Simona Paraschiv

Enhanced HIV-1 surveillance using molecular epidemiology to study and monitor HIV-1 outbreaks among intravenous drug users (IDUs) in Athens and Bucharest

BACKGROUND A significant increase in HIV-1 diagnoses was reported among Injecting Drug Users (IDUs) in the Athens (17-fold) and Bucharest (9-fold) metropolitan areas starting 2011. METHODS Molecular analyses were conducted on HIV-1 sequences from IDUs comprising 51% and 20% of the diagnosed cases among IDUs during 2011-2013 for Greece and Romania, respectively. Phylodynamic analyses were performed using the newly developed birth-death serial skyline model which allows estimating of important epidemiological parameters, as implemented in BEAST programme. RESULTS Most infections (>90%) occurred within four and three IDU local transmission networks in Athens and Bucharest, respectively. For all Romanian clusters, the viral strains originated from local circulating strains, whereas in Athens, the local strains seeded only two of the four sub-outbreaks. Birth-death skyline plots suggest a more explosive nature for sub-outbreaks in Bucharest than in Athens. In Athens, two sub-outbreaks had been controlled (Re<1.0) by 2013 and two appeared to be endemic (Re∼1). In Bucharest one outbreak continued to expand (Re>1.0) and two had been controlled (Re<1.0). The lead times were shorter for the outbreak in Athens than in Bucharest. CONCLUSIONS Enhanced molecular surveillance proved useful to gain information about the origin, causal pathways, dispersal patterns and transmission dynamics of the outbreaks that can be useful in a public health setting.

Recent HIV-1 Outbreak Among Intravenous Drug Users in Romania: Evidence for Cocirculation of CRF14_BG and Subtype F1 Strains

Since 2011, Romania has faced an HIV outbreak among injecting drug users (IDUs). Our aim was to identify and describe clinical and epidemiological patterns of this outbreak. A cross-sectional study enrolled 138 IDUs diagnosed with HIV infection between 2011 and 2013 with 58 sexually infected individuals included as the control group. The IDUs had a long history of heroin abuse (10 years) and a recent history of new psychostimulant injection (3-4 years). Classical epidemiological data and molecular techniques were used to describe the transmission dynamics. A high prevalence of hepatitis C virus (HCV) coinfection was noted (98.6%) compared to the control group (10.3%) (p<0.001). IDUs had initially been infected with HCV. HIV infection was more recent, linked to starting injecting stimulants. HIV subtype analysis showed a predominance of the local F1 strain in both IDUs and sexually infected patients; in IDUs it also identified 28 CRF14_BG recombinants and six unique recombinant forms (URFs) between F1 and CRF14_BG. A few patients from both risk groups were infected with subtype B. Among IDUs, CRF14_BG was associated with a lower CD4 cell count and more advanced stages of disease, which correlated with CXCR4 tropism. Phylogenetic analysis revealed the spread of HIV through three major IDU clusters of recent date. Among IDUs with CRF14_BG, some reported travel abroad (Spain, Greece). By identifying clusters of IDUs with related viruses, molecular epidemiologic methods provide valuable information on patterns of HIV transmission that can be useful in planning appropriate harm reduction interventions.

RegaDB: community-driven data management and analysis for infectious diseases

Summary: RegaDB is a free and open source data management and analysis environment for infectious diseases. RegaDB allows clinicians to store, manage and analyse patient data, including viral genetic sequences. Moreover, RegaDB provides researchers with a mechanism to collect data in a uniform format and offers them a canvas to make newly developed bioinformatics tools available to clinicians and virologists through a user friendly interface. Availability and implementation: Source code, binaries and documentation are available on http://rega.kuleuven.be/cev/regadb. RegaDB is written in the Java programming language, using a web-service-oriented architecture. Contact: pieter.libin@rega.kuleuven.be

Molecular typing of the recently expanding subtype B HIV-1 epidemic in Romania: evidence for local spread among MSMs in Bucharest area.

Highlights ► The most frequent route of infection in subtype B patients was MSM transmission. ► Many of the patients acquired the infection abroad, in Western European countries. ► A local transmission network has been identified. ► This network includes mainly MSM living in the Bucharest area. ► Subtype B local transmission network is recent, dating at the beginning of the 90s.

Combined Analysis of the Prevalence of drug Resistant Hepatitis B Virus in antiviral therapy Experienced patients in Europe (CAPRE)

BACKGROUND European guidelines recommend treatment of chronic hepatitis B virus infection (CHB) with the nucleos(t)ide analogs (NAs) entecavir or tenofovir. However, many European CHB patients have been exposed to other NAs, which are associated with therapy failure and resistance. The CAPRE study was performed to gain insight in prevalence and characteristics of NA resistance in Europe. METHODS A survey was performed on genotypic resistance testing results acquired during routine monitoring of CHB patients with detectable serum hepatitis B virus DNA in European tertiary referral centers. RESULTS Data from 1568 patients were included. The majority (73.8%) were exposed to lamivudine monotherapy. Drug-resistant strains were detected in 52.7%. The most frequently encountered primary mutation was M204V/I (48.7%), followed by A181T/V (3.8%) and N236T (2.6%). In patients exposed to entecavir (n = 102), full resistance was present in 35.3%. Independent risk factors for resistance were age, viral load, and lamivudine exposure (P < .001). CONCLUSIONS These findings support resistance testing in cases of apparent NA therapy failure. This survey highlights the impact of exposure to lamivudine and adefovir on development of drug resistance and cross-resistance. Continued use of these NAs needs to be reconsidered at a pan-European level.

Diversity of HIV-1 subtype C strains isolated in Romania

Two unique aspects particularities of the HIV-1 epidemics in Romania are the high prevalence of subtype F1 strains and the large pediatric population infected in the late 1980s and early 1990s. During recent years, more infections with other subtypes have been seen in newly diagnosed patients. After subtype B, subtype C was the most frequent one. This subtype is prevalent in countries from sub-Saharan Africa and India, being responsible for half of the total HIV-1 infections in the world. We have identified 37 patients infected with subtype C, sequenced the reverse transcriptase and protease regions of their pol genes, and applied phylogenetic analyses to the sequences. We have also included 20 subtype F1 strains isolated from both teenagers (children at the time of diagnosis) and adults. The phylogenetic analysis was performed by using the PhyML method, the GTR (general time reversible) model of evolution and gamma distribution of variability of rates between sites, empirically calculated from the data. The epidemiological data indicates that the main route of transmission for the adult subjects was by heterosexual contact and a relatively small number of patients were possibly infected abroad. In three cases, blood transfusion prior to 1989 or surgical procedures at early ages were suspected to be the cause of the HIV infection and three other patients were most probably parenterally infected. The phylogenetic analyses showed that the Romanian C strains are very diverse overall, clustered in several groups characterized by common transmission route (transfusion/surgical procedures) or local geographical relatedness. The HIV-1 epidemics in Romania apparently followed different patterns for subtypes F and C. While subtype F1 seems to have been monoclonally introduced and extensively spread in the 80s, the subtype C strains, although present in the late 80s, failed to spread to the same extent.

Nucleoside reverse transcriptase inhibitor resistance mutations in subtype F1 strains isolated from heavily treated adolescents in Romania

OBJECTIVE To examine the nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations in the reverse transcriptase gene of HIV-1 F1 subtype strains isolated from heavily treated adolescents. METHODS Three hundred and fifty reverse transcriptase (RT) genotypes with at least three NRTI resistance mutations were included in this study; the corresponding strains were isolated from adolescents with a complex history of antiretroviral treatment. Subtyping was done using the publicly available algorithm REGA HIV-1&2. Resistance genotyping was performed using Big Dye Terminator chemistry provided by the ViroSeq genotyping system. The RT gene carrying the K65R mutation and thymidine analog mutations (TAMs) was cloned into pGEM-T vector (Promega), followed by sequencing. In order to identify mutational clusters we calculated the binomial (phi) correlation coefficient using SPSS 11.0 software. RESULTS The analyzed sequences all belonged to the F1 subtype and were frequently carrying TAMs associated with substitutions at position 184. TAM-2 was the pathway more frequently encountered, and the demarcation between TAM-1 and TAM-2 was rather weak. Although the combination of K65R mutation with TAMs has rarely been reported because of their antagonistic effects on NRTI resistance, its presence was confirmed by clonal analysis of one strain. Four percent of the studied genotypes presented insertions and deletions in the region 67-70 of the RT gene and they were frequently associated with particular TAMs. Most of the NRTI resistance mutations were found to belong to one of three distinct clusters. CONCLUSION Although the overall resistance mutations were not different from those described for subtype B, the subtype F1 HIV-1 NRTI mutation patterns displayed same specificities with possible therapeutic consequences.

Epidemic dispersion of HIV and HCV in a population of co-infected Romanian injecting drug users

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

NGS combined with phylogenetic analysis to detect HIV-1 dual infection in Romanian people who inject drugs

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

A21 HIV-1 sub-subtype F1 outbreak among MSM in Belgium

Current antiretroviral therapies for HIV-1 are not curative because a small number of CD4þT-cells remain infected with a latent, replication-competent provirus that contributes to viral rebound after the cessation of therapy. Several approaches to purge persistent HIV-1 reservoirs are in the beginning phases of clinical trials. To ensure future curative therapies target replication-competent HIV-1 proviruses for eradication, a thorough understanding of the distribution of replication-competent HIV1 within T-cell subsets and how activation and proliferation of these cells contribute to the maintenance of the replicationcompetent HIV-1 reservoir is required. This study will employ a full-length single-proviral sequencing assay based on Next Generation Sequencing (NGS) techniques to sequence the entire HIV-1 genome of proviruses isolated from CD4þT-cell subsets (central, transitional, and effector) sorted from peripheral blood and lymphoid tissue after 5–15 years of suppressive therapy from two groups of participants (1) three participants who initiated therapy during acute/early infection and (2) three participants who initiated therapy during chronic infection. Replication-competent proviruses will be identified by the absence of deletions and APOBEC3G induced hypermutation. The infection rates of replication-competent proviruses located in specific cell populations between participants will be compared along with the frequencies of replication-competent proviruses between different T-cell populations and within tissuederived cells from these participants. This important study will allow us to determine whether specific cellular compartments harbour replication-competent HIV-1 and will provide valuable information for future curative HIV-1 clinical trials.