays and provide recommendations regarding tropism testing in daily clinical management.

HIV DRUG resistance genotyping – an Australian perspective of its role and utilisation in contemporary HIV practice

Resistance to individual drugs within combination antiretroviral therapies (cART) regimens has been a growing international problem. HIV drug-resistance genotyping has been widely, but not universally, available in Australia for over a decade, initially through state funded reference laboratories, and more recently since 2011 by Medicare through the Pathology Services Table. An assessment by the Medical Services Advisory Committee (MSAC) found both in-house and commercial kit testing to be cost effective in a complex modelling of outcomes. Currently standard resistance genotyping provides a predicted resistance profile for three classes of antiretroviral agents. Individual state-based service data show moderate prevalence of primary resistance to at least one antiretroviral agent from the three main classes of NRTI, NNRTI and PI. Non RTI and non-PI therapies are increasingly being used with little evidence of primary resistance. Laboratories will need to develop a broader repertoire of gene sequencing capability to accommodate the clinical need in those exposed to these newer drugs. CCR5 blockers require pre-use tropism testing of an individual’s virus. The early reported experience of laboratories performing tropism genotyping from integrated proviral DNA sequences in virally suppressed patients highlights the need for caution in interpreting tropism in the setting of viral suppression.

Factors Influencing the Sensitivity and Specificity of Conventional Sequencing in Human Immunodeficiency Virus Type 1 Tropism Testing

Human immunodeficiency virus type 1 (HIV-1) V3 loop sequence can be used to infer viral coreceptor use. The effect of input copy number on population-based sequencing of the V3 loop of HIV-1 was examined through replicate deep and population-based sequencing of samples with known tropism, a heterogeneous clinical sample (624 population-based sequences and 47 deep-sequencing replicates), and a large cohort of clinical samples from phase III clinical trials of maraviroc including the MOTIVATE/A4001029 studies (n = 1,521). Proviral DNA from two independent samples from each of 101 patients from the MOTIVATE/A4001029 studies was also analyzed. Cumulative technical error occurred at a rate of 3 × 10(-4) mismatches/bp, without observed effect on inferred tropism. Increasing PCR replication increased minority species detection with an ~10% minority population detected in 18% of cases using a single replicate at a viral load of 1,072 copies/ml and in 44% of cases using three replicates. The nucleotide prevalence detected by population-based and deep sequencing were highly correlated (Spearman's ρ, 0.73), and the accuracy increased with increasing input copy number (P < 0.001). Triplicate sequencing was able to predict tropism changes in the MOTIVATE/A4001029 studies for both low (P = 0.05) and high (P = 0.02) viral loads. Sequences derived from independently extracted and processed samples of proviral DNA for the same patient were equivalent to replicates from the same extraction (P = 0.45) and had correlated position-specific scoring matrix scores (Spearman's ρ, 0.75; P << 0.001); however, concordance in tropism inference was only 83%. Input copy number and PCR replication are important factors in minority species detection in samples with significant heterogeneity.

Which HIV Tropism Test Is Preferred: Genotypic or Phenotypic?

The use of maraviroc, a CCR5 antagonist, is restricted to patients who have only CCR5-using HIV as determined through a tropism test. In the U.S., HIV

No significant association between patient self‐reported non‐adherence to antiretrovirals and HIV‐tropism: a preliminary analysis

Nonadherence to antiretroviral therapy (ART) may cause virologic failure and disease progression has been associated with switch of viral coreceptor usage from CCR5 to CXCR4. We aimed to assess the association between patient-reported non-adherence and HIV tropism. This is a cross-sectional analysis. HIV-tropism was performed within routine clinical practice either at start of ART or at virological failure. Adherence questionnaire includes: how many times ART has been taken during the last month, missed doses in the last week, timing deviation, refill interruption, drug holidays. Demographics, epidemiological data, HIV and ART history, CD4 and HIVRNA were collected. To assess co-receptor tropism, env V3 genotyping from viremic plasma HIVRNA was performed. For the analysis, dual/mixed viruses were considered as X4. We included 102 individuals: 76% males; median age 42 y (IQR, 37–46); transmission was heterosexual 37%, homosexual 31%, intravenous drug use 29%. Median nadir of CD4 154/mmc (IQR, 53–274), median zenith of HIVRNA 5.26 (4.72–5.70), 46% had AIDS. 124 tropism tests were: 78% R5, 17% X4, 5% dual/mixed. In cases with previous ART, mono/dual ART was found in 26%, median number of regimens was 5 (IQR, 2–10), median time on triple-ART was 54 months (IQR, 0–123) with median time of HIVRNA <50 c/ml of 16 months (IQR, 6.5–34.9). At HIV-tropism, median CD4 and HIV RNA were 321/mmc (IQR, 210–436) and 2.65 (IQR, 2.65–4.91), respectively. Median time between adherence questionnaire and HIV-tropism was 68 days (IQR, 23–116). At adherence questionnaire, median percentage of ART taken during the last month was 100% (IQR, 90–100), 39% reported missed doses in the last week, 40% timing deviation, 7% refill interruption, 17% drug holidays. At univariate analysis, no statistically significant association between non-adherence and dual/mixed-X4 viruses was found (p>0.1). Also gender, age, HIV transmission, AIDS, CD4 nadir, HIVRNA zenith, mono/dual ART, and number of ART regimens were not associated with type of tropism. Only longer time with undetectable HIVRNA before tropism test showed a lower probability of dual/mixed-X4 viruses (OR for each month 0.95; 95% CI 0.90–1.00; p=0.06). No significant association between adherence and HIV-tropism was found in this preliminary analysis. It is possible that patient self-reported adherence is not able to capture nonadherence behaviors that underlie more pronounced viral replication which may be necessary for tropism switch.

Analysis of HIV‐1 primary drug resistance in Kazakhstan

Purpose of the studyMonitoring of primary resistance in HIV‐1 variants circulating in Russia and FSU countries is the important task of HIV infection molecular epidemiology. The data of HIV molecular epidemiology are absent or limited in many FSU countries. IDU‐A variant of HIV‐1 subtype A has been dominating in Russia (&gt;90%) and other FSU countries since 1996. Additionally, the Central Asian region (e.g. Kazakhstan and Uzbekistan) is characterized by relatively wide spread of CRF02_AG recombinant. The aim of our study was the analysis of HIV primary drug resistance in HIV‐1 variants from Kazakhstan.MethodsBlood collection from the HIV‐infected naïve patients was carried out by local specialists. The study was performed with the informed consent of patients. All sequences of pol gene were analyzed by COMETv0.1 and HIVdb on‐line programs. The phylogenetic analysis and tropism testing were carried out by MEGA4.0 and geno2pheno.Summary of results51 PBMC samples were analyzed. According to pol gene phylogenetic analysis and genotyping 24 (47.0%) samples belonged to recombinant CRF02_AG; 25 (49.0%)‐to subtype A1; 2 (3.9%)‐to CRF03_AB circulating in Russia and some FSU countries. Only one subtype A1 sample had D30N mutation in Pro‐region that cause high‐level resistance to NFV. All AG‐samples had K20I substitution which is characteristic for HIV‐1 subtype A and G and is believed to be associated with resistance to LPV and NFV. In addition, we found that 2 AG‐samples had L10V and L76I substitutions, accordingly. Two A1‐subtype samples studied had L10I and K43T, accordingly. A62V characteristic mutation in RT‐region was found in 12 (48%) subtype A1‐samples. One of them had M184I mutation as well. Besides, we found 3 A1‐samples harboring NNRTIs resistance mutations‐K103N, G190S, K238N, accordingly. Further we analyzed IN region of pol‐gene in 16 and env‐gene in 23 samples studied. We found only one E157Q mutation (in CRF03_AB sample) in IN region. As to tropism, only two subtype A1 and 1 CRF03_AB samples belonged to X4/X4R5 variant, all these A1‐subtype samples harbored A62V and one of them‐G190S in RT region of pol‐gene; the other samples were treated as R5 variants.ConclusionsOur data demonstrated the low level of HIV primary drug resistance in Kazakhstan. HIV‐1 subtype A variant dominates on this territory, but CRF02_AG recombinant is rather widespread as well. CRF02_AG variant studied has characteristic features in Pro region compared with IDU‐A.

PIN2 Assessing the Diagnostic Performance of Human Immunodeficiency Virus Type 1 (HIV-1) Tropism Testing Methods: A Systematic Review of Genotypic Sequencing of the Third Hypervariable (V3) Loop and Enhanced-Sensitivity Phenotypic Assay Technology

To evaluate the relative diagnostic performance of genotypic sequencing and enhanced-sensitivity phenotypic testing methods for: i) identifying the tropism of HIV-1 infected patients, and ii) predicting virological response to CCR5-antagonist therapy. A systematic review of the literature (via EMBASE and Medline databases) initially identified 359 publications. Based on a priori defined eligibility criteria, five studies were included in the final analysis. Four of the five studies assessed the diagnostic performance of genotypic testing in identifying viral tropism, with the enhanced-sensitivity Trofile assay (ESTA) as the reference standard. Using the geno2pheno bioinformatic algorithm, genotypic testing maintained a high concordance with ESTA (range: 70.6%-85.3%). At a false-positive rate (FPR) of 5%, specificity for R5-tropic virus was also high (range: 85.7%-95.3%), but came at the expense of sensitivity for X4-using virus (range: 36.7%-66.7%). One study compared the effectiveness of both genotypic tropism testing and ESTA in predicting virological response to the CCR5-antagonist maraviroc. The study found in each screening group, a similar proportion of patients achieved a viral load < 50 HIV-1 RNA copies/mL by Week 48. In the absence of a ‘gold standard', clinical response to CCR5-anatagonist therapy offers the best measure of diagnostic performance in HIV-1 tropism testing. The results of this review indicate that genotypic sequencing of the V3 loop is as capable of predicting response to CCR5-antagonist therapy as the current diagnostic standard, ESTA. In addition, of the bioinformatic algorithms reviewed here, the geno2pheno model set at 5-10% FPR offered the best balance between sensitivity and specificity. This evidence provides further support for the use of genotypic tropism testing in routine clinical practice.

Is there a relationship between HIV tropism and historical genotypic resistance in treatment‐experienced patients?

Purpose of the studySince X4/DM HIV‐1 tropism is associated with poorer prognosis and worse response to treatment, the aim of this study was to assess whether X4/DM HIV‐1 tropism is also related with a higher accumulation of resistance in patients experiencing treatment failure.MethodsHIV protease (PR) and reverse transcriptase (RT) resistance mutations and tropism test results were extracted from a national database. Viral tropism data included enhanced sensitivity Trofile assay (ESTA) and geno2pheno results at 10% false positive rate. Historical resistance mutations (HRM) for PI, NRTI and NNRTI, detected in all genotypic tests performed during patient treatment history, were selected according to IAS‐USA indications.Summary of resultsOverall, 1280 patients were included: males 65%, median age 45 years (IQR: 40–50), median CD4 nadir 116 (IQR: 34–272), median past regimens 7 (IQR: 3–12), median previous NRTI used 5 (IQR: 4–7), median previous NNRTI used 1 (IQR 1–2) and median previous PI used 3 (IQR 1–5). HIV tropism was assessed by ESTA in 271 patients (21.2%) and by geno2pheno in 1009 patients (78.8%). Four hundred and fifteen patients (32.4%) carried X4/DM virus and 321 (25.1%) had ≥1 HRM for each antiretroviral class. The mean number of HRM was higher in patients harboring X4/DM virus than in patients harboring R5 virus (5.1±6.4 vs. 4.3±5.9, p = 0.02 at ANOVA test). X4/DM strains also harbored a higher mean number of PI‐related HRM (1.8±2.8 vs. 1.5±2.6, p = 0.003) and NNRTI‐related HRM (1.2±1.6 vs. 0.9±1.4, p = 0.001), but not of NRTI‐related HRM (2.2±2.6 vs. 2.0±2.6). At logistic regression, patients with HRM for all the 3 classes had a significant higher risk of also harboring X4/DM virus (OR: 1.6, 95% CI: 1.0–2.4, p = 0.04). Moreover, X4/DM virus was found to be associated with previous use of NNRTI‐containing regimens (OR: 1.4, 95% CI: 1.1–1–9, p = 0.03) and lower CD4 nadir (OR: 0.9, 95% CI: 0.9–1.0, p = 0.001, per 50‐CD4 increase). The analysis was adjusted by number of genotypic tests, number of treatment lines, age and HV subtype. Single mutations significantly associated with X4/DM tropism were: for PI, V32I and L76V; for NRTI: M41L, K70R, L74V and T215Y and for NNRTI: E138G and V179T.ConclusionsOur data suggest that X4/DM tropism is associated with accumulation of resistance mutations during treatment history. X4/DM tropism is also confirmed to be a marker of a more compromised clinical and immune‐virological condition.

Test for CCR5 tropism and treatment with maraviroc in Sicily: an observational retrospective multicentre study

Purpose of the studyMaraviroc (MVC) is the first CCR5 inhibitor licensed for clinical use. A pre‐treatment test is mandatory to identify R5 tropic patients. Aim of this study is to detect indications and results of tropism test and to evaluate efficacy and tolerability of MVC‐based regimen.MethodsAn observational retrospective multicentre study was performed in Sicily in 15 Infectious Diseases Units. Clinical records of 213 screened for tropism HIV+ subjects were reviewed for age, sex, risk, clinical stage (CDC, CD4 cell count, HIV RNA viral load), therapeutic line, indication and result of test for tropism; within subjects treated with MVC, HIV RNA, CD4 cell count and metabolic parameters trend and adverse events were analysed.Summary of resultsMedian age 44 (IQR 30–50) years, 67.1% males; 46.3% heterosexuals, 28.6% MSMs, 21.4% IVDUs; 23.7% CDC A, 32.1% CDC B, 44.2% CDC C; median CD4 was 217 (IQR 121–374) cells/µl and mean of HIV RNA was 4.72 (Cl 95% 4.07–4.67) log10 copies/ml; median therapeutic line was 4 (IQR 2–7). 80.8% were submitted to Trofile™ test, 19.2% to genotypic test, 75.5% after a therapeutic failure. 56.8% of subjects screened were R5, 7.5% X4, 21.6% DM, 14% undefined. All X4 patients were tested after a therapeutic failure; patients screened for toxicity were more frequently R5 (75%) (p&lt;0.01). 76 (35.7%) multi‐experienced (at baseline 8% HIV RNA&lt;50 copies/ml, median CD4 cell count 219 (IQR 124–345) cells/µl) subjects were treated with MVC plus an optimized background treatment: MVC was associated in 74% of cases with a protease inhibitors (56% darunavir/ritonavir), in 42% with raltegravir, in 56% with a NUC‐sparing regimen. After 12 months of treatment 56.8% (ITT analysis) and 61.7% (AT) of patients had HIV RNA&lt;50 copies/ml; median CD4 cell count was 387 (IQR 222–455) cells/µl. After 24 months 64.8% (ITT) 80% (AT) had HIV‐RNA&lt;50 copies/ml. Median CD4 cell count was 381 (IQR 218.515) cells/µl with a median increase of 168 (IQR 54–274) cells/µl. At 24 months median value of total and HDL cholesterol and triglycerides were within the normal range. 7 patients stopped the treatment: 2 died, 1 adverse event, 4 virological failure.ConclusionsAlthough the test has been proposed to patients with long treatment history and failure, only 3/5 of R5 tropic patients were treated with MVC. An high number of multi‐experienced subjects treated with a MVC‐based regimen obtained HIV RNA&lt;50 copies/ml and a satisfactory increase of CD4 cell count.

A phase 4, single‐arm, open‐label, pilot study of maraviroc, raltegravir and darunavir/r in HIV‐1 adults with triple class failure: TERCETO study

The purpose of this phase 4, single‐arm, open‐label study was to evaluate the safety, tolerability, efficacy, antiviral and immunological activity of maraviroc (MVC) in combination with raltegravir (RGV) and darunavir/r (DRV/r) in adult HIV‐1 infected patients (pts) with limited treatment options. HIV‐1 pts with documented virologic triple class failure or multi‐drug class resistance defined as the presence of Q151 complex, 69 insertion complex and/or≥3 TAMs for NRTIs and K103N, G190S+Y181C or Y188L mutants for NNRTIs and≥3 RAMs (L10F/I/R/V; M46I/L; I54V/M/L; V82A/F/T/S; I84V; L90M) for protease inhibitors (PIs) were offered a triple drug regimen consisting of MVC 150 mg BID, RGV 400 mg BID and DRV/r 600/100 mg BID. Safety, lipid profile and virologic efficacy were evaluated at week 4, 12, 24, 36 and 48. Between January 2010 and March 2012, 27 pts were enrolled. Screening failure rate was 52% due to undetectable viral load (pVL) or non R5 tropism type (Trofile™). Despite being heavily pre‐treated pts, only 26% had negative tropism test at SCR. Baseline characteristics of 13 included pts were: 77% male, median age 43 years (IQR: 40.1–48.6), 38% had a prior AIDS‐defining condition. Median BSL pVL was 23,350 cps/mL (4.4 log10) (IQR: 11,236–55,785) and median CD4 was 222 cells/mm3 (IQR: 179–318). Median time on NRTIs, NNRTIs and PIs were 10.7 (8.6–13.7), 1.7 (1.3–7.6) and 5.4 (4.7–10) years respectively. Pts had received a median of 2 PIs (IQR: 2–3). 8/13 pts showed thymidine analogue‐associated mutations (TAMs), and≥2 were present in 5/13. Detectable NNRTI resistance‐associated mutations (RAMs) were present in 10/13 patients. 9/13 had≥4 primary PI RAMs. At 48 weeks, 2 pts had discontinued therapy (OIs related death (cryptococcal meningitis)=1, withdrawn from the study on W36 due to blips despite not achieving criteria for virologic failure=1) and the remaining pts (11/13) achieved undetectable pVL and increased CD4 in 133 cell/mm3 from BSL (IQR: 81–174.5). Median total cholesterol levels increased from 162 mg/dL (IQR: 135‐188) to 215 mg/dL (IQR: 182–237) between BSL/W48; median change in cholesterol levels: 40 mg/dL (IQR: 6.5‐66). Salvage therapy including MVC, RGV and DRV/r achieved sustained reductions in pVL (&lt;50 copies/mL) through 48 weeks of therapy in this pilot study with no treatment limiting toxicity.AcknowledgementsTERCETO study was sponsored through an investigator‐initiated research (IIR) grant from Pfizer Inc. Merck provided raltegravir.

Optimisation of baseline genotypic testing for safe and efficient maraviroc administration

Different diagnostic parameters may affect the tropism prediction reliability. The impact of usage of FPR cut‐offs&lt;20%, use of viral RNA versus proviral DNA samples, single versus triple amplification, and presence of MVC resistance mutations on tropism prediction at baseline were analysed on 101 patients receiving maraviroc (MVC) and correlated with their clinical outcome. This was a non‐interventional, retrospective study. 82 RNA and 54 DNA samples from the 101 patients receiving MVC were obtained. The V3 region was sequenced and the tropism predicted using the geno2pheno[coreceptor] and T‐CUP tools with FPR cut‐offs of 5%, 7.5%, 10%, 15% and 20%. Additionally, 27/82 RNA and 28/54 DNA samples were analysed in triplicate and 34/82 samples with the ESTA assay. The influence of 16 MVC resistance mutations on clinical outcome was studied. The genotypic susceptibility score (GSS) of the concomitant drugs was mapped to numerical values: susceptible to 1 (or 0.5 for NRTIs), intermediate to 0.5 (0.25 for NRTIs) and resistant to 0. Detection of baseline R5 viruses in RNA (by geno2pheno[coreceptor] and T‐CUP) or DNA (by T‐CUP) samples correlated with MVC‐treatment success. Both tools performed very similarly, with PPVs close to 90%, even with FPR cut‐offs as low as 5%. The use of triple amplification did not improve the prediction value but reduced the number of patients elegible for MVC treatment. No influence of the GSS or MVC resistance mutations on the clinical outcome was detected. Genotypic tropism testing from viral RNA and proviral DNA using the geno2pheno[coreceptor] and T‐CUP systems is valid to select candidates for MVC treatment. Our data suggest that the use of FPR cut‐offs of 5–7.5% and single amplification from RNA or DNA would assure a safe administration of MVC without excluding many patients who could benefit from this potent antiretroviral drug.

A Genotypic Test for HIV-1 Tropism Combining Sanger Sequencing with Ultradeep Sequencing Predicts Virologic Response in Treatment-Experienced Patients

A tropism test is required prior to initiation of CCR5 antagonist therapy in HIV-1 infected individuals, as these agents are not effective in patients harboring CXCR4 (X4) coreceptor-using viral variants. We developed a clinical laboratory-based genotypic tropism test for detection of CCR5-using (R5) or X4 variants that utilizes triplicate population sequencing (TPS) followed by ultradeep sequencing (UDS) for samples classified as R5. Tropism was inferred using the bioinformatic algorithms geno2pheno[coreceptor] and PSSMx4r5. Virologic response as a function of tropism readout was retrospectively assessed using blinded samples from treatment-experienced subjects who received maraviroc (N = 327) in the MOTIVATE and A4001029 clinical trials. MOTIVATE patients were classified as R5 and A4001029 patients were classified as non-R5 by the original Trofile test. Virologic response was compared between the R5 and non-R5 groups determined by TPS, UDS alone, the reflex strategy and the Trofile Enhanced Sensitivity (TF-ES) test. UDS had greater sensitivity than TPS to detect minority non-R5 variants. The median log10 viral load change at week 8 was −2.4 for R5 subjects, regardless of the method used for classification; for subjects with non-R5 virus, median changes were −1.2 for TF-ES or the Reflex Test and −1.0 for UDS. The differences between R5 and non-R5 groups were highly significant in all 3 cases (p<0.0001). At week 8, the positive predictive value was 66% for TF-ES and 65% for both the Reflex test and UDS. Negative predictive values were 59% for TF-ES, 58% for the Reflex Test and 61% for UDS. In conclusion, genotypic tropism testing using UDS alone or a reflex strategy separated maraviroc responders and non-responders as well as a sensitive phenotypic test, and both assays showed improved performance compared to TPS alone. Genotypic tropism tests may provide an alternative to phenotypic testing with similar discriminating ability.

Pitfalls of HIV genotypic tropism testing after treatment interruption

The genotypic method is reliable enough for the determination of tropism and largely preferred in Europe. However, careful interpretation is essential when assessing HIV genotypic resistance during treatment interruption (TI) due to the possible disappearance of resistant strains. The results of HIV genotypic tropism testing in such a context remain unknown. First, we studied changes in tropism in patients included in a structured TI assay: the Reverse study. Second, we investigated the unexpected tropism switches from X4 to R5 recorded in our routine database. Tropism determination was possible in 21 patients of the Reverse study, 9 of whom had an X4 virus (43%) at baseline. Two patients displayed a change of tropism during TI, both switching from X4 to R5. Regarding the database investigation, 7 of the 222 patients with at least two plasma tropism determinations recorded in the database displayed a switch from X4 to R5. TI due to non-compliance at the time of the tropism change was reported for five of these seven patients. We have shown that the redistribution of the HIV population caused by TI could potentially result in X4 viruses becoming undetected and inappropriate prescription of a CCR5 receptor antagonist. Therefore, genotypic tropism results should be interpreted with caution in such a context.

Maraviroc Observational Study: The Impact of Expanded Resistance Testing and Clinical Considerations for Antiretroviral Regimen Selection in Treatment-Experienced Patients

Maraviroc (MVC) use has trailed that of other post-2006 antiretroviral therapy (ART) options for treatment-experienced patients. We explored the impact of free tropism testing on MVC utilization in our cohort and explored barriers to MVC utilization. The Maraviroc Outcomes Study (MOS) is an investigator-initiated industry-sponsored trial where consecutive ART-experienced patients receiving routine care with viral loads ≥1,000 copies/ml, and whose provider requested resistance testing and received standardized resistance testing (SRT; phenotype, genotype, coreceptor/tropism). Sociodemographic, clinical, and ART characteristics of those receiving SRT were compared to a historical cohort (HC). Subsequently, providers were surveyed regarding factors influencing selection of salvage ART therapy. The HC (n=165) had resistance testing 7/08-9/09, while prospective SRT (n=83) patients were enrolled 9/09-8/10. In the HC, 92% had genotypes, 2% had tropism assays, and 62% (n=102) changed ART after resistance testing (raltegravir 37%, etravirine 25%, darunavir 24%, MVC 1%). In the SRT cohort, 57% (n=48) changed regimens after standardized resistance testing (darunavir 48%, raltegravir 40%, and etravirine 19%). CCR5-tropic virus was identified in 43% of the SRT group, and MVC was used in 10% [or 20% of R5 tropic patients who underwent a subsequent regimen change (n=25)], a statistically significant (p=0.01) increase in utilization. The factors most strongly influencing utilization were unique patient circumstances (60%), clinical experience (55%), and potential side effects (40%). The addition of routine tropism testing to genotypic/phenotypic testing was associated with increased MVC utilization, raising the possibility that tropism testing may present a barrier to MVC use; however, additional barriers exist, and merit further evaluation.

HIV population genotypic tropism testing and its clinical significance

HIV co-receptor tropism testing is recommended before therapy when the C-C chemokine receptor type 5 antagonist maraviroc is initiated. This review addresses the use of population genotypic tropism testing in relation to clinical practice. Genotypic tropism tests predict viral co-receptor tropism based on the sequence of the V3 loop of the viral envelope. HIV occurs in a swarm of variants in the patient's body, called quasispecies. As next-generation sequencing techniques are not generally accessible to date, triplicate testing is often performed to improve sensitivity of population-based approaches, but no prospective studies assessing the performance of single and triplicate procedures related to clinical outcome have been performed yet. For interpretation of the genotype several web-based algorithms have been developed. Varying the cut-off of the commonly used geno2pheno[co-receptor] algorithm changes the sensitivity and specificity of the tropism prediction. In retrospective analyses of clinical studies and cohorts genotypic population tropism testing demonstrated equal correlation with clinical outcome on maraviroc compared with phenotypic assays.In patients with suppressed viraemia, proviral DNA testing is a well tolerated alternative to HIV-RNA testing. Population genotypic methods have greater accessibility, lower cost, and faster turnaround time than other methods. Despite limited sensitivity for minority variants HIV genotypic population tropism testing showed good correlation with clinical outcome.