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INTRODUCTION: The human immunodeficiency virus (HIV) infection leads to immune activation, senescence and exhaustion of T cells. Co-stimulatory molecules play important roles in controlling these processes. The CD28 signaling triggers efficient T cell activation, while CD27 provides survival signals to CD28- T cells. Loss of these molecules was associated with senescent phenotype and resistance to checkpoint inhibitors. Romania has faced an HIV outbreak among people who inject drugs (PWID), most of them chronically infected with hepatitis C virus (HCV). HIV/HCV co-infection was associated with increased immune activation and rapid disease progression. METHODS: We evaluated by flow cytometry the expression of CD27, CD28, CD38, HLA-DR, CD57 and PD-1 on CD4 and CD8 T cells from 34 subjected infected with HIV (22 PWID and 12 people who acquired HIV by sexual route – PWHS) and 18 HIV-negative individuals (controls). RESULTS: We found that as compared to controls, HIV patients, regardless of infection route, have high percentages of intermediately differentiated (CD27+CD28-) and low percentages of less differentiated (CD27+CD28+) CD8 T cells. Significantly higher levels of CD8+CD27+CD28- T cells were found in PWHS than in PWID. A lower percentage of intermediately and highly differentiated (CD27-CD28-) CD8 T cells express CD57 in people living with HIV (PLWH) than in controls. Increased levels of less and intermediately differentiated CD4 and CD8 T cells expressing PD-1 were identified in PLWH, especially in PWID; these directly correlated with HIV viral load and T cell activation and negatively correlated with CD4 counts. CONCLUSIONS: Our data show that induction of PD-1 on T cells expressing co-stimulatory molecules CD27 and/or CD28 might contribute to poor control of HIV infection and to immune activation.

Dual HIV infections are possible and likely in people who inject drugs (PWID). Thirty-eight newly diagnosed patients, 19 PWID and 19 heterosexually HIV infected were analyzed. V2V3 loop of HIV-1 env gene was sequenced on the NGS platform 454 GSJunior (Roche). HIV-1 dual/multiple infections were identified in five PWID. For three of these patients, the reconstructed variants belonged to pure F1 subtype and CRF14_BG strains according to phylogenetic analysis. New recombinant forms between these parental strains were identified in two PWID samples. NGS data can provide, with the help of phylogenetic analysis, important insights about the intra-host sub-population structure.

Romania has faced an HIV outbreak among people who inject drugs (PWID) since 2011. The introduction of so-called ‘legal highs’ (amphetamine-type stimulants) on the drug market a few years prior contributed substantially to this outbreak. Next-generation sequencing (NGS) provides the possibility to detect drug resistance mutations with higher sensitivity than Sanger sequencing. The aim of this study was to search for transmitted drug resistance (TDR) mutations in strains from PWID recently diagnosed with HIV infection by parallel use of Sanger sequencing and NGS. The study was conducted on strains from 34 PWID diagnosed with HIV infection between 2016 and 2017. Sequencing was performed for the pol (PR, RT, and INT) and env (V2-V3 loop) regions. Sanger sequencing was performed with the commercial ViroseqTMHIV-1 Genotyping system (Abbott Laboratories) and with an in-house protocol for the env gene. NGS was performed in the same genomic regions using Nextera DNA Library Preparation Kit (Illumina) and the Miseq instrument (Illumina). NGS data were processed for error correction, read mapping, and detection of drug resistance mutations with HIV-1 Deepchek analysis software. Geno2pheno algorithm was used for viral tropism prediction and the WHO 2009 list for TDRM analysis. By using NGS, we detected seven cases (20.6%) of TDR in PWID and only two cases (5.8%) with standard sequencing. The TDR mutations detected by NGS were K103N, K101EN, Y181C, T215S in RT gene, I54V and M46L in PR, and none in INT. Two NNRTI mutations (K103N and K101EN) were detected in the same sample. Most of the TDR identified were present in the minority population (between 1% and 2% of the total reads) explaining the higher sensitivity of NGS method compared with standard sequencing. No significant differences were observed between these two methods when tropism prediction was analyzed. The majority of the viruses circulating in this group were R5-tropic. All strains showed more resistance mutations when analyzed by deep sequencing than by Sanger sequencing and more than previously observed in other risk groups. NGS proved to be a sensitive tool to detect TDR in newly infected PWID.

Aim: The study aimed to evaluate the clinical utility of the chemiluminescent HCV core Ag test compared to viral load assessment in the management of patients with chronic hepatitis C.Methods: A retrospective study was performed at a tertiary-care infectious diseases hospital on samples collected from anti-HCV positive patients. Seventy-six samples were tested with the Architect HCV core Antigen kit and Cobas AmpliPrep/Cobas Taqman HCV kit. The HCV Ag test accuracy was estimated using data from all the HCV RNA tested samples received between January 2011 and December 2012.Results: The HCV Ag test showed a good correlation between the logarithmic values of HCV RNA and HCV Ag (R=0.98), with a 100% specificity and PPV, but with reduced sensitivity for viral loads lower than 1,000 UI/ mL. In a model using data from 2,478 HCV RNA tested samples and a cut-off of the Ag assay corresponding to 1,000 UI/mL HCV RNA, the Ag test would have a sensitivity of 82.4%, a NPV of 80.9% and a high specificity and PPV (100%) compared to the viral load. The sensitivity would be higher for baseline evaluation compared to on-treatment samples (98.5 vs. 50%). The highest NPV (98%) would be obtained at 48 and 72 weeks after the initiation of treatment, with a sensitivity of 88.2% and 96.1%, respectively.Conclusion: The Architect HCV core Ag assay might be an alternative for the diagnosis of active HCV infection if molecular tests are not available, and a useful method for the evaluation of sustained virological response in treated patients.

Co-infections with HIV and HCV are very frequent among people who inject drugs (PWID). However, very few studies comparatively reconstructed the transmission patterns of both viruses in the same population. We have recruited 117 co-infected PWID during a recent HIV outbreak in Romania. Phylogenetic analyses were performed on HIV and HCV sequences in order to characterize and compare transmission dynamics of the two viruses. Three large HIV clusters (2 subtype F1 and one CRF14_BG) and thirteen smaller HCV transmission networks (genotypes 1a, 1b, 3a, 4a and 4d) were identified. Eighty (65%) patients were both in HIV and HCV transmission chains and 70 of those shared the same HIV and HCV cluster with at least one other patient. Molecular clock analysis indicated that all identified HIV clusters originated around 2006, while the origin of the different HCV clusters ranged between 1980 (genotype 1b) and 2011 (genotypes 3a and 4d). HCV infection preceded HIV infection in 80.3% of cases. Coincidental transmission of HIV and HCV was estimated to be rather low (19.65%) and associated with an outbreak among PWID during detention in the same penitentiary. This study has reconstructed and compared the dispersion of these two viruses in a PWID population. © 2017 Paraschiv et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.