Background: WAVES (GS-US-236-0128) was a double-blind, phase 3b study among treatment-naïve HIV-1-infected women that demonstrated that elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (E/C/F/TDF; N=289) was superior to atazanavir+ritonavir+F/TDF (ATV/r+F/TDF; N=286) for HIV-1 RNA <50 copies/mL by FDA Snapshot analysis at Week 48 (87% vs 81%). Participants with HIV-1 strains A or A1 in Russia represented 29% (168/575) of all study participants. Some accessory resistance substitutions have been shown to be highly prevalent in Russian subtype A strains. The substitution A62V in reverse transcriptase (RT) is a fitness compensatory mutation for K65R and other multidrug-resistant viruses, and L74I in integrase (IN) has been implicated in virologic failure on cabotegravir (CAB)+rilpivirine (RPV). Here, a detailed analysis of baseline genotypes and treatment outcomes of the A and A1 subgroups from Russian sites was performed.

Methods: At screening, protease (PR)/RT were analyzed by population sequencing (Monogram Biosciences, Inc.). Baseline samples were also analyzed by deep sequencing for PR/RT and IN with a frequency cut off of 15% (Seq-IT, Germany). Resistance analyses were performed on samples with HIV-1 RNA ≥400 copies/mL at confirmed suboptimal virologic response (HIV-1 RNA ≥50 copies/mL and <1 log10 reduction from baseline at Week 8), confirmed virologic failure (HIV-1 RNA ≥50 copies/mL after achieving HIV-1 RNA <50 copies/mL), discontinuation, or Week 48.

Results: In the WAVES study, 192 participants were located in Russia; 88% (168/192 participants) had HIV-1 subtype A or A1. At baseline, all 168 had PR/RT genotypic data available and 102 had IN data. Some amino acid substitutions at drug resistance sites were highly prevalent and appear fixed in the circulating A/A1 strains in Russia: 46% (78/168) had A62V in RT and 99% (101/102) had L74I in IN. Primary NRTI, NNRTI, and PI resistance substitutions were present in 1%, 11%, and 1% of participants, respectively. Notably, RPV resistance substitutions were present in 9% of participants, all with E138A/G/Q (n=15). Through 48 weeks of treatment, virologic suppression (HIV-1 RNA <50 copies/mL) was observed at similar frequency for the Russian subtype A/A1 participants compared to the overall study population (81% vs. 84%, respectively). By treatment arm, more Russian subtype A/A1 participants were suppressed on E/C/F/TDF (90%; 82/91) than ATV/r+F/TDF (70%, 54/77). A total of 14 Russian A/A1 participants met the criteria for resistance analysis, 7 participants each from the E/C/F/TDF and ATV/r+F/TDF groups. No participant in the E/C/F/TDF group had emergent resistance, and 2 participants in the ATV/r+F/TDF group developed M184V/I in RT, neither of whom had preexisting A62V.

Conclusions: The WAVES study had a high proportion of participants from Russia with HIV-1 subtype A or A1. The HIV-1 from these participants had polymorphic A62V in RT and L74I in IN. These substitutions did not lead to virologic failure with drug resistance in either treatment group.

Background: Everyday, 1 million sexually transmitted infections (STIs) are acquired daily, of which youth aged 20 -24 years account for the highest rates. These statistics are comparable in the United States of America (USA) where youth account for 50% of all new STIs. A significant influence of the incidence of STIs among youth is the low rates of STI testing. The majority of STI testing is recommended by a clinician following assessment of sexual health and sexual risk behaviors. However, this strategy of testing has been ineffective in comprehensively reaching youth due to barriers like low knowledge, cost, access, and fear of judgment and discrimination. Innovative strategies to promote STI testing are urgently needed among youth. To address this gap, the purpose of this qualitative study was to explore the preferences, feasibility, and acceptability of a home-based STI testing self-collect kit.
Methods. Data was collected using a demographic and sexual history questionnaire, STI knowledge questionnaire, and individual audio-recorded interviews using a semi-structured interview guide from 30 youth aged 18 to 24 in Central Pennsylvania, USA. The semi-structured interview guide contained questions on the feasibility and acceptability of using home-based STI testing self-collect kits, and the preferences for access, packaging and instructions, cost of self-collect kits, sending self-collect kits and receiving test results. Descriptive data were analyzed using the SPSS statistical software, and qualitative data (audio-recorded interviews) were analyzed using qualitative content analysis.
Results. The findings from this study revealed that the availability of home-based self-collect kits could reduce experienced youth barriers related to STI testing. Youth expressed willingness to use home-based self-collect kits. Some described preferences include access to pick-up kits without a prior clinician visit, clear instructions on using kits using packaging instructions or video explanations, discrete packaging, self-collecting testing specimen (i.e., urine, rectal swabs, vaginal swabs) at convenient locations, and dropping off kits at specified locations.
Conclusion. With low rates of STI testing as one of the significant prevention challenges, findings from this study provide insights to structuring alternative ways to offer STI testing services for youth. Recommendations from this study include locations where home-based self-collect kits could be accessed by youth, cost considerations and methods of designing user instructions. Suggestions for future research include the examination of facilitators, barriers, and predictors of the use of home-based self-collect kits and evaluating the accuracy of self-collected test specimens outside clinical settings. Furthermore, findings from this study provide guidelines for policy creations that will enhance the uptake of STI testing.

Background
In Bulgaria 1242 cases with HIV/AIDS were diagnosed from 2012 to 2017. The number of HIV-infected men who have sex with men (MSM) has increased in recent years, while the populations of people who inject drugs (PWIDs) with HIV have been decreasing. Epidemiological data indicated great heterogeneity of HIV-1 positive populations, including 493 (39.7%) heterosexuals (HET), 560 (45.1%) MSM, 151 (12.2%) PWIDs, 28 (2.3%) MSM who inject drugs and 10 (0.8%) infants infected by vertical transmission. The aim in this national representative study was to analyze the diversity among newly diagnosed HIV-1 infected individuals in Bulgaria for the period 2012 - 2017.

Materials & Methods
HIV-1 pol sequences were generated with TruGene and/or ViroSeq Genotyping Systems. HIV-1 subtypes were defined using COMET v2.2. The sequence alignment contained Bulgarian sequences and reference sequences from the Los Alamos database. Manual phylogenetic analysis and possible transmission clusters were inferred by ML analysis using IQ-Tree program.

Results
642 (51.7%) HIV-1 pol sequences were obtained and analyzed. Men dominated significantly with 536 (83.5%) while women were 106 (16.5%). The main transmission groups were HET with n=241 (37.5%), 311 (48.4%) MSM, 70 (10.9%) PWIDs, 13 (2.0%) were MSM who inject drugs and 7 (1.1%) infants infected by vertical transmission. The main HIV-1 clade was defined as subtype B n=388 (60.4%) followed by 11 different HIV-1 subtypes including: 85 (13,2%) CRF 01_AE, 40 (6.2%) CRF 02_AG, 38 (5.9%) A1, 33 (5.1%) F1, 31 (4.8%) unclassified and 7 other subtypes and CRFs representing 4.2% of all individuals in the study. Тhe most prevalent subtype among HET was B with 53.5%, followed by CRF 01_AE 10.8%, and CRF 02_AG 7.9%. Тhe most prevalent subtype in MSM was B with 80.4%, followed by subtypes A1 and F1 respectively with 7.5% and 4.5%. Among PWIDs most widespread were CRF 01_AE, CRF 02_AG and subtype B respectively with 64.3%, 14.3% and 7.1%. Among MSM who inject drugs the prevailing subtypes were CRF 01_AE 46.2%, CRF 02_AG 38.5% and subtype B with 15.4%. In the infected infants the most widespread HIV-1 subtypes were CRF 01_AE 42.9%, subtype B 28.6% and CRF 02_AG 14.3%.

Conclusions
Our analysis identified a significant proportion of diagnosed MSM with HIV-1 in recent years. The most prevalent subtypes were B and the recombinants: CRFs 01_AE and 02_AG. High genetic diversity and unequal distribution of HIV-1 subtypes in the vulnerable groups were found. The biggest subtype diversity was introduced and disseminated in HET transmission group, while among MSM subtype B dominated with higher proportion. Our study highlights the importance of sustained molecular-epidemiological surveillance of the dynamics of HIV-1 infection among the most vulnerable groups.

Background: Protease/reverse transcriptase genotypic resistance test (GRT) performed on peripheral blood mononuclear cells (PBMCs) is a useful tool to detect resistance in HIV-1 infected patients with low/undetectable plasma viral load. Despite the increasing usage of integrase inhibitors (INIs) in clinical practice, few data about integrase resistance on PBMCs are available. Thus, this study aims to explore INI-resistance detected in PBMCs in comparison to plasma compartment, as previous or contextual GRT.
Materials & Methods: Patients with low (<1000 copies/mL) or undetectable plasma HIV-RNA (<50 copies/mL) with an available integrase GRT on both PBMC and plasma compartments were included. INI major (MRM) and accessory (ARM) resistance mutations were evaluated according to the Stanford resistance list 2018. Mutations detected in PBMCs were compared to those detected in contextual and/or previous cumulative plasma GRTs. The presence of stop codons and/or APOBEC-associated substitutions was also considered.
Results: Among 150 patients included in the analysis, 51% had a plasma HIV-1 RNA <50 copies/mL at the moment of PBMC GRT, while in the remaining 49% of patients, the median (IQR) viremia was 124 (78-259) copies/mL. The majority of patients was infected with HIV-1 B subtype (73.9%) and was Italian (80.7%), showing median (IQR) viremia zenith and nadir CD4 count of 5.44 (4.95-5.75) log10 copies/mL and 163 (50-292) cells/mm³, respectively. One-hundred and two (67.9%) patients were previously exposed to an INI before PBMC GRT, while 82 (54.7%) were under an INI-based regimen at the moment of GRT.
Regarding INI-resistance, the proportion of patients with at least one MRM detected in PBMCs was lower (n=4, 2.7%) compared to that with MRMs in plasma GRTs (n=14, 10.0%; P=0.009, by Chi Squared test). Among 15 patients harboring INI-resistance in at least one compartment, 11 (61.1%), 3 (16.6%) and 1 (5.6%), showed resistance only in plasma, in both compartments or only in PBMCs, respectively. Concerning the specific INI-resistance mutations, the unique patient harboring resistance only in PBMCs previously failed a raltegravir-based regimen, and showed the E138K MRM together with several stop codons and APOBEC-associated substitutions. All the other three patients who showed resistance in PBMCs were previously exposed to INIs and showed the same mutations in plasma (1: G140S+Q148H; 2: Y143C/H; 3: N155H). No stop codons were found in these three cases.
Considering the INI-ARMs, the proportion of patients with at least one ARM was similar in both compartments (33.3% in PBMCs vs. 28.7% in plasma, P=0.454) and no difference in the median [IQR] number of ARMs detected per patient was observed (PBMCs: 0 [0-1] vs. plasma: 0 [0-1], P=0.684).
Conclusions: Major resistance to INI in patients with low/undetectable plasma HIV-1 RNA is low. Integrase GRT performed in PBMCs might be useful for patients without any previous therapeutic and/or resistance information, revealing with good reliability polymorphisms potentially associated with resistance. Further investigation, preferably through ultra-sensitive technology, are needed to clarify the clinical impact of INI-resistance present in PBMCs.

Background

Antiretroviral combination therapy (cART) normally contain two nucleotide reverse transcriptase inhibitors (NRTIs) and an integrase inhibitor (INSTI). But there is still a lack of studies showing how baseline resistance to NRTIs affects the risk of virologic failure in this constellation.
Prospective clinical trials do not enable cases with resistance at baseline. Older retrospective studies in this form exist, but those don’t include current INSTI drug combinations, or include only few cases. Now we are at the point to receive meaningful results from retrospective studies for these newer regimens. For a smaller dataset, namely the ARCA database, such data already exists. Here we present the analysis on the Europe wide database EuResist.
We assess whether NRTI resistance can impair INSTI based treatment with NRTI backbone. As noted in the ARCA paper, resistance for NRTI reduces the success of INSTI/2NRTI based therapies which signifies a need for NRTI resistance testing in INSTI based therapies. A suggested, we perform a larger study here.
The study’s aim is to determine if this relation of significantly higher risk for virologic failure holds true in the larger and Europe wide context of the EuResist database (i.e. in the scope of multiple European countries and an increased sample size).

Materials & Methods

This retrospective study uses the EuResist Integrated Database. EuResist is a meta database of HIV related data. At present, the central database contains data from >81.000 patients, including >100,000 genotypes, >170,000 treatments, >1 million viral load and >1 million CD4 data.
Selected patients had a recorded firstline therapy and a baseline sequence within 1 month of the therapy start. Only therapies after 2009 were selected, the onset of dual therapies in the data set. NRTI resistance mutations where obtained by the Stanford HIVdb program. The cases were then classified as either no NRTI mutation or >1 NRTI mutation. Virologic failure was defined as after 6 months or after first virologic suppression a VL viral load measurement of over 1000 cp/mL. This resulted in 497 INSTI therapies without NRTI resistance and 38 INSTI therapies with NRTI resistance.
Kaplan Meier plots were generated both for the overall risk of viral failure stratified by NRTI mutation and specifically for INSTI based ARTs.

Results

The Log-rank test for the Kaplan Meier curve depicting overall viral failure shows a significant difference between no NRTI mutation and >=1 NRTI mutation p=0.048. The log-rank test for INSTI-based ARTs was p=0.003. For INSTI based ARTs the risk of viral failure was 13% in the resistant group and 4% in the non-resistant group.

Conclusions

Our results point in the direction that NRTI resistance in firstline integrase therapies does indeed increase the risk of viral failure.

Background
Multi-experienced patients are considered more challenging to treat, especially in places where potent drugs as well as drug resistance testing are hardly available. In this scenario, an optimized regimen for patients failing raltegravir (RAL) is often the best and only approach considering the evidence from clinical trials. Despite this, there is limited data regarding the outcome of this approach in real life cases. The aim of the study was to measure the virologic outcome in multi-treated patients failing raltegravir following optimized salvage therapy compared to patients non-failing to integrase inhibitors.

Material & Methods
We conducted a retrospective case-control analysis of multi-treated HIV-1 positive patients failing integrase inhibitors under salvage therapy with an optimized regimen. The control group weas randomly selected from multi-treated patients failing a regimen without integrase inhibitors experience that were paired by the number of previous regimens and by the date of lost of viral control. Susceptibility was analyzed with the Stanford HIVdb Genotypic Resistance Interpretation Algorithm version 8.8. Viral control was defined as having a viral load <40 copies/mL after 6-months under salvage therapy. Time-to-event analysis, Cox regression hazard analysis model and correlation statistics were performed with SPSS. A p<0.05 was considered statistically significant.

Results
22 subjects were included in total (11 in each group). Nine (82%) subjects of the RAL-failing group achieved viral control at 6 months compared to four (36.4%) in the control group. From the case group only 7 subjects were sequenced with an integrase inhibitor, whilst only 4 isolates harbored integrase mutations: The most frequent was N155H. We found a higher probability of viral control in the case group compared to the control group despite having optimized regimens in both groups (Log rank test, p=0.008), with the same number of active drugs. Not having an integrase inhibitor in the salvage regimen was associated with a lower probability of achieving viral control (hazard ratio [HR], 0.049; 95% CI 0.01-0.247; p=<0.05), whereas the viral load level was not related to the outcome. Only in the case group the probability of patients achieving viral control was higher for subjects undergoing antiretroviral combinations based on integrase inhibitors compared to protease inhibitor (p=0.009) and also for the use of DTG compared to the other regimens (p=0.06).

Conclusions
We documented higher levels of viral control in our multi-treated subjects in response to salvage therapy including an integrase inhibitor compared to regimens without integrase inhibitors. Moreover, the use of dolutegravir in these real-life cases was associated with higher levels of response. However, we emphasize the importance of assembling a salvage regimen based on the virtual susceptibility of the drugs, always taking into account the clinical context of each patient. We believe that the use of integrase inhibitors can offer an improved probability of viral outcome in multi-treated patients, despite the previous use and failure to RAL . Close surveillance of cases requires to be implemented in order to observe mid and long-term viral control.

Ongoing combinations of antiretroviral drugs for the treatment of Human Immunodeficiency Virus (HIV) infection can successfully maintain long-term suppression of HIV-1 replication in plasma. but an effective vaccine against this virus has not still found. It is desirable to develop multifunctional strategies that improve coverage of epitope diversity and allow for understand conformational changes that occur during attachment and membrane fusion. For this purpose, in relation to the pivotal role played by gp41 in these polyvalent vaccine approaches, the conservation of the gp41 protein was evaluated, using a large dataset of sequences retrieved from person to drug-naïve or ART-treated.

To genetically characterize gp41 in terms of amino acid variability, the Los Alamos databases were used and 24.505 full-length Env sequences derived from HIV-1 subtype-B infected individuals at all stages of infection were analyzed. To select the B-subtype strains and to analyze gp41 mutations, multiple alignments were obtained using ClustalW2 manually edited with Bioedit software. The final resulting dataset was composed by 546 drug-naïve infected individuals and 2.746 antiretroviral drugs (ARVs)-treated patients, respectively: these 3.292 sequences were then used for the entire study (Table 1).
To calculate the average hydropathy of sequences Grand average of hydropathy (GRAVY) calculator was used.

In drug-naïve patients, among the 231 gp41 variable residues (variability >1%), 141 were mutated in >5% of patients and 48 of them (13.9%) were highly variable (substituted in >25%). Therefore, 114 out of 345 gp41 amino acid residues (33.0%) were highly conserved (≤1% variability), and 13 never muted (11.4%) (Table 1).
In ARV-treated patients, among the 224 variable residues (variability >1%), 143 were mutated in >5% of patients and 53 of them were highly variable (substituted in >25%). Consequently, 121 out of 345 gp41 amino acid residues (35.1%) were highly conserved (≤1% variability) and just 1 never muted (0.9%) (Table 1).

The study of gp41 variability shows similar sequence variability between drug-naïve and ARV-treated infected individuals thus strengthening the growing appreciation for the identification of specific single sensitizing mutations in the control of HIV infection. Overall, these results shed light on the specific mechanism related to host cell antiviral control and provide important implications for the therapy of HIV infection.

Background
Based on genetic divergence, HBV has been classified into 9 genotypes (GT) designated A to I, defined by >8% divergence at the nucleotide level, and several sub-genotypes. Within every genotype, due to the absence of proofreading activity, the HBV polymerase/RT leads to the introduction of random mutations into HBV genome, creating a genetic variability described as viral quasi-species. These variants include escape mutants and antiviral drug-resistance mutants.
The aim of this study was to evaluate the prevalence of genotypes, HBV escape mutants, treatment-resistance mutants in a cohort of HBV chronic patients.

Materials & Methods:
From 2014 to 2018, in a cohort of 143 HBV chronic patients treated with Entecavir (ETV) and/or Tenofovir (TDF) from Infectious Diseases Unit, San Martino Polyclinic Hospital, sequencing assay for detection of RT/S-Gene mutations was performed using Trugene® HBV Genotyping kit (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) or Abbott HBV Sequencing (Abbott Molecular) according to manufacturer’s instruction.

Results:
All HBV chronic patients received ETV e/o TDF. Forty-six/143 (32.6%) had been sequenced prior to treatment, 8/143 (5.6%) after treatment start. GT D was found in 36/54 (66.7%), A in 11/54, (20.4%), B in 3/54 (5.6%), C in 2/54 and E in 2/54 (3.7%).
In 9/54 (16.7%) samples at least one HBsAg escape mutant was found: 8 had pre-therapy escape mutations, while in 1 sequencing was performed after ETV was started. The presence of escape mutations was not GT-restricted (4/36, 11%, vs. 5/18, 27.7%, p = 0.14, escape mutations in GT D vs. nonD). The following escape mutants were found: 129H, 120S, 120P/T, 123A+130R, 120T, 145A, 122K, 100C, 144G.
With regard to RT mutation prevalence, 9/54 (16.7%) sequences revealed the presence of at least one mutation conferring drug resistance to either Lamivudine or Adefovir or, partially, ETV. No mutation impacting susceptibility to TDF was found. The RT mutations were the following: V173V+L180M+M204V in 1/9, 181T in 1/9, L180M+M204I in 1/9, 180V in 1/9, 180M+204V in 3/9 and 181V in 2/9 patients.

Conclusions:
The present study showed that D and A GT are currently the most prevalent in our Region, not differently from the rest of Europe.
The regular therapeutic response in patients with escape mutations does not seem to per se justify differences in the approach of treatment.
In comparison to data from literature, this study highlighted that over the last years a progressive decrease of resistance-associated mutations is being observed, likely due to the latest international guidelines that recommend the use of drugs with high antiviral potency and high genetic barrier such as ETV or TDF. Moreover, no mutations conferring relevant resistance to ETV and TDF were found before the start of treatment.

Background: A rare safety finding of neural tube defect (NTD) was reported among babies born to women living with HIV in Botswana, who were exposed to the antiretroviral dolutegravir (DTG) at the time of conception (Zash et al; NEJM 2018). As folate deficiency has been linked to NTD, we investigated the potential inhibition of folate binding to folate receptor by HIV integrase strand transfer inhibitors (INSTIs), including DTG, as a possible mechanism for folate deficiency and the formation of NTDs.

Methods: KB cells were chosen for their selective expression of folate receptor alpha (FOLR1) and for their lack of expression of folate receptor beta (FOLR2), reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), as determined by FACS analysis. The in vitro folate receptor binding assays were performed using 3H-folic acid in folate free-media, in the presence and absence of INSTIs at supra-therapeutic concentration. The INSTIs tested were bictegravir (BIC), cabotegravir (CAB), dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). Following incubation with drugs, cells were washed with PBS and lysed with 1% SDS. Lysates were transferred to scintillation vials; levels of 3H- folic acid were measured. After Kd and Bmax determination for 3H-folic acid binding, IC50 and Ki values were determined for all INSTIs. The anti-folate methotrexate was used as positive control.

Results: Bmax and Kd values were established by titration with 3H-folic acid. A Kd value of 5.0 nM was determined, with a Bmax corresponding to 1.4 x 10^7 receptors/cell. Folic acid and methotrexate yielded Ki values of 2.9 nM, and 4.0 μM, respectively, all values being consistent with previously published data in the literature. For all INSTIs (BIC, CAB, DTG, EVG, and RAL), the maximum concentration used (10 μM) was supra-therapeutic. None of the five INSTIs showed inhibition of 3H-folic acid binding to FOLR1; the Ki values for five INSTIs were >3.3μM.

Conclusions: In this in vitro study, none of the INSTIs evaluated showed inhibition of folate binding at supra-therapeutic concentrations. This is in agreement with the recent pharmacovigilance review, showing no evidence of increased risk of NTDs with the use of EVG- or BIC-containing products during pregnancy (Farrow et al; HIV Drug Therapy 2018, Glasgow UK), and in agreement with the lack of NTD among prospective cases for 3 INSTI evaluated (EVG, DTG, RAL) (Albano et al; CROI 2019, Seattle WA, USA). These data are highly relevant to inform reproductive age women living with HIV and their treating physicians.

Hepatitis E is clinically occurs similarly with other types of acute viral hepatitis. There is a particular risk group with immunodeficiency state, among them are the HIV infected. The purpose of this study was to determine the markers of HEV infection among of HIV positive patients in Republic of Belarus.

Material and methods. Were study 126 patients with HIV infection and icteric form of acute hepatitis. Serum samples were tested for anti-HEV IgM and IgG by ELISA and for HEV RNA (PCR).

Results and its discussion. The analysis of the survey allowed to identify the presence of anti-HEV Ig M in 3.17% of patients, indicating acute infection of hepatitis E at the time of the study. In 7.14% of patients were detected anti-HEV IgG, indicating previous contact with HEV and the presence of immunity to the infection. It was revealed that the frequency of anti-HEV Ig M and Ig G were higher in men as compared to frequency of circulation rate of these antibodies among women i.e. anti-HEV Ig G (8: 1) and Ig M (3: 1), respectively. It was also revealed that anti-HEV Ig G was found in combination with anti-HCV in 56% of cases. All patients with acute infection in the past year did not leave the territory of the Republic of Belarus and consumed dried meat of pigs. Only one patient was infected outside the country, in Thailand. The patient was diagnosed with hepatitis E, hepatitis A and acute HIV infection. However, due to the fact that immunity is not lifelong, this category of people is at risk. It can be argued that these autochthonous cases are caused the virus.

Conclusions: The conducted research allow to claim that the cases were caused by autochthonous virus (patients were infected in the Republic of Belarus, resulting from the consumption of dried meat of pigs) circulating on the territory of the Republic of Belarus. All patients, including HIV patients, diagnosed with Hepatitis or with elevated levels of alanine and aspartic transaminase should be tested by ELISA for detection of antibodies to hepatitis E virus. All cases of hepatitis E in the Republic of Belarus were associated with HEV genotype 3.

Background:
With new therapeutics for treatment of Hepatitis B on the horizon there is an increased need for reliable diagnostics to identify genotype, nucleoside/nucleotide inhibitor resistance pattern, escape mutations and mutations in the pre-core and core region of HBV. While in-house solutions are available for many different viral pathogens, commercial NGS solutions are at the moment only available for HIV and HCV. We compared the Vela Sentosa HBV genotyping and resistance assay (Vela) which is in development to previously characterized samples.
Materials & methods:
17 samples were analysed with the Vela assay. We compared the results to the previous characterization of the Hepatitis B virus RT-domain by Sanger sequencing. Viral load of the samples was between 2 200 IU/ml up to >100 000 000 IU/ml. Genotypes included were A, B, C, D and E. A reference set of sequences consisting of 4267 HBV full genome sequences was generated form HBVdb (https://hbvdb.ibcp.fr/HBVdb/HBVdbDataset). Sequences were generated from the precore, core surface antigen and RT-Domain region using our established NGS pipeline.
Results:
Sequencing was performed with high efficacy and nearly 3.2 million final reads could be analyzed. The sample set consisted of 3 A, 5 B, 2 C, 4D and 3 genotype E samples. All previously determined genotyping results could be confirmed. Phylogenetic distance calculation showed a high pairwise homology of the generated sequences with differences mainly resulting from increased sequence quality in the leading and trailing ends of the sequences. Reverse Transcriptase mutations conferring resistance to RT-inhibitors could be recovered in all the appropriate samples with an additional minority mutation in the Vela assay. All mutations in the Hepatitis B surface antigen gene associated with escape could be verified.

Conclusion:
The Vela Sentosa HBV genotyping and resistance assay showed excellent performance for all tested genotypes and viral loads. All relevant mutations could be reliably detected. The assay is planned to be combinable with the Vela Sentosa HIV, HCV and CMV genotyping and resistance assays to allow short turn-around times even in laboratories with lower sequencing requests.

Title: Use of Rapid HIV Antibody Tests in Chile as prevention strategy
Ferrer Pablo1 Afani Alejandro1, Bastias Carla1, and HCUCH HIV Rapid Test Group
(1) Sección Inmunología, VIH y Alergias, Hospital Clínico Universidad de Chile
Santos Dumont 999, Piso 5, Independencia, Santiago, Chile.
Introduction: Today Chile has the highest rate of new HIV infections in Latin America. Although in Chile the first rapid test for HIV was registered in 1999, it has never been used massively in the general population as a prevention strategy for HIV infection.
Method: 4th generation rapid HIV test (BTNX, Ontario Canada) and an anonym epidemiologic survey were realized to 1337 people in Santiago between November 28 of 2017 and March 31 of 2018. In accordance with the national HIV/AIDS law, a complete confirmation process was made to the people who were reactive in the rapid test.
Results: A total of 1,337 people were tested of which 20 were reactive for antibodies against HIV. The 20 people were confirmed as HIV positive by the confirmation algorithm carried out by the Institute of Public Health of Chile (ISP-Chile), the technical agency mandated at the national level to confirm all new cases of HIV infection that are being screened in Chile. Of the 20 people detected, 18 were men with an average age of 32.4 years and 2 were women with an average age of 42.5 years. According to these data, the current prevalence of HIV in the people studied is 1.5%. A total number of 1200 surveys were collected. Responders were 52% male and 48% women. During the last year: only 20% of the respondents answered that they had always used condom, only 40% reported having had an HIV test and same period time the average number of sexual partners was 3. An 80% responded that they would be willing to use a drug as pre-exposure prophylaxis. A 90% of the respondents answered that the test was not carried out for the following reasons: difficult access, lack of time, lack of interest, excessive price and fear of the result.
Conclusions: In Chile during the five-year period 2010-2015 new cases of HIV have increased up to 96% in the age group that goes from 14 to 29 years. This reality has the country in a situation of epidemiological alert. The prevalence of 1.5% reported by our work is three times higher than that estimated by UNAIDS for Chile. This discrepancy has its origin in the difficult access that the population has to the diagnosis of HIV, which is performed exclusively in public and private health centers. Our experience tells us that massive testing by means of rapid fourth-generation tests is an excellent opportunity to bring the diagnosis closer to people. The limited use of condoms by the majority of the population studied is evidence that could explain the high number of new HIV cases in Chile.

INTRODUCTION
Sanger/Population sequencing is widely for resistance testing in clinical settings. Slow, labor-intensive manual sequence editing, subjective variant mixture determinations, and the lack of resistance interpretation can contribute to erroneous reporting. An automated sequence analysis tool is needed to facilitate the use of resistance information for HCV management.

METHODOLOGY
The RECall program (https://hcvshared.hcvdb.ubc.ca) originally designed for HIV genotyping was modified and adapted for HCV analyses. Over 200 reference sequences from ICTV were incorporated for GT/subtype determination. Seven HCV “prototype” sequences (GT1a, 1b, 2-6) were used as references for mutation reporting. A rule-based algorithm to interpret clinically relevant resistance mutations was introduced.

To validate accuracy of GT/subtype determination, 1000 HCV sequences with annotated GT/subtype information for NS3 and NS5A were downloaded from LANL and aligned with the reference sequences. To examine accuracy of basecalling, 1046 raw ABI HCV chromatograms were submitted to RECall. The sequencing trace files were processed with the software, Phred, which trimmed sequence ends and excluded regions with <20 quality scores from the contig assembly. Mixture calls were identified based on the primary and secondary peaks of the chromatograms. The resistance-associated substitutions within NS5A were evaluated.

RESULTS
ICTV HCV sequences provide a good reference to accurately determine HCV GT/subtype. For NS5A, the concordance was 100% at the GT level and 99.2% at the subtype level; 8 GT1b were identified as GT1a using the references. For NS3, the concordance was 100% and 99.3% at the GT and subtype levels, respectively: 4 annotated GT1a were identified as GT1b, one GT4b was identified as 4w, one 6u was identified as 6xa, and one 1a was identified as 1 only.

HCV ReCall can automatically select the correct region of HCV (e.g., NS3, NS5A or NS5B) and process raw chromatogram data. For example, a collection of 1046 raw ABI chromatograms were automatically processed for NS5A codons 20 to 95 in a total of 8 mouse clicks, and the aligned FASTA sequence results compared with those generated by an independent reference laboratory. Of the 100 NS5A samples compared, there were a total of 116 basecalling discordances out of 22,500 bases (0.5%); the vast majority of the discordances were caused by differences at "mixed" bases. The correct HCV genotype was chosen in all cases. For samples derived from virologic failures, mutations were observed at the known resistance-associated positions. The HCV ReCall analysis report also provided a summary of quality scores for each of the primers, pairwise assessments of genetic distance of each sample from the others to identify contamination, a list of mutations relative to the prototype sequence, and an assessment of the relative peak height of every mixture.

CONCLUSIONS
HCV RECall web application provides a comprehensive analysis for HCV drug resistance; directly from raw ABI sequencing trace files without manual intervention. This tool enables objective and consistent interpretation of HCV genotype data, improves processing speed, and decreases labor and software costs. Consensus on appropriate “reference” strains for genotyping and “prototype” strains for resistance mutation reporting will be important for consistency in inter-laboratory comparisons.

Objectives:

Chronic Hepatitis C treatment is no longer challenging in the era of DAAs with an SVR of up to 97%. Triple infection treatment with HCV, HIV and Hepatitis B has not been explored in real life situations. HCV Genotype 3 is still the most challenging clinical state in Hepatitis C treatment. Regardless of concomitant triple infection, shorter duration of therapy revealed favorable outcome with the highest retention, fewer side events, and cost containment. This study evaluates the efficacy and safety of Sofosbuvir, Velpratasvir, and Veloxpravir in the treatment of triple infection with HBV, HIV, and HCV (Genotype 3).

Methods;

Twenty-two (n = 22) HCV treatment-naive patients with Triple Infection (HIV HBV HCV Genotype 3) were recruited for the study.

Patients with HIV were on Atripla for over three years with HIV with Undetectable Viral load and HBV Viral load Undetectable. HCV infected patients had a Median Viral load of 3 million IU and Genotype 3 prior to treatment.

Demographics:

HCV Genotype Genotype 3 Genotype 3a Genotype 3c Genotype 3b
No of people 22 10 9 3

Patient Characteristics
Race No of Patients Mode of transmission
Males Females IVDU MSM Blood transfusion
African-American 1 0 1 0 0
Caucasian 1 0 0 1 0
Haitian 2 0 0 0 2
Asian 0 18 1 0 17
India 0 4 1 0 3
Pakistan 0 12 0 0 12
Bangladesh 0 2 0 0 2
Total 4 18 2 1 19

Mean Age 56 (44 – 68)
Mean BMI 27 (21 – 29.6)
Mean Fibrosis F3

Patient HBV characteristics
Race No of Patients Genotype
Males Females A B C D G H
Asian 0 18 0 1 5 12 0 0
India 0 4 0 1 0 3 0 0
Pakistan 0 12 0 0 3 9 0 0
Bangladesh 0 2 0 0 2 0 0 0
Caucasian 1 0 1 0 0 0 1 0
African-American 1 0 1 0 0 0 1 0
Haitian 2 0 0 0 0 0 2 2

Infection Mean years of acquisition
HIV 20
HBV 15
HCV 7

HBV Characteristics

HBeAg Negative 19
HBeAg Positive 3
HBsAg Positive 22
HBcAb Positive 22

Exclusion Criteria:

Active Drug Abuse or excess Alcohol intake, CHF NY heart Type IV, Cardiomyopathy, Arrhythmia, COPD, Renal Failure with creatinine clearance less than 30 %, Decompensated Cirrhotic HCC, Transplant recipients,

Results:

Results
Duration of treatment HCV Viral load
Viral load - Undetectable Viral load detectable
a. Fourth week 18/21 3/21 detectable, 200 copies mean
b. Eighth week 18/21 3/21 detectable
c. Twelfth week 18/21
d. Twenty fourth week 18/21

Resistance-associated substitution Pre-therapy Post-therapy
RAS 31 1 3
RAS 36 0 1
RAS 93 1 1

Conclusion:
The study demonstrates the efficacy of DAAs in 12-week treatment with an SVR of 87% in a very challenging triple infected cohort, with significant efficacy, tolerability, and safety. A larger trial is needed to validate the results.

Background: Prevalence of pretreatment drug resistance (PDR) to widely used nonnucleoside reverse transcriptase inhibitors (NNRTIs) has risen considerably in recent years in low- and middle-income countries such as Uganda, where the majority of individuals on first-line antiretroviral therapy are prescribed NNRTI-containing regimens. Although integrase inhibitor dolutegravir is now a recommended component of first-line regimens by the World Health Organization (WHO), this option excludes women of reproductive age due to the potential risk of neural tube defects in infants. Standard genotypic resistance tests detect drug resistance-associated mutations (DRMs) present at ≥15-20% of the viral quasispecies population and are unable to detect potentially rare clinically-relevant DRMs present at lower frequencies. In this study, we examine the prevalence of pretreatment low frequency DRMs in Uganda using next-generation sequencing, and assess its impact on viral suppression.

Materials & Methods: Participants were treatment-naive individuals ≥18 years of age, initiating NNRTI-based regimens as part of the Uganda AIDS Rural Treatment Outcomes cohort. The 90-234 amino acid region of the reverse transcriptase gene was amplified, and consensus-based sequences were obtained on an Illumina MiSeq. Low frequency DRMs were defined as resistance-associated substitutions detected at a threshold of 2%, 5%, and 10% of viral population. Resistance was defined as ≥1 DRM(s) which resulted in cumulative low-, intermediate-, or high-level resistance to NNRTIs, as defined by a score ≥15 based on the Stanford University HIV Genotypic Resistance Interpretation Algorithm v8.5. Individuals contributed to the prevalence count of PDR if they had study-defined NNRTI resistance detected in their pretreatment genotypic drug resistance test. We assessed the impact of low frequency DRMs at different viral frequencies on viral suppression at one-year post-therapy initiation using univariate and multivariate binomial logistic regression models with age at enrollment, baseline CD4 count and HIV-1 RNA viral load, and sex treated as covariates.

Results: In total, 234 unique individuals (68% female; 60% subtype A1) contributed data from 2005-2013. Prevalence of PDR was 12%, 14%, 16%, and 18% at 20%, 10%, 5%, and 2% viral frequency, respectively. The most prevalent NNRTI DRMs were E138A and K103N. Individuals harbouring DRMs at higher viral frequencies had higher odds of having detectable viral load (≥400 copies/mL) at one-year post-therapy initiation (20% viral frequency: adjusted odds ratio [aOR] 1.20; 95% confidence interval [CI] 0.5-2.9; p=0.64) compared to lower viral frequencies (10% viral frequency: aOR 1.10; CI 0.4-2.4; p=0.87). Age was a statistically significant protective variable (OR 0.7; CI 0.5-0.9; p=0.03). There were five instances of DRMs detected at higher viral frequencies not being detected at lower viral frequencies in individuals, an artefactual result of mixtures interpreted using standard interpretation algorithms.

Conclusions: The prevalence of NNRTI PDR detected at 20% is similar to those previously reported in Uganda by the WHO. Overall, pretreatment low frequency DRMs did not have a statistically significant impact on viral suppression, potentially due to small sample size. Analysis of low frequency DRMs using consensus-based sequences present potentially significant complications using standard genotypic interpretation algorithms due to the ambiguous interpretations of nucleotide mixtures detected at low viral frequencies.

Background. In Uzbekistan twice a year conducts epidemiological surveillance of HIV infection among at-risk groups. The collection of plasma samples is complicated by the hot climate and geographical remoteness of the regions. The aim of the study was to adapt the method of using DBS to conduct epidemiological surveillance of HIV among at-risk groups in Uzbekistan using available diagnostic reagents.
Materials & Methods. 123 samples of a DBS and blood plasma obtained from HIV-positive patients and 200 blood plasma samples and a DBS from HIV-negative patients were examined by PCR. The Interlabservice reagents "AmpliSens® DNA-HIV-FL" were used for detection of proviral DNA of HIV by PCR.
Results. Testing of DBS according to the manufacturer’s instructions showed a result of 74.8% (plasma samples were taken as 100%). The addition of extra-time during extraction significantly increase the sensitivity of the method to an acceptable level of 97.5%. Replacing the extraction kit offered by the manufacturer, the “Ribo-prep” with the “DNA sorb B” with the addition of the exposure time of the DBS discs with lysing solution, made it possible to raise the sensitivity of the method to 99.1%.
Conclusions. Changing the extraction method and extra-time for lysis of DBS disks allows using this method for epidemiological surveillance in Uzbekistan.

Hepatitis B virus (HBV) infection is a major public health problem affecting approximately 360 million people globally. Mother-to-child transmission (MTCT) is responsible for more than one third of chronic HBV infections worldwide. Mothers who are co- infected with HBV/ Human Immunodeficiency virus (HIV) and are antiretroviral therapy (ART) naïve have a high tendency of transmitting the two viruses during pregnancy, delivery or postnatally. This study aimed to determine the prevalence and associated risk factors of HBV infections among Highly Active antiretroviral therapy (HAART) receiving HIV-infected mothers and their exposed infants at the Kenyatta National Hospital (KNH) in Kenya. Eligible mothers and their exposed infants were recruited from a cohort enrolled in a Prevention of mother to child transmission of HIV (PMTCT) program in KNH.
A structured questionnaire was used to capture the socio-demographic data of the participants and information on associated factors to HBV infections. Four milliliters (ml) sample of paired whole blood were obtained from HIV positive mothers and their exposed infants. Whole blood was separated into plasma and stored at -80oC. HBV infection was determined using Euromedi Equipp (EME) rapid kit for Hepatitis B surface antigen (HBsAg) test and confirmed by a HBsAg Enzyme linked immune sorbent assay (ELISA). The HBsAg sero reactive samples were further screened for HBV envelope antigen (HBeAg) using ELISA (Accubiotech co.ltd). Samples which turned positive with ELISA and rapid tests were subjected to Polymerase-chain reaction (PCR) targeting the preS1 region using nested primers. HBV infection was presented as a proportion with 95% confidence interval and the associations tested using chi-square tests. A total of 534 HIV-infected mothers and their highly exposed infants were recruited. The mean age of the mothers was 31.2 years (SD 5.4 years) and the infants had a median of 6 months (IQR 3-10 months). Four hundred and thirty-three (81.1%) of the mothers were married, 272 (50.9%) having tertiary education and 113 (59.5%) were employed. One hundred and thirteen (21.2%) of the mothers were aware of HBV infection and HBV vaccination. Most of the mothers were currently receiving HAART with 502 (94%) of the mothers taking TDF/3TC/NVP and 32 (6%) on AZT/3TC/ NVP or AZT/3TC/EFV. Out of 534 mothers, 19(3.6%) were positive for HBV. All the 19 samples that gave positive HBsAg results tested negative for HBeAg. Out of the 19 samples that tested positive with ELISA, also gave positive results with PCR targeting the preS1 gene. All exposed infants tested negative for HBV with the HBsAg rapid, ELISA and PCR tests. History of dental surgery was associated with increased rate of HBV infection among the HIV-infected mothers (OR 3.3 (95% CI 1.1-9.6). In conclusion, the results of this study suggest that the HAART regimen received by the HIV infected pregnant mothers may have prevented vertical transmission of HBV infections to exposed infants.

Background. The aim of the study was to evaluate the characteristics of isolates causing the occult form of hepatitis B. We have previously studied the occult form of hepatitis B among patients with viral hepatitis C and cryptogenic liver cirrhosis by identifying ccc DNA of HBV in samples;
Materials & Methods. 51 liver biopsies and blood plasma was collected from patients admitted to the intensive care department in a serious condition, including 6 patients with hepatitis B + C (control group), 20 biopsies from patients with hepatitis C, 25 biopsies from patients with cryptogenic cirrhosis were collected. All patients were from different regions of the country. The nucleotide sequences of Pre-S1 / Pre-S2 / S regions for 32 isolates were obtained (6 from patients with hepatitis B + C, 26 from patients with occult form of hepatitis B). The primary analysis of the obtained fragment was performed using the NCBI Blast program in comparison with the nucleotide sequences presented to the GenBank. To align the nucleotide sequences and phylogenetic analysis used the Mega 6 software.
Results. The phylogenetic analysis of all 32 isolates showed the prevalence of D genotype, which is the most common in Central Asia. At the same time, the D1 subtype prevailed - 84.38% compared with the D2 subtype - 3.12% and the D3 subtype - 12.5%. The nucleotide identity in the group was 97.65±0.4%. There was no correlation between genotype of virus and the geographic region. Thus, patients with the D3 subtype, whose intragroup percent nucleotide identity was more than 99%, came from different regions of the country. The prevalence of genotypes and subtypes in different groups was associated with the paths of transmission.
Conclusions. The prevalence of subtype D1 was detected among patients in serios condition. Genotype D of HBV than other genotypes can cause a more severe disease and a higher level of drug resistance. Subtype D1 is characterized by low viral load and early HBeAg seroconversion, which can create problems for the well-timed detection of the virus and lead to the development of a more serious condition in patients.

We hypothesized that HIV drug resistance (HIVDR) is a wicked problem with several causes which find roots in different fields of science. Molecular medicine, unraveling the molecular basis of HIVDR, pharmacy and the development of antiretroviral therapy (ART) with a higher genetic barrier and anthropology and psychology aiming to improve adherence and reduce stigmatization are all crucial fields of science in the quest to prevent HIVDR, next to several other disciplines. To our knowledge no transdisciplinary overview of all causes leading to HIVDR has been made before. A well-documented approach to clarify complex causes of wicked problems is the development of systems maps. Such maps can be used to identify key causes to the problem that can and should be addressed, but also to develop guidelines specifically for certain populations. We therefore aimed to develop the first transdisciplinary systems map of causes leading to HIVDR.
The systems map was gradually developed by literature study combined with a series of interviews with experts from different disciplines (epidemiology, pharmacy, psychology, public health, medicine, bioinformatics, virology, anthropology, visual methodology) all working in the field of HIVDR in Sub Saharan Africa. The interviews were transcribed and factors leading to HIVDR and their links with each other were extracted and entered into a systems map with the KUMU software. The map was adapted throughout a series of discussions with experts from several disciplines.
73 drivers of HIVDR and 130 connections between those elements were identified and visualized in one systems map. To structure the map, the elements were divided in four categories according to their relation to: 1) access to treatment, 2) adherence, 3) healthcare system 4) biology and pharmaceutics. When organizing the elements according to discipline, we found that disciplines are strongly interconnected and disciplinary boundaries are vague with regards to causes leading to HIVDR. For example, adherence is not only influenced by psychological factors but also pharmaceutical aspects (pill design), comorbidities, religion, gender equality, culture, education and the financial status of the person living with HIV (PLHIV).
We conclude that HIVDR is indeed a wicked problem with causes related to different disciplines which are interconnected with each other. Rather than through a multidisciplinary approach, HIVDR should thus be approached from a transdisciplinary point of view. The map will be further optimized with insights from people living with HIV and experts from several other disciplines and can be used as a tool for researchers to orient their research question and discover new connections between their results and other elements which might at first seem unrelated.

Background: The development of the diagnostic and treatment process has increased an average age of people living with HIV (PLHIV). Therefore, an assessment of comorbidity is necessary for planning and organizing medical care for them.
Materials & Methods: 537 outpatient cards have been analyzed by descriptive and comparative statistics of Statistica 10.0 software, Microsoft Excel 2013.
Results. The study group consisted of 537 people including 324 men (272 – 83.9% with co-morbidity) and 213 women (162 – 76.1% with co-morbidity). Distribution by age: up to 34 years 102 (19.0%), 35-44 – 236 (43.9%), 45-54 – 82 (15.3%), 55-64 – 73 (13.6%), 65 years and older – 44 (8.2%). It is noteworthy that the general morbidity in study group is 1.5 times higher than that of the adult population of the Krasnodar region (1918.1 and 1272.4 per 1000 people, respectively). A comparative analysis of the structure of these indicators demonstrates the following differences: diseases of the hepato-biliary system in the study group were more common by 8.9 times, skin by 3.4 times, the urogenital system 1.6 times, respiratory organs in 1.5 times, neuropsychiatric diseases 1.5 times more often.
The structure of overall incidence of liver diseases was represented by viral hepatitis C - 78%, Toxic hepatitis - 13%, viral hepatitis B - 4%, viral hepatitis D - 2%, liver cirrhosis - 3%. The overall incidence of the listed groups progressively decreased in the range of 35-65 years.
Assessment of the general morbidity in age groups brought us some interesting results for practical work. The general morbidity in most of the listed groups progressively decreased in the range of 35-65 years. At the same time, the incidence of CVDs increased in the age groups of 35–65 years from 3.7 to 89.4 per 1000 people.
The problem of polypragmagia in PLHIV is associated with the need for a combination of ART and treatment for concomitant diseases. Most often ART included lamivudine (99.2%), tenofovir (56.4%), efavirenz (57.7%). The ART regimen contained 4.7 ± 1.7 tab., The treatment regimen for comorbidity treatment - 4.4 ± 0.8 tab.
Conclusions: 1. The overall incidence of PLHIV in comorbidities is 1.5 times higher than that of the adult population of the Krasnodar region in 2017. The difference in the structure of comorbidity among PLHIV is in the prevalence of diseases of hepato-biliary system (8.9 times higher than the regional level). 2. Dynamics of the incidence of CVD in the study group by age (steady growth with maximum rates in the group of 65+ years). 3. The predominance of tenofovir in the ART regimen is the basis for the dynamic monitoring of renal function and bone density, especially in older age groups. 4. The combination of ART drugs and therapy for comorbidities should be under the obligatory control of drug-drug interactions.

Background: HCV genotyping before treatment remains crucial for optimal selection of direct acting antiviral therapy. Due to high genetic variability of HCV, assays can struggle to provide clear genotyping and subtyping results. Our aim was to evaluate the performance of the Abbott RealTime HCV Genotype II assay (Abbott Molecular, Illinois, USA) in a real-life setting.
Materials & Methods: In total 539 samples were genotyped using the Abbott assay between September 2016 and November 2018 at the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Slovenia. Samples yielding undetermined results (no genotype result, no 1a/1b subtype result or reactivity with another genotype) were further evaluated by using either universal core PCR or subtype-specific PCRs, followed by sequencing.
Results: The Abbott assay provided a clear genotype or subtype in 95.2% of samples (513/539), namely subtype 1a (n=190), subtype 1b (n=70), genotype 2 (n=13), genotype 3 (n=232), and genotype 4 (n=8). In 12 samples (2.2%), subtype of genotype 1 could not be determined by the Abbott assay; eight of these samples were resolved by sequencing as subtype 1a, three as subtype 1b and one as subtype 6i. Co-infection of subtype 1a and genotype 2 was detected by the Abbott assay in one sample and later confirmed by subtype-specific PCRs and sequencing. In nine samples, reactivity with another genotype was observed. In two of the nine samples co-infection with two HCV genotypes was confirmed (1a+3 and 1b+4) by sequencing, while the remaining seven showed only one genotype in the genotype-specific PCRs, indicating a probe cross-reactivity or contamination issue. In addition, two samples yielded an HCV indeterminate result and two HCV inhibition by the Abbott assay; all four were further resolved by sequencing as genotype 3.
Conclusions: Our study shows that the Abbott RealTime HCV genotype II assay provides unambiguous genotype/subtype information in a vast majority of samples. However, the laboratory should still have back-up assay(s) prepared as well as sufficient knowledge to resolve unclear genotyping results. Since the Abbott RealTime HCV genotype II assay erroneously identified the subtype 6i sample as genotype 1, resolving samples with initial indeterminate 1a/1b subtyping results is crucial.

Introduction. Hepatitis C virus (HCV) infection is the most important cause of liver failure and the second cause of death in cART (combination antiretroviral therapy) treated HIV patients. Although direct acting antivirals (DAA) have revolutionized HCV treatment, HIV-HCV co-infected patients are still considered a difficult-to-treat population due to comorbidities and potential drug-drug interactions.

Aim. In this study, we reported our real-life experience of DAA treatment in a cohort of Sardinian HIV-HCV co-infected patients.

Materials and Methods, Results. All consecutive HIV-HCV patients who underwent DAA treatment at two Sardinian liver units between 2016 and 2018 were included. Different DAA combination were used depending on HCV genotype, liver disease severity, cART and concomitant drugs, in according with current guidelines. The primary endpoint was treatment efficacy defined as a sustained virological response (SVR) at 12 weeks after therapy. Secondary endpoints were treatment safety and tolerability. We recruited 131 patients: males 74,80%, median age 54 years (range 30-66). The most common genotype was 1a in 41 (31,29%). Cirrhosis was detected in 53 (40,45%) patients. A positive history of injective drug use was present in 108 subjects (82,44%). All patients were receiving antiretroviral treatment, while 32 (25.4%) were on methadone maintenance therapy and 15 (11,45%) were on psychotropic drugs therapy. 39 (29,77%) patients had a history of interferon-based treatment. Among those who completed the 12-week follow-up post DAA (n=128), SVR was observed in 124 (96,87%). Only 1 (0.78%) patients was non responder to the treatment, and 3 (2,34%) relapsed at the end of therapy. The most common side effects included pruritus (8,59.%), headache (7.81%) and fatigue (6.25%). None of our patients discontinued the DAAs for adverse reactions or drugs interactions.

Conclusions. Our data confirm that DAA therapy is highly effective and well tolerated among co-infected HIV-HCV patients, without or with advanced liver disease.

Materials and methods. Investigated 224 samples of peripheral blood of patients with HIV-infection. The average age of patients was 36.8±0.6 years. Patients were divided into two main groups: 1st –– taking ARVT (n=135), 2nd –– not receiving ARVT (n=89). The duration of ART was on average 9.0±0.5 years. The key parameters of the cellular immunity were determined by the cytometric method using labeled monoclonal antibodies. The RealTime PCR method was used to determine the concentration of HIV RNA.
Results. In the 1st-group of patients with HIV-infection, the proportion of naive T-cells CD4+CD45RA+CD45R0- was 14.1±1.4%, in the 2nd - 16.4±2.1% (p>0.05 ). The population of naive T-cells with a phenotype of 62L (CD4+CD45RA+62L+) also prevailed in the 2nd group compared with the 1st (p<0,05). T-cells of “memory” CD4+CD45RA-CD45R0+ were determined at the level of 51.4±1.9% in patients of the 1st-group and 49.4±1.9% in the 2nd group (p>0.05). The level of activated T-cells (CD45RA+CD45R0+) was significantly higher in the 2nd group of patients compared to the 1st: 0.3±0.1% versus 0.06±0.05% (p<0.05). The marker of late activation of immunity CD3+HLA-DR+ in the 1st group was at the level of 50.8±1.5%, which turned out to be significantly lower than in the 2nd-group - 61.1±2.3%, (p<0.001). Mean levels of activated CD8+Tcells were significantly higher for HIV patients who did not take ART. The share of CD3+CD8+CD38+ in the 1st-group was 56.7±1.7%, in the 2nd-group - 68.6±2.5% (p<0.001); in the 1st-group, the level of CD3+CD8+HLA-DR+cells averaged 17.7±1.0%, and in the 2nd - 32.3±1.9% (p<0.001). As expected, HIV-infected patients who did not receive ART did have a higher proportion of CD3+CD8+CD38+HLA-DR+cells than patients with ART (p<0.001). Moreover, in patients with complete suppression of HIV RNA concentration against the background of ART, the level of CD3+CD8+CD38+HLA-DR+ was significantly lower than in patients with a detectable level of HIV viral load (more than 10,000 copies/ml), p<0.001. Also, the level of CD8+cells with coexpression of CD38+ and HLA-DR+ was significantly lower in patients with a high concentration of CD4+lymphocytes, p<0.05. An inverse correlation between the absolute number of CD4+cells and the level of CD3+CD8+CD38+HLA-DR+cells (r=0.4), as well as a direct relationship between the concentration of HIV RNA in the blood and the levels of CD3+CD8+HLA-DR+ and CD3+CD8+CD38+HLA-DR+cells (r=0.35).
Findings. HIV infection causes a pronounced activation of both innate and adaptive immunity, induces and supports systemic inflammation in the body. In HIV-infected patients with ARVT, there is a decrease in the proportion of naive T-cells and an increase in the level of memory T-cells. ART reduces the activation of T-cells, but does not normalize it. The proportion of CD8+T-lymphocytes carrying CD38 and HLA-DR on their surface remains elevated even with complete (less than 50 copies / ml) viral load suppression. The levels of CD8+CD38+HLA-DR+T-cells correlate with the concentration of HIV RNA, decreasing with the suppression of HIV replication against the background of ART. Thus, persistently high levels of CD38+HLA-DR+cells can be an indicator of HIV replication.

Background: The time passed since the infection is an important epidemiological and prognostic indicator but often is undefined. Recent Infection Testing Algorithm (RITA) is an approach based on laboratory methods that allow to differentiate recent and established HIV infection. Laboratory tests include detection of antibody titer, avidity index (AI), viral load (VL), and CD4 cell count. A recent infection is defined as the period during the first 6-12 months after infection, depending on the diagnostic tests used. Today, knowledge of molecular biology of the virus gives an opportunity to estimate the time period after infection using additional technique. The proportion of variable positions in HIV genome can be used as a marker of the duration of infection because the heterogeneity of the viral population in human organism increases with time. This parameter can be successfully used in practice to evaluate a recent infection. The aim of this study was to assess the effectiveness of serological and molecular tests for determination of HIV infection duration.

Materials & Methods: Plasma samples (n=91) was obtained from ARV-naïve HIV patients: 34 samples from patients with infection duration up to 6 months (recent infection samples) and 57 samples from patients with duration more than 9 months (established infection samples). The duration of infection was determined by epidemiological and clinical data and indicators of seroconversion. Determination of antibody titers was carried out with DS-EIA-HIV-Ab-TERM kit (Diagnostic Systems, Russia) and antibody avidity was estimated by Architect HIV Ag/Ab Combo kit (Abbott, USA). Nucleotide sequences of pol region including protease gene and fragment of reverse transcriptase gene (according to HXB2, positions 2052-3345) were obtained using AmpliSens HIV-Resist-Seq kit (CRIE, Russia).

Results: According to RITA on the first step all samples were analyzed by the Sensitive-Less Sensitive and antibody avidity assays. The concurrence of DS-EIA-HIV-Ab-TERM results and epidemiological data were obtained for 23/34 (67.6%) of recent infection samples and for 54/57 (94.7%) of established infection samples. The concurrence data for Architect HIV Ag/Ab Combo were 25/34 (73.5%) and 43/57 (75.4%) respectively. The concordance of two tests was 82.4% (28/34) for recent infection samples and 80.7% (46/57) for established infection samples. On the second step were done the sequencing of pol region. The reliable differences in a number of variable positions in sequences were found in comparing of patients with infection duration less 6 months and more than 9 months (0.26% vs 0.37%). There was no significant difference between groups in CD4 cells number, but VL in the early stages of infection (up to 6 months) was significantly higher than in the later period (mean 1.9x10^5 vs 3.6x10^4 copies/ml).

Conclusions: Study results showed that serological tests (DS and Abbott) correctly identified the duration of HIV infection in 84.6% and 74.7% respectively. It was also found that cohorts of patients with recent and established HIV infection differ in viral load and degree of heterogeneity of the viral population. The inclusion of these laboratory parameters in diagnostic algorithm will increase the accuracy of determining the recent HIV infection.

Background&aims:This study is aimed at i)evaluating HBsAg levels in different HBV genotypes (D, A and E) in HBeAg-negative individuals with chronic HBV infection, and ii)the correlation of specific mutations in HBsAg C-terminus (critical for a proper HBsAg-release) with HBsAg levels in vivo, iii)their impact on HBsAg-secretion in vitro and on structural stability in silico.
Methods:HBsAg levels were investigated in 323 drug-naïve HBeAg-negative patients chronically infected with HBV genotype D (N=228), A (N=65) and E(N=30).
In 228 genotype-D infected patients, association of mutations in HBsAg C-terminus with HBsAg<1000 IU/mL (N=130) is assessed by Fisher’s Exact test. Association among mutations in C-terminus is assessed by binomial correlation coefficient (phi). Impact of mutations on HBsAg-secretion is analyzed by transfecting HepG2 cells with plasmids encoding WT- and mutated-HBsAg, linked to a streptavidin-tag (StrepTag). The StrepTagged-HBsAg amount in supernatants is quantified by an ELISA targeting StrepTag, not affected by HBsAg-antigenicity and thus, capable to identify defects in HBsAg-secretion. HBsAg-structures and their stability are predicted by I-Tasser (∆∆G[WT-mutated]<0 indicating reduced stability in presence of mutations).
Results:HBV genotype D is characterized by HBsAg levels lower than genotypes A and E (2,016[520-6,173]IU/ml, 6,416[3,140-14,587]IU/ml, 9,937[4,566-16,032]IU/ml, respectively P<0.001 for all comparisons). Results confirmed by ANOVA multivariable analysis (P<0.0001 for genotype D vs A, P=0.02 for genotype D vs E). In order to unravel factors contributing to lower HBsAg levels in genotype D, we focused on HBsAg C-terminus known to play an important role in HBsAg-secretion. In particular, we found that specific C-terminus mutations (V190A, S204N, Y206C, Y206F and S210N) significantly correlated with HBsAg<1,000IU/ml (P from <0.001 to 0.04).
These mutations lie on divergent pathways involving other mutations in HBsAg C-terminus: V190A with F220L (Phi=0.41, P=0.003), S204N with L205P (Phi=0.36, P=0.005), Y206F with S210R (Phi=0.47, P<0.001) and S210N with F220L (Phi=0.40, P=0.006). Notably, patients with these pairs of mutations are characterized by HBsAg levels 1log lower than patients without them (P=0.003-0.02). Some pairs of mutations also decrease serum HBV-DNA levels (V190A+F220L:2.2[1.6-2.7]logIU/ml; S204N+L205P:2.3[1.9-2.9]logIU/ml, WT: 3.4[2.7-4.1]logIU/ml for wt, P=0.01-0.04), suggesting a detrimental impact also on the release of viral particles.
Similarly, in vitro, all the above-mentioned pairs of mutations determine a significant decrease (up to 90%) in the amount of extracellular HBsAg compared to wt (P values ranging from 0.022 to <0.001). Notably, for the pairs of mutations S204N+L205P, Y206F+S210R, Y206F+S204T and Y206F+M197T, the decrease in HBsAg-release is significant also compared to the single mutation S204N (P<0.001) or Y206F (P=0.007, P=0.001 and <0.0001, respectively).
Finally, by structural analysis, these pairs of mutations determine a relevant reduction in the stability of HBsAg C-terminus and a profound rearrangement of this domain.
Conclusions:HBsAg levels in HBV genotype D are significantly lower than in genotype A and E in different phases of HBeAg-negative chronic infection. In genotype D infected patients, specific clusters of mutations in HBsAg C-terminus correlate with lower HBsAg levels in vivo, hamper HBsAg-release in vitro and affect HBsAg structural stability, supporting their detrimental role on HBsAg-secretion. Knowledge of these mutations can help in optimizing the clinical interpretation of HBsAg levels in HBV genotype D.

Introduction
Human cytomegalovirus (CMV) causes significant complications in immunocompromised patients. CMV reactivation can be prevented and treated with the antiviral drugs (val)ganciclovir, foscarnet, and cidofovir. However, frequent treatment is complicated by drug toxicity, antiviral resistance and hampered by intravenous drug administration. Letermovir, a novel CMV antiviral which was recently approved by the FDA and EMA for prophylactic use in hematopoietic stem cell transplantation recipients, has several advantages over currently used CMV antivirals. Letermovir can be administrated either orally or intravenously and shows mild toxicity. In addition, there is no risk of cross-resistance with existing anti-CMV drugs due to a different mechanism of action.
Case
Here we report off-label prophylactic use of letermovir in a patient with a severe B- and T-cell deficiency and multiple episodes of CMV end-organ disease (colitis and retinitis) and repeated CMV reactivations with selection of a ganciclovir resistant virus with the M460I in pUL97. Thus, the patient received several courses of foscarnet i.v over 18 months, which were complicated by foscarnet related side-effects (anaemia, leukopenia, hypomagnesaemia, and renal impairment.). Therefore, the patient was switched to off-label oral letermovir prophylaxis of 480mg per day while CMV replication was suppressed. Letermovir was well tolerated. Unfortunately, the patient experienced several CMV breakthroughs during letermovir prophylaxis and the predominant CMV variant in blood acquired the C325Y letermovir resistance mutation in pUL56 after 119 days of treatment, which confers high-level resistance to letermovir. Letermovir was stopped and replaced with foscarnet i.v. CMV DNAemia returned to below 50 IU/mL.
Conclusions
This is the first report on the use of letermovir in primary immunodeficiency. Although letermovir was well tolerated, a viral breakthrough with the development of the C325Y mutation in pUL56 conferring resistance after 119 days occurred. More insight is needed regarding the resistance profiles of letermovir encountered in clinical practise. Therefore, we propose to gain more in-depth knowledge on letermovir resistance profiles by starting a letermovir resistance registry, to aggregate letermovir resistance data across multiple centers in Europe. Furthermore, efficacy of letermovir in other patient groups such as solid organ recipients should be investigated as well as its use for pre-emptive therapy and treatment of CMV end organ disease.

Background: HIV-1 DNA resistance genotyping is useful in patients with low or undetectable HIV plasma RNA either to explore a virological failure or to guide a treatment simplification. Ultra-deep sequencing (UDS) techniques have improved the detection of resistant minority variants in HIV plasma RNA. Our aim was to validate the Sentosa platform (Vela DX) for automated deep sequencing for HIV DNA genotypic resistance testing by comparison with direct sequencing.
Materials & Methods: We evaluated the Sentosa SQ HIV genotyping assay on 40 prospective DNA samples isolated from treatment-experienced patients with undetectable or very low plasma RNA load (<200 copies/mL). Automated DNA extraction was performed on MagnaPure 96 (Small volume kit - Roche). HIV-1 DNA was quantified using the Generic HIV DNA cell assay (Biocentric). Direct sequencing was performed using the ANRS protocol (http://www.hivfrenchresistance.org/ANRS-procedures.pdf). The Sentosa SQ HIV Genotyping Assay generated protease (PR), reverse transcriptase (RT) and integrase (IN) sequences. HIV drug resistance was interpreted using the ANRS algorithm v29 (http://www.hivfrenchresistance.org/2018b/Algo-nov2018-HIV1.pdf).
Results: The success rate of Sentosa SQ HIV genotyping assay according to HIV DNA load was evaluated on 20 samples and was 100% for PR and RT and 86% for IN when the HIV DNA load was over 2.5 log copies/million cells. The success rate decreased to 70% for PR and RT when the HIV DNA load was comprised between 1.6 and 2.5 log copies/million cells. The global success rate of prospective UDS on 40 DNA samples was 72%. The prevalence of resistance to at least one antiretroviral drug was 55%. Patients were infected with viruses resistant to protease inhibitors (5%), nucleos(t)idic and non nucleosidic RT inhibitors (42% and 23%, respectively) and integrase inhibitors (15%) according to UDS. PR and RT genes were analysed in parallel using direct sequencing. UDS identified more samples harbouring viruses resistant to nucleos(t)idic and non nucleosidic RT inhibitors (19/40) than direct sequencing (7/40), while both assays were concordant for predicting resistance to protease inhibitors. Resistance to PR and RT inhibitors was consistent with treatment history. Three patients had been treated with integrase inhibitors (prevalence of resistance =3/15 patients previously treated with integrase inhibitors) among the six patients harbouring viruses resistant to integrase inhibitors.
Conclusions: Automated deep sequencing using the Sentosa SQ HIV genotyping assay performed better in predicting HIV DNA drug resistance than direct sequencing. Thus, UDS would be useful for evaluating patients eligible to a strategy of treatment simplification.

Background: In some low- and middle-income countries, include Armenia, directly acting antiviral agents (DAAs) are still not registered and health care budgets are not cover treatment expenses. Take into consideration published studies and expert consensus in 2018, it was estimated that 4.0% (2.9-6%) of the adult population of Armenia were anti-HCV positive. Using a viremic rate of 70% (65-72%) there were 68,000 Armenians have chronic hepatitis C in 2018, correlating to a prevalence of 2.8% among all ages.
Based on the mathematical model of disease progression, which was calibrated using reported Armenian specific epidemiologic data, if there is no change through 2030 under the current treatment paradigm liver related deaths, HCC, and decompensated cirrhosis will increase by 1-8%. New cases of hepatocellular carcinoma will increase by 6% to 330 in 2030. Management of HCC is huge challenge, because of problems with diagnostic and therapeutic procedures (RFA, TACE, liver transplantation) and insurance system covered expenses. From August of 2018 we set up checking of PIVKA-II (Protein Induced by Vitamin K Absence or Antagonist-II) or DCP (Des-γ-carboxy-prothrombin) as an early bio-marker of HCC.
Materials & Methods: 80 HCV-infected patients from 18 to 73 years old (62.5%male, 48.6±13.2 years old, BMI 26.3±5.23 kg/m2, viral load 118–27485300 IU/ml) treated with DAA-contain regimens with/without IFN (8/69). Genotype distribution: genotype 1b –43.8%, genotype 1b+2 – 5%, genotype 1b+3 – 1.3%, genotype 2 – 6.3%, genotype 3 – 40.0%. IFN-free SOF contain regimens were following: SOF+RBV in 5 patients; SOF+DCV±RBV in 35 patients, SOF/LDV±RBV in 25 patients and SOF+VLP±RBV in 7 patient. In 7 naïve patients with 1b genotype and viral load <6000000 IU/ml duration of SOF/LDV treatment were shortened to 8 weeks. AFP checked in all patients with F 3 and 4 (normal ≤8.78ng/mL). PIVKA-II checked in patients with elevated AFP or liver nodules on US. Serum levels of PIVKA-II were measured using the chemiluminescent assay (Architect, Abbott, USA) with cut of 50.9 mAU/mL.
Results: F4 diagnosed in 46.3% of patients, F3 in 13.8%. Three patients with decompensated cirrhosis died despite SVR. AFP in average 14.9±3.9 ng/mL (range 1.4-135.3) was elevated in 44% of patients with F3 or F4. In majority of patients AFP decreased after antiviral therapy, except 3 patients. In one of them with Child-Pugh A liver cirrhosis, genotype 1b, obesity (BMI=34.3) AFP elevated from 26 to 51.6 ng/mL, despite SVR on SOF/LDV. Patient despite awareness not pass US control. After 2 years US revealed nodules, biomarkers PIVKA-II 95658.28 mAU/mL, AFP 77308.58 ng/mL. Another experienced patients with Child-Pugh A liver cirrhosis, genotype 3, obesity (BMI=35.7), metabolic syndrome, dyslipidemia, NAFLD/NASH treated with SOF+PEG+RBV with SVR (in 2016) after two years develop 3 nodules in liver. AFP dynamic: 8.56 (12.05.2016.), 5.08 (07.07.2017.), 20.55 (27.11.2018.), 181.72 (12.03.2019.) ng/mL. PIVKA-II in 01.12.2018. – 577.18 mAU/mL, elevated 644.42 (12.03.2019.).
Conclusions: Awareness to HCC development is mandatory in all patients with F3-F4. Patients with HCV cirrhosis required close monitoring even after SVR. Serum levels of PIVKA-II in combination with AFP and imaging technics can help in early diagnosis of HCC.

Background: The EMA label for elbasvir/grazoprevir (EBR/GZR) recommends that in chronic hepatitis C (HCV) GT1a patients, to minimise the risk of treatment failure, a 16 weeks plus ribavirin (RBV) regimen should be considered in patients with baseline HCV RNA level >800,000 IU/ml and/or the presence of specific NS5A polymorphisms causing at least a 5-fold reduction in the activity of elbasvir.

Materials and Methods: Retrospective/prospective, observational, multi-centre chart review of patients with HCV GT1a or GT4 who initiated EBR/GZR ± RBV between 01-Dec-16 and 30-Sep-17 in 3 large public hospitals in England. We examined a real-world (RW) cohort of patients to determine if the EMA label recommendations around baseline viral load (BVL) and Resistance Associated Substitutions (RAS) testing were followed and what impact this had on outcomes.

Results: We identified 130 patients eligible for inclusion. Mean age was 54 years, 85% male, 94% GT1a, 6% GT4, 60% high BVL (≥ 800,000 IU/ml), 19% cirrhosis, 27% P/R or NS3/4A treatment-experienced, 70% were ex- or active PWID (people who inject drugs) and 43% drink alcohol.

Overall per protocol (PP) SVR12 (patients who completed therapy and had an SVR assessment) was 93.1% (94/101 patients). The rest 22% of patients were lost to follow-up (did not attend their last-visit or SVR12 appointments).

Among the GT1a patients 57% (74/129) had high BVL, of whom 58% (43/74) did not have baseline RAS testing. 91% (39/43) of these received the recommended 16 weeks ELB/GZR + RBV achieving SVR12 100% (34/34). For 9% (4/43) patients the EMA label was not followed as they had just 12 weeks EBR/GZR with SVR12 100% (2/2).

Of the GT1a patients with high BVL, 42% (31/74) underwent baseline RAS testing. EBR-specific NS5A RAS were detected in 26% (8/31) of patients: M28/A/G/T/S (2/8), Q30/H/K/Y/R (6/8), L31/F/M/I/V (2/8). These patients received: 12 weeks EBR/GZR (1/8) with the patient achieving SVR12; and 16 weeks EBR/GZR + RBV (7/8) with 5 patients achieving SVR12. Both patients not achieving SVR12 were ex-PWIDs, and one had 2 identified EBR-specific RAS and was treatment-experienced.

GT1a patients with low BVL: as per EMA label, 64% (25/39) were given 12 weeks ELB/GZR with SVR12 100% (18/18). Against EMA label, two patients received 12 weeks ELB/GZR + RBV, with SVR12 100% and the remaining 31% (12/39) patients were given 16 weeks ELB/GZR + RBV, with 9/10 achieving SVR12; the patient not achieving SVR12 had prior null response.
Although not mandated by EMA label, 28% (11/39) of GT1a patients with BVL < 800,000 IU/ml underwent RAS testing but no EBR-specific RAS were detected.

SVR12 in patients reporting weekly alcohol consumption above UK-recommended safe limits (>14 units; range 20 –186 units) was 100% (10/10).

Conclusions: In this English RW cohort EBR/GZR was highly effective in GT1a. Treatment extension and addition of RBV was common in GT1a patients with high BVL irrespective of baseline RAS testing, which only identified a few EBR-specific RAS; but often also in GT1a patients with low BVL, in both cases leading to very high SVR12 regardless of alcohol abuse.

Background. Virologic failure and development of HIV drug resistance can hinder the results of combined antiretroviral therapy (cART), causing progression of HIV infection. Our goal was to assess the efficacy of cART in a Romanian cohort of young adults, parenterally infected with HIV clade F in the late 1980s and exposed to cART for more than a decade.
Materials & methods: 170 HIV+ subjects ( males: 47%, mean age: 24 years) with a median duration of HIV infection of 24 year and of cART of 13 years were analyzed. Currently, 67.3% of the participants receive a NRTI- based regimen, 13.6% a NNRTI- based regimen and 19% 3-class regimen. HIV viral load was tested by quantitative RT-PCR (Cobas TaqMan HIV-1 Test Roche Molecular Systems, USA) and plasma samples with HIV RNA level > 1,000 copies /mL were sequenced in the pol gene (ViroSeq HIV-1 Genotyping System,Abbott Laboratories, USA); drug resistance mutations were assessed using the Stanford HIV-Drug Resistance algorithm.
Results: 58% of the participants have achieved viral suppression and 45% showed no sign of immunosuppression (CD4 count >500 cells/ul). Lower CD4 T-cell counts (p=0.02), longer time on cART (p=0.04) and longer exposure to monotherapy regimens (p=0.03) were significantly associated with virological failure. No correlation was found between VF and current treatment regimens. Only 24% of the subjects presented HIV viral load > 1000 copies/m. The rate of antiretroviral resistance was 15%, with 6% of the subjects presenting resistance to two drug classes, 4% having triple class resistance and 1% having multiple resistance mutations to all currently available drugs. Reverse-transcriptase inhibitors mutations were predominant, followed by PIs resistance mutations. The presence of HIV-drug resistance was associated with lower nadir CD4 count (p=0.01) and longer duration of HIV-infection (p=0.04).
Conclusions. Our results show a high rate of virological suppression in the Romanian cohort of HIV-infected patients, long term survivors, parenterally infected in early childhood; a low prevalence of virologic failure and ARV drug resistance was recorded despite the fact that these patients were exposed to suboptimal treatment and sequential monotherapies in the past.

*Presenting on behalf of the authors.
Background: Abbott Realtime assay measures quantitative HIV-1 RNA viral load (VL) from 40 to 10,000,000 c/mL and generates qualitative target detected (TD) or target not detected (TND) for VL<40 c/mL. The US FDA snapshot algorithm uses 50 c/mL as the cut-off. Clinical significance and subject management implications of low-level quantitative and qualitative VL data remain controversial. We assessed the number of participants having 40 c/mL ≤ VL <50 c/mL, and TD/TND over 48 weeks for DTG+RPV 2-drug regimen vs CAR (PI-, NNRTI-, or INSTI-based 3-drug current antiretroviral regimen).
Materials & Methods: SWORD-1 and SWORD-2 are identical open-label, multicenter, global, phase III, non-inferiority studies evaluating the efficacy and safety of switching from CAR to DTG+RPV once daily in HIV-1-infected adults with HIV-1 RNA <50 c/mL (VL <50 c/mL) for ≥6 months and no history of virologic failure. We explored VL shifts from baseline, cumulative, and per visit classification of participants into >50 c/mL, 40 c/mL ≤ VL< 50 c/mL, or TD/TND when <40 c/mL across arms throughout 48 weeks.
Results: 1024 participants were randomized and exposed (DTG+RPV, 513; CAR, 511) across both studies. At Week 48, 95% of participants in both arms had Snapshot VL <50 c/mL in the intention-to-treat–exposed population. Confirmed virologic withdrawal (CVW) rates were <1% in both arms. Similar proportion of participants at Week 48 had Snapshot VL <40 c/mL and TND in the 2 arms (84% vs 80%, adjusted difference 3.1%, 95% confidence interval, −2.2 to 8.3). Participants with Baseline TD had similar and low occurrence of ≥1 VL ≥50 c/mL (DTG+RPV 14%; CAR 17%) or at ≥1 VL between 40 c/mL and 50 c/mL (DTG+RPV 4%; CAR 8%). Those participants with Baseline TND had similar and low occurrence of ≥1 VL ≥50 c/mL (DTG+RPV 5%; CAR 5%), or a similar percentage of ≥1 VL between 40 and 50 c/mL (DTG+RPV 3%, CAR 1%), or ≥1 VL<40 c/mL and TD (DTG+RPV 44%; CAR 41%) through Week 48.
Conclusions: DTG+RPV was non-inferior to CAR at Week 48 by Snapshot <50 c/mL. The two groups were similar with Snapshot <40 c/mL and TND as endpoint. Proportions of TD at Baseline and over time were similar between arms with higher rates of TD post-Baseline in those with TD at Baseline. Incident viremia (≥40 and ≥50 c/mL) was similar between arms by Baseline TD vs TND, but it was more common with TD. However, this had limited clinical consequence, because efficacy rates were high (95%) and equal between arms and CVW numbers were low and equal between arms.
Data included in this abstract have been previously presented at HIV Drug Therapy Glasgow; October 28-31, 2018; Glasgow, UK.

Background: At 48 weeks in the GEMINI-1 and GEMINI-2 studies (NCT02831673 and NCT02831764), the 2-drug regimen (2DR) dolutegravir (DTG) + lamivudine (3TC) was non-inferior to the 3-drug regimen (3DR) DTG + tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) in achieving plasma HIV-1 RNA <50 c/mL in treatment-naive adults with baseline HIV-1 RNA ≤500,000 c/mL. To better understand the potency of DTG+3TC compared with the 3DR, we explored the rapidity of initial viral load (VL) decline and efficacy response rates in those with baseline VL >100,000 c/mL.

Materials & Methods: Participants were randomized 1:1 to receive DTG 50 mg + 3TC 300 mg once daily or DTG 50 mg + TDF 300 mg/FTC 200 mg once daily (stratified by baseline HIV-1 RNA and CD4+ cell count). The primary endpoint was the proportion of participants with HIV-1 RNA <50 c/mL at Week 48 (using snapshot algorithm, intention-to-treat–exposed population), with a 10% non-inferiority margin. As a post hoc analysis, mean change log10-transformed HIV-1 RNA from baseline and 95% confidence intervals (CIs) were calculated at Weeks 4, 8, 12, 16, 24, 36, and 48. Proportions of participants with plasma HIV-1 RNA <50 c/mL at Week 48 (using snapshot) for the 2DR vs 3DR therapy by baseline HIV-1 RNA strata ≤100,000 c/mL, >100,000 c/mL, >250,000 c/mL, and >400,000 c/mL were also analyzed.

Results: In the pooled analysis at Week 48, 91% (655/716) of participants in the 2DR vs 93% (669/717) in the 3DR group achieved HIV-1 RNA <50 c/mL (adjusted treatment difference, −1.7%; 95% CI, −4.4 to 1.1). 20% (140/716) in the 2DR and 21% (153/717) in the 3DR group had baseline HIV-1 RNA >100,000 c/mL (including 2% with baseline VL >500,000 c/mL). Similar rapid VL log decline was observed in both treatment groups overall (median change from Baseline at Week 4: −2.77 log10 c/mL in the 2DR and −2.80 log10 c/mL in the 3DR group) and in participants with baseline VL >100,000 c/mL. (median change from Baseline at Week 4: −3.38 log10 c/mL in the 2DR and −3.40 log10 c/mL in the 3DR group). High and similar response rates were seen in participants across baseline VL strata < and >100,000 c/mL. For participants with baseline VL ≤100,000 c/mL, 91% (526/576) in the 2DR vs 94% (531/564) in the 3DR group achieved HIV-1 RNA <50 c/mL (adjusted treatment difference, −2.8%; 95% CI, −5.8 to 0.2). For participants with baseline VL >100,000 c/mL, 92% (129/140) in the 2DR vs 90% (138/153) in the 3DR group achieved HIV-1 RNA <50 c/mL (adjusted treatment difference, 1.9%; 95% CI, −4.5 to 8.4). A consistent response pattern was also observed in the HIV-RNA strata >250,000 c/mL and >400,000 c/mL.

Conclusions: Viral load decline with the 2DR DTG+3TC was rapid and comparable with that of the 3DR DTG+TDF/FTC. Response rates in participants with baseline HIV-1 RNA >100,000 c/mL were high with DTG+3TC, consistent across strata, including participants with HIV-1 RNA >400,000 c/mL, and similar to the 3DR group. These data demonstrate a high potency of DTG+3TC, similar to that of standard-of-care 3DR.

Background: Until recently the rate of transmitted drug-resistance mutations (TDR) was relatively high mainly to NNRTIs. The prevalence of TDRs is regularly evaluated in treatment-naïve patients in Tel-Aviv. This study evaluated the rate and pattern of TDR among HIV-1 treatment-naïve patients in Tel Aviv from 2010 to 2018.
Material and methods: We analyzed TDR mutations prevalence in blood samples of treatment-naïve patients in Tel-Aviv (2010-2018). Integrase region was sequenced in samples from 2016. Transmission dynamics were analyzed by reconstructing viral phylogenies from pol sequences of HIV-1 subtype A, B and C viruses the most common subtypes in Tel Aviv.
Results: 862 Viral sequences revealed 12.9% TDR. Over three-fourths (76%) of men who have sex with men (MSM) were born in Israel, and 81.6% harbored subtype B viruses. Other groups include intravenous drug users (IVU), 78% of whom were born in the former Soviet Union countries and 88% of whom harbored subtypes A viruses. The heterosexual group was very heterogeneous in origin, including patients born in Israel, Ethiopian immigrants, immigrants from the former Soviet Union, and worker immigrants mainly from Africa. NNRTIs TDR was the major class resistance (40%) followed by PIs (30%), NRTIs (23%) and 7% more than one class. Integrase inhibitors resistance mutations were represented only by minor mutations: L74m (n=1), T97A (n=4), E138K (n=1), E157Q (n=4) and G163K (n=1) .
Phylogenetic analysis of subtype A and B viruses supported clustered transmission of TDR among men who have sex with men. These clusters were represented by the mutation K103N in RT and L90M in PR. No major integrase inhibitors TDR were found in 2016-2018.
Conclusion: TDRs among patients followed in Tel-Aviv were represented by clusters in MSM. These clusters contained resistance associated mutations to drugs less frequently prescribed in recent years, so their effect on treatment strategy is not straightforward.
Characterization of evolving clusters and transmission networks is useful to concentrate prevention and control efforts where they are most needed and to assess the impact of these interventions.

Background: According to the Israeli ministry of health (MOH), women comprise 37% of the reported HIV-1 positive individuals. However, in depth analysis of the characteristics of this population has not been reported. Here we studied women diagnosed with HIV-1 in 2010-2018.
Materials & Methods:
The data-base of the National HIV reference center, which covers all HIV-1 reported cases in the country, was screened for records of all newly diagnosed HIV patients identified between January 2010 and December 2018. Men, women <16 years and women diagnosed in years other than 2010-2018 were excluded. Women who are foreign citizens were also excluded due to lack of reliable data. The final cohort included 727 women. Sequencing of early samples collected from treatment naïve carriers was performed using 41.4% (301) of the samples selected by a stratified random selection design. Demographic (age, country of origin and risk factor for HIV acquisition)) and viral (time of HIV diagnosis, HIV-1 subtype and TDRM including RT-A138) characteristics were recorded and examined by Chi-Square test. Logistic regression was used to assess the associations between these variables. Yearly trends of TDRM rates were examined by segmented Poisson regression.
Results: Median age was 38 at diagnosis. 17.1% (124) were older than 50. The prevalence of women from Africa (OGE-IL, n=227) decreased while that of women from former Soviet Union (FSU, n=307) increased significantly (p<0.001) over the years. Statistically significant yearly decrease in the rate of subtype C (Africa origin) was also observed (p=0.004). 23.6% (71 of 301 patients) had TDRM; 3.8%, 9.6% and 16.2% had PI, NRTI and NNRTI TDRM, respectively. Significant yearly increase in TDRM rate (1.6% per year, mainly NNRTI) was observed (p<0.001). The NRTI A62 (6.0%), NNRTI E138 and K103 (5.3% and 4.3%, respectively) were the most prominent mutations. Age of diagnosis (>50) was associated with a higher TDRM rate (p=0.04; 95% CI of OR 2.00).
Conclusions: The epidemiology of HIV infected women in Israel is changing, shifting to women from FSU infected through heterosexual contact with persistent escalation in the rate of TDRM. Delayed age of diagnosis contributes to the higher TDRM rate. These results re-enforce the national policy of resistance testing at baseline and call to apply appropriate preventive measures to women at risk.

Introduction: Uptake of antiretroviral therapy (ART) is expanding rapidly in low-income and middle-income countries. Monitoring of virological suppression is recommended at six months of treatment and annually thereafter. In case of confirmed viral rebound a switch to second-line ART is indicated. We report the first multicentre assessment of suppression over time and clinical response to viral rebound under programmatic conditions.
Methods: 104719 patients on first-line ART at 52 South African centres were studied. Virological suppression, switch to second-line ART, death, and loss to follow-up were analysed. Multistate models and Cox proportional hazard models were used to assess suppression over time and predictors of treatment outcomes.
Findings: In on-treatment analysis, suppression below 1000 copies/mL was 89·0% at month 12 and 90·4% at month 72. Suppression below 50 copies/mL was 73·1% at month 12 and 77·5% at month 72. Intention-to-treat suppression was 75·0% and 64·3% below 1000 and 50 copies/mL at month 72 respectively. Viral rebound occurred in 19·8% (20766/104719) of patients during an average follow-up of 152 [61-265] weeks. Male, young, and late presenting patients were at highest risk. After rebound, confirmatory testing took 29 weeks [IQR: 16-54]. Viral resuppression without switch of ART occurred frequently (45·6%; 6030/13210) but was associated with renewed viral rebound and switch. Of patients with confirmed failure who remained in care, only 41·5% (1872/4510) were switched. The median time to switch was 68 weeks [35-127], resulting in 12325 person-years spent with a VL above 1000 copies/mL.
Interpretation: 90% virological suppression was achieved using the threshold of 1000 copies/mL in on-treatment analysis. However, this target was not met at the 50 copies/ml threshold, nor in intention-to-treat analysis. Clinical management in response to rebound was profoundly delayed prolonging the duration of viraemia and potential for transmission. Diagnostic tools to establish the cause of rebound are urgently needed to accelerate clinical decision-making.