Background: Hepatitis C virus (HCV) causes a major health burden. HCV is classified into 7 major genotypes and further into subtypes (GT). GT1 represents 69% of the infections in the P.E.P.S.I. Study, which analyses HCV infections in several German centres.
HCV can be effectively treated by direct-acting-antivirals (DAAs). The viral NS5A protein, which plays a key role in viral replication, is one of the DAAs targets. Resistance-associated-viruses (RAVs) harbouring NS5A resistance-associated-mutations (RAMs) have been described at baseline and after DAA therapy failure. Q30R is a major RAM in GT-1a. Surprisingly, R30 corresponds to the wild type in the GT-1b, and still, GT-1b strains are susceptible to NS5A-inhibitors.
Material & Methods: 2777 GT-1b sequences of from Los Alamos database and 364 sequences from patients from the P.E.P.S.I. cohort were used in this study. Sequences were aligned in a codon-aware manner with MACSE and mutations that were statistically significantly correlated with the R30Q polymorphism were identified. In order to confirm the epistatic nature of this association, a maximum likelihood phylogenetic tree was reconstructed with RAxML using a GTR + Gamma model of substitution, and ancestral sequences were reconstructed in all internal nodes. Amino acid substitutions correlated with the R30Q polymorphism in several independent branches of the phylogenetic tree were identified and mapped into the three-dimensional model structure of the NS5A dimer in complex with the Daclatasvir inhibitor.
Results: We show that R30Q is a polymorphism in GT-1b appearing in around 5% of the NS5A sequences retrieved from Los Alamos database. These strains often display other specific NS5A substitutions, particularly in positions Q24K and V34I, but also in positions L28M, Q54H and V138L. We demonstrate that secondary substitutions usually happen after initial R30Q development in the phylogeny of the GT-1b NS5A sequences, both in vivo and in vitro, and that the chemical properties of the corresponding amino acids either serve to restore the positive charge in this region or affect the movement of the N-terminal portion of the protein, acting as compensatory mutations.
Conclusions: We have demonstrated epistatic intragenic interaction in the NS5A gene of GT-1b, which lead to compensating amino acid changes affecting physical and chemical properties of the protein after introduction of substitutions in the residue 30. These findings may have implications for RAVs treatment.

Background: Guidelines for modern treatment of HIV-1 infection recommend triple therapy consisting of an integrase strand transfer inhibitor (INSTI) plus 2 nucleoside/tide reverse transcriptase inhibitors (NRTIs). Recently, controlled clinical trials have evaluated the efficacy and safety of an INSTI + 1 NRTI. To simulate regimen “forgiveness” of triple therapy versus experimental 2-drug combinations, in vitro experiments monitoring viral breakthrough and emergence of resistance were conducted under conditions simulating drug exposures at full adherence or suboptimal adherence to treatment.

Methods: Viral breakthrough experiments were performed in MT-2 cells infected with wild-type HIV-1 (IIIb strain). Infected cells were cultured in the presence of fixed doses of bictegravir + emtricitabine + tenofovir alafenamide (BIC+FTC+TAF) or dolutegravir + lamivudine (DTG+3TC), split every 3-4 days with fresh media containing drug, and monitored for viral breakthrough by cytopathic effect for up to 5 weeks. Drug concentrations of BIC and DTG were calculated using their human plasma clinical trough concentrations (Cmin) according to their prescribing information and adjusted for plasma protein binding. The TAF Cmin concentration generated tenofovir-diphosphate at its physiological concentration in PBMCs from TAF-treated individuals. FTC and 3TC concentrations were set at their human plasma-free adjusted clinical maximum or minimum concentrations. In breakthrough experiments that simulate 2 consecutive missed doses, the drug concentrations were adjusted by their plasma half-lives for BIC and DTG and active metabolite half-lives for the NRTIs (TAF, FTC, and 3TC). Emergent HIV-1 variants were characterized using standard genotyping methods.

Results: At trough drug (Cmin) concentrations corresponding to full adherence in adults, no viral breakthrough occurred with BIC+FTC+TAF or DTG+3TC through 5 weeks in culture (0 of 24 replicate wells for each drug combination). At trough drug concentrations corresponding to two consecutive missed doses (Cmin minus 2), no viral breakthrough occurred for BIC+FTC+TAF (0 of 24 replicate wells) through 5 weeks in culture but viral breakthrough occurred as early as 9 days post-infection for DTC+3TC (23 of 24 replicate wells through 5 weeks). Using higher concentrations of FTC and 3TC in the Cmin minus 2 doses experiments, no viral breakthrough occurred for BIC+FTC+TAF (0 of 24 replicate wells) but viral breakthrough occurred as early as 11 days post-infection for DTG+3TC (21 of 24 replicate wells through 5 weeks). When analyzed by population sequencing, all viruses lacked drug resistance mutations at the time of viral breakthrough.

Conclusions: Consistent with high efficacy rates for BIC/FTC/TAF in treatment-naïve and virologically suppressed individuals and for DTG+3TC in carefully selected treatment-naïve adults, no viral breakthrough occurred in vitro using clinical drug trough concentrations corresponding to full adherence. However, our in vitro studies suggest that the higher drug levels provided by triple drug therapy may be more protective than two drug combinations in individuals with suboptimal adherence particularly in the real world where imperfect drug adherence is frequent.

Background

Primary integrase inhibitor (INI) resistance transmission is rare. INI are recommended as first-line therapy for HIV infection on both the DHHS and IAS guidelines. INI resistance testing has not been routinely performed in ART-naïve HIV-1 infected individuals.

The CDC has reported that among newly diagnosed individuals in Florida, INI Resistance increased significantly from 2015 to 2016, with specific significant increases for raltegravir and elvitegravir.

This report describes an antiretroviral-naïve individual discovered in a Rapid Initiation of Highly Active Antiretroviral Therapy (HAART). Baseline resistance testing later revealed a G140 and Q148 mutations. A retrospective survey of resistance in an urban ambulatory clinic in The Bronx was performed to survey for the same resistance combination. The Stanford University drug resistance database reports the G140S/A/C usually occur with Q148 mutations. Alone, they have minimal effects on INSTI susceptibility. However, in combination G140 and Q148 mutations are associated with high-level resistance to RAL and EVG and intermediate reductions in DTG and BIC susceptibility


Methods

The Rapid Initiation of HAART and discovery of Primary INI resistance was at a community health center in NY, NY. Subsequently, a retrospective resistance surveillance was performed at a Bronx, NY clinic for similar cases of G140 & Q148 INI substitutions. The data mining effort identified ten individuals. Medical providers then performed retrospective review of clinical history and resistance testing through chart abstraction.

Results

A 38 year-old man tested positive for HIV on 10/11/2018. Baseline viral load (VL) was 40,140 copies/ml and CD4 count was 541. Genotypic resistance testing was sent and he was rapidly initiated on dolutegravir and tenofovir alafenamide and emtricitabine (TAF/FTC). Pre-HAART resistance testing later revealed INI mutations G140S and Q148H, the reverse transcriptase mutation K70R, the non-nucleoside reverse-transcriptase inhibitor mutation V90I, and protease mutations I62V, and I13V On 10/19/2018 hIs regimen was switched to darunavir 800 mg/cobicistat 150 mg and TAF/FTC. His VL on 10/26/2018 was 280 copies/mL. He subsequently suppressed to less than 20 copies/mL.

Our retrospective survey of resistance in a Bronx clinic revealed ten similar G140 and Q148 combination mutations. Five (50%) of these patients did not achieve durable viral suppression and continued to have high levels of viremia with concern for transmitting INI resistance.


Conclusion

While primary transmitted INI resistance has been thought to be rare, the Florida CDC report of 2015 to 2016 reports that it is increasing. We believe that Rapid Initiation of HAART needs to be followed by Rapid Follow Up. Here we report that Fifty percent of patients who engendered G140 and Q148 INI resistance mutations had viral loads at levels known to be transmissible.

The NYS Dept. of Health, AIDS Institute reported at CROI 2019 that while INI drug resistance is low, the level of clinically significant INI resistance mutations observed suggested that transmitted INI resistance is emerging. Our data is consistent with this suggestion.

As INIs are recommended first line therapy, baseline INI resistance testing should be considered in high-risk demographic settings and Rapid Initiation of HAART should be followed by Rapid Clinical Follow Up.

Background and Aims
The World Health Organization recently called for the elimination of hepatitis C virus (HCV) and has identified people who inject drugs (PWID) as a key target population. Clinical trials analyzing currently available all-oral regimens have demonstrated a high degree of efficacy in this population. There is an urgent need to confirm these data in a harm reduction and active consumption setting.
The aim of this study was to assess the efficacy of HCV therapy among drug users followed at low-threshold mobile harm reduction units (LTMHRUs) included in a program of coordination between a mobile harm reduction unit and a Hospital.
Method
We included active drug users (persons who had smoked or injected heroin/cocaine in the previous 6 months) who received HCV treatment and were attended at 2 LTMHRUs. Participants received HCV treatment while in active consumption or recent abstinence in a low-threshold setting. Recent abstinence was defined as cessation of drug consumption for at least 15 days and no more than 6 months. The program implemented through a patient navigator consisted of: Take the patient to the healthcare Centre, remind the patient about their next appointment, accompany the patient to Fast Track Clinic (2 dedicated vehicles available), contact hospital by phone to receive notification of appointments, collect medications from pharmacy and deliver them like supervised treatment.
Sociodemographic variables and characteristics related to drug use were collected at initiatiation of HCV treatment and during follow-up. End-of-treatment response (ETR) was defined as an undetectable HCV viral load at the end of treatment. SVR was defined as undetectable viral load at the first available HCV RNA measurement obtained a minimum of 12 weeks after the end of treatment. Virological failure was defined as detectable plasma HCV RNA before the date of SVR.
Results:
A total of 165 individuals initiated interferon-free therapy for HCV infection with DAA at the 2 LTMHRUs between January 2016 and July 2018. Notably, 120 patients (72.7%) were homeless, and 122 (73.9%) and 88 (53.3%) reported IDU in the 6 months and 30 days prior to HCV treatment, respectively. In addition, 142(86.1%) were receiving OST, 59(36%) had HIV coinfection, 19(13.6%) had cirrhosis, 31(18.9%) had mental disorders, and 71(43%) started therapy in recent abstinence. Of those who started therapy in recent abstinence, 39(55%) relapsed during follow-up.
159 patients had achieved ETR, 1 had discontinued treatment and was lost to follow up, and 5 were still on treatment. Virological failure was recorded in 3 participants. Eight participants were lost to follow-up after achieving ETR. Thirty participants with ETR were waiting for an SVR analysis.The overall SVR rate was 80% (108/135) in the ITT analysis (reinfection=failure) and 97.5% (118/121) in the mITT analysis. SVR rates showed no differences according to HIV status, IDU status, or OST status.
Conclusions:
Given the high efficacy of treatment, active drug users attended at LTMHRUs should be treated for HCV.Models of care must be adapted to the circumstances and the needs of the target population. Low-threshold access and a flexible model are essential for PWIDs.

Background: The introduction of direct-acting antiviral agents (DAAs) in clinics has revolutionized the management of HCV infection. Even if today, with the new recommended regimens, we have a very high rate of sustained virologic response (SVR, >95%) and viral failures are much less frequent than in the past, they still represent a concern, especially when resistance is present. This study aimed to evaluate the presence of natural resistance-associated-substitutions (RASs) and other pre-treatment risk-factors for failure in the Italian Vironet C cohort group of HCV-infected patients, starting a first-line treatment with a NS5A inhibitor-containing regimen
Materials & methods: RASs in NS3/NS5A/NS5B (N=1748/1560/1239) were analysed in 2010 DAA-naïve patients. Of them, 769 patients with a baseline NS5A resistance-test and available outcome after a first-line NS5A-containing regimen recommended by the 2016 or 2018 European Association for the Study of the Liver (EASL) guidelines, were also analysed. HCV Sanger-sequencing was performed by home-made protocols in 7 different Italian centers. Potential differences between the SVR and virological-failure group were evaluated by Fisher’s exact test. A multivariable logistic-regression analysis was performed to define risk-factors associated to treatment-response.
Results: Overall, 596/2010 (29.7%) patients showed at least one natural-RASs, particularly NS5A-RASs were observed in 18.5% patients. 769 patients (GT1a/b/g[239/222/1]-GT2a/c[87]-3a[153]-4a/d[67]) had an available outcome (720 with a SVR and 49 with a virological-failure) after the following recommended NS5A-containing regimen: daclatasvir/ledipasvir/velpatasvir+sofosbuvir+/-ribavirin (N=125/134/179), 3D/2D (paritaprevir/ritonavir+ombitasvir±dasabuvir)+/-RBV (N=125/44), grazoprevir+elbasvir+/-ribavirin (N=96), glecaprevir+pibrentasvir (N=66). Analysing retrospectively the baseline samples, a higher prevalence of natural NS5A-RASs was observed before treatment in DAA-failures (38.8%) vs SVR-patients (18.6%; p=0.01). Notably, ≥2 risk-factors for failure were more frequently observed at baseline among patients who experienced a virological-failure to a DAA treatment (69.4%) vs those achieving SVR (34.2%, p<0.001). By multivariable logistic-regression high HCV-RNA, natural RAS, cirrhosis, previous IFN-failure were negatively associated with SVR.
Furthermore, by multivariable logistic-regression, baseline HCV-RNA >800.000 IU/ml, presence of at least 1 natural RAS regimen-related cirrhosis and previous IFN-treatment were all negatively associated to SVR (adjusted odd ratios [95%C.I.]: 0.42 [0.20-0.89], P=0.002; 0.47 [0.23-0.95], P=0.035; 0.43 [0.22-0.86], P=0.017; 0.39 [0.20-0.78], P=0.007; respectively). No other risk-factors were associated to SVR.
Interestingly, all 66 GT1/GT2/GT3 patients treated with glecaprevir+pibrentasvir achieved SVR, with the exception of 1 GT3 breakthrough, having at baseline the NS5A-RAS A30K and HCV-RNA>800.000 IU/ml. Regarding velpatasvir+sofosbuvir+/-ribavirin, all 179 GT1/GT2/GT3/GT4 treated patients achieved SVR with the exception of 2 relapser (GT1a and GT3a), none showing baseline RASs regimen-related.
Conclusions: In this study, around one third of DAA-naïve patients showed at least one natural-RASs. NS5A-RASs were particularly observed with higher prevalence before treatment in DAA-failures. The presence of one or more specific pre-treatment risk factors, such as RAS regimen-related, baseline HCV-RNA >800,000 IU/ml and cirrhosis was associated with virological failure for some specific regimens and genotypes. Further analyses are needed to confirm these observations, particularly for the new current regimens and in the context also of shorter treatment durations (8 weeks).

Background
Two-drug regimens (2DR) are largely prescribed in Italy as maintenance therapy, nowadays mainly based on DTG. While many data have been reported about PI-based 2DR, the impact of resistance mutations and duration of virological suppression on DTG-based 2DR remains to be elucidated. The aim of this study was to evaluate the impact of resistance mutations on virological outcome of DTG-based 2DR maintenance ART.

Material and methods
Virologically suppressed patients switching to DTG+3TC or DTG+RPV with pre-baseline (time of switch=baseline, BL) resistance genotype (at least PR/RT) were selected from the ARCA database. Primary endpoint was virological failure (VF: an HIV-RNA, VL, >200 cps/mL or 2 consecutive >50 cps/mL). The probability of VF was estimated by Kaplan-Meier analysis. Resistance to 2DR was defined as occurrence of at least Stanford HIVdb (v.8.5) low-level resistance (LLR) to at least one drug included in the current 2DR, based on cumulative genotype. CD4 changes were assessed using Student’s t-test for paired samples. A secondary analysis comparing 2DR with DTG-based 3D regimens was also performed.

Results
A total of 318 2DR patients were analysed: 260 (82%) switching to DTG+3TC, 58 (18%) to DTG+RPV; 68% were males, median age was 51 (44-56) years, 12 (6-23) years of HIV infection, 5 (3-8) years of virological suppression, nadir CD4 cell count 231 (121-329), 5 (3-9) previous ARV lines, 59% previously exposed to INSTI, 11% with resistance to current 2DR. The integrase sequence was available in 14% of patients, none harbouring resistance to DTG. 20 VF were observed, of whom 4 (3/17 VF in DTG+3TC, 1/3 in DTG+RPV) in patients with at least LLR at BL (M184V+K219Q; D67N+K70R+K219Q; D67N+K70R+T215Y+219Q; E138A), in a median FU of 1.3 years (IQR 0.6-2). The 2-year estimated probability of VF was 8.7% (95% CI 4.4;13); 8.6% (4.1;13.1) in those without resistance and 9.7% (-4.4;23.8) in those with resistance (Log rank: p=ns). No factor was significantly associated with VF at multivariate analysis, however in patients with <6 years of virological suppression, baseline resistance was associated with a higher probability of VF (log rank p=0.003). After 48 weeks, a statistically significant increase in CD4+ cells was detected (+56 cells/mmc, p<0.001), independently from baseline resistance. The 2-year estimated probability of VF in the reference 3DR group (n=564) was not different from that for the 2DR group: 8.8% (5.9;11.7) in the whole case file and 9.7% (6.6;12.8) in the presence of baseline resistance. Longer time of virological suppression was the only factor associated with a lower risk of VF in the 3DR dataset.

Conclusions
DTG-based 2DR maintenance regimens show high virological efficacy, even in the context of predicted incomplete activity, at least within a short-term follow-up. A long duration of virological suppression seems to decrease the impact of resistance on virological outcome, however further studies are warranted to confirm this hypothesis and possibly define a clinically useful threshold.