Strategies to overcome HIV drug resistance-current and future perspectives.

The availability of combined antiretroviral therapy (cART) has revolutionized the course of HIV infection, suppressing HIV viremia, restoring the immune system, and improving the quality of life of HIV infected patients. However, the emergence of drug resistant and multidrug resistant strains remains an important contributor to cART failure, associated with a higher risk of HIV-disease progression and mortality. According to the latest WHO HIV Drug Resistance Report, the prevalence of acquired and transmitted HIV drug resistance in ART naive individuals has exponentially increased in the recent years, being an important obstacle in ending HIV-1 epidemic as a public health threat by 2030. The prevalence of three and four-class resistance is estimated to range from 5 to 10% in Europe and less than 3% in North America. The new drug development strategies are focused on improved safety and resistance profile within the existing antiretroviral classes, discovery of drugs with novel mechanisms of action (e.g., attachment/post-attachment inhibitors, capsid inhibitors, maturation inhibitors, nucleoside reverse transcriptase translocation inhibitors), combination therapies with improved adherence, and treatment simplification with infrequent dosing. This review highlight the current progress in the management of salvage therapy for patients with multidrug-resistant HIV-1 infection, discussing the recently approved and under development antiretroviral agents, as well as the new drug targets that are providing a new avenue for the development of therapeutic interventions in HIV infection.

Neurocognitive Impairment in the Combined Antiretroviral Therapy Era in a Romanian Cohort of Young Adults with Chronic HIV Infection.

HIV-associated neurocognitive disorders (HAND) continue to be reported even in patients with successful antiretroviral treatment. We investigated the prevalence of neurocognitive impairment and possible HIV-associated determinants of cognition in a Romanian cohort of young adults, parenterally infected with HIV during their first years of life. Two hundred fourteen treatment-experienced HIV-positive individuals [median age: 24 years, males: 48%, median duration on combined antiretroviral therapy (cART): 12 years] underwent standard immunologic and virological monitoring and antiretroviral resistance testing using pol gene sequencing in both plasma and, when available, cerebrospinal fluid (CSF) paired samples. Neurocognitive impairment was assessed using a comprehensive neuropsychological test battery, and a global deficit score (GDS) was calculated (cutoff ≥0.5). Cognitive impairment was detected in 35% of the study participants, without any association with sex, median age, CD4 cell count (actual or nadir), CSF and plasma viral load (actual or zenith), AIDS diagnosis, duration of HIV infection, and cART characteristics. Participants carrying resistant viruses tended to be more frequently cognitively impaired (p = 0.36), with a higher median GDS value (p = 0.06) compared with participants harboring wild-type HIV, although the figures did not reach statistical significance. No signs of virological compartmentalization were observed based on CSF versus plasma viral load and on the profile of pol sequences. A moderate rate of mild neurocognitive impairment is still present in young adults with chronic HIV infection acquired in early childhood despite successful cART, without any association with classic markers of HIV infection. New biomarkers reflecting persistent central nervous system inflammation and neuronal injury may be more relevant for the development of HAND.

HIV transmission clusters among injecting drug users in Romania.

Injection drug use is increasingly an important route of HIV transmission in Romania (from 1.5% of the newly diagnosed cases prior to 2010 to 31% in 2013). In this study we investigated the viral characteristics and relationships in newly HIV infected persons who inject drugs in Bucharest, Romania. RESULTS: HIV-1 pol sequencing, followed by phylogenetic and clustering analysis was performed on blood from 37 injecting drug users (IDUs) newly diagnosed with HIV infection. While HIV subtype F1, the dominant strain in Romania since 1990, remains prevalent, new subtypes were found including G, B, B/G and B/F recombinants. Overall, 27 of the available sequences (72.9%) clustered with at least one other. Network and phylogenetic analysis revealed tight monophyletic clusters for both subtypes F and G, with short genetic distances between sequences, suggesting recent numerous acute to acute transmissions or single burst-type episodes. No transmitted drug-resistance mutations were identified. Greater immunosuppression was present in subjects forming the subtype G cluster, possibly indicating a faster rate of progression associated with this subtype. CONCLUSIONS: The recent increasing numbers of IDU related HIV transmissions in Bucharest, has resulted in closely-knit transmission networks that maychange the genetic profile of the local HIV epidemic.

Immunoassay and molecular methods to investigate DNA methylation changes in peripheral blood mononuclear cells in HIV infected patients on cART.

This study aimed to investigate the influence of antiretroviral therapy on methylation markers, in a group of HIV infected, heavily treated patients. Immune and molecular methods were used to investigate potential changes in methylation profile in DNA isolated from peripheral blood mononuclear cells collected from antiretroviral-experienced HIV infected patients and healthy controls. The percentage of 5-methylcytosine was inversely correlated with proviral DNA and active replication while DNMT1 (p = 0.01) and DNMT3A (p = 0.004) independently correlated with active viral replication. DNMT3A expression increased with total treatment duration (p = 0.03), number of antiretroviral drugs ever used (p = 0.003), and cumulative exposure to protease inhibitors (p = 0.02) even in currently HIV undetectable patients.

miR-29a associates with viro-immunological markers of HIV infection in treatment experienced patients.

MicroRNAs (miRNAs) are small, non-coding RNA species essential for the post-translational regulation of gene expression. Several miRNA have been proposed to contribute to Human immunodeficiency virus-1 (HIV-1) infection establishment, progression and latency. Among them, miR-29a seems to be of particular interest. The aim of this study was to investigate the association between miR-29a expression and immunologic and virologic markers of HIV infection progression in long-term antiretroviral-treated individuals. In a homogenous group of 165 young adults, with chronic HIV infection, parenterally acquired during childhood, the expression level of miR-29a was found to be inversely correlated with HIV viral load and the degree of immunosuppression, expressed by both CD4 cell count and the CD4/CD8 ratio. There was a significant difference in miR-29a expression according to the patient's response to treatment, with the lowest levels expressed by patients with treatment failure, defined as detectable viremia and CD4 < 350 cells/mm3 . No significant correlation was found between miRNA level and the nadir CD4 count or zenith HIV viral load. This study establishes the association between miR-29a expression and markers of HIV infection in long-term survivors, treatment-experienced patients, suggesting its potential use as an indicator for the on-treatment disease evolution. J. Med. Virol. 88:2132-2137, 2016. © 2016 Wiley Periodicals, Inc.

HIV-1 circulating subtypes in Romania.

The Romanian HIV epidemic is characterized by the prevalence of a single particular HIV1 subtype, called F, a minor form, previously reported only in South America and Central Africa. Initially reported in the early '90s by serotyping studies in the large cohort of parenterally infected children, this subtype remained dominant during the following two decades, despite the continuous growth in the number of heterosexually-acquired infections in adults. A steady prevalence of F subtype was further demonstrated by genotyping and molecular epidemiology studies. This article reviews the hypothesis on the origin and the unusual steady persistence of this HIV strain and discusses the recent changes in the molecular epidemiology of the epidemic, associated to the emergence of new infection routes. Phylogenetic and phylogeography studies conducted through the epidemic seem to indicate that HIV F subtype originated in the 1950s in the Democratic Republic of Congo and was separately spread by immigration waves to Brazil, Angola and Romania. Data released at the end of 2012 report F1 subtype as the dominant HIV-1 clade in Romania in all categories of patients: recently infected or late presenters, antiretroviral- naive or heavily treated, but signal the emergence of other subtypes (B--the most frequent non-F subtype among the newly diagnosed individuals, followed by subtypes C, A and several circulating recombinant forms). In this context, it is of outmost importance to follow the spreading of new emerging subtypes in the predictable setting of new infection waves in the Romanian HIV epidemic.

Transmitted HIV drug resistance in treatment-naive Romanian patients.

Transmitted HIV drug resistance (TDR) remains an important concern for individuals unexposed to antiretroviral treatment. Data on the prevalence of TDR, available mainly for HIV-1 subtype B, are now also emerging for other subtypes. In Romania, a steady predominance of subtype F was reported among both long-term survivor children and newly infected adults. The pol gene of 61 drug-naïve patients infected with HIV, diagnosed between 1997 and 2011 was sequenced in order to analyze the prevalence of primary resistance mutations and to correlate these with the infecting genotype. Only 5/61 specimens were classified as infected recently using the BED-Capture Enzyme Immunoassay. Subtype F1 was prevalent (80.3%), however, other HIV-1 clades are increasingly identified, especially in the group of subjects infected recently. An HIV transmission cluster, associated to injecting drug use was identified by phylogenetic analysis. The overall prevalence of TDR was 14.75%, mainly associated with NRTI resistance (13.11%), TAMs and M184V being the most common mutations. A declining trend of TDR was recorded from 26.08% in 1997-2004 to 7.89% in 2005-2011. No primary resistance was identified among recent seroconvertors. All HIV-1 strains had minor mutations in the protease and RT genes, often detected at polymorphic positions. The declining rates of TDR might be related to the high efficacy of HAART and to the increasing number of treated patients with virological success who have a low risk of transmission. The recent increase of HIV-1 infections which involve other subtypes impose a continuous surveillance of the genetic composition of the epidemic.

Correlation between resistance profile and immunosuppression in heavily treated HIV-1 infected Romanian patients.

BACKGROUND AND OBJECTIVE: As available data on HIV-1 strains from Romania indicate the prevalence of a particular subtype- F, not found in other European countries, we aimed at investigating the impact on drug susceptibility of mutations associated with drug resistance and their correlation with the virological and immune response to therapy. METHODS: 38 long term survivors, HIV-1 parenterally infected in childhood, with repeated virological failures, were genotyped for drug resistance and subtype determination. A phylogenetic tree of aligned reversetranscriptase sequences was built. RESULTS: 94.7% of all the patients'strains were subtype F1, clustering together with other Romanian and Angolan F1 strains. Despite the long and complex treatments, 15.8% of patients had wild type virus, 68.4% were fully susceptible to protease inhibitors, 47.3% to non-nucleoside reverstranscriptase inhibitors, 28.9% to nucleoside reverstranscriptase inhibitors. Only 13.2% were resistant to all antiretroviral drug classes. A significantly higher total number of mutations were encountered in severely immunosuppressed patients, who presented also major mutations in the protease gene (V82A, I54V, G48V) and the major M184V mutation associated with type 2 thymidine analogs mutations in reverstranscriptase gene. CONCLUSION: A good immune status seems to be associated with a low range of mutations, indicating the impact of immune restoration or preservation on the therapeutic success rate. The slower post-HAART progression of mutational pattern of HIV- 1 subtype F1 in long term survivors may also influence the viral replicative fitness, a fact that can explain its steady prevalence in Romania.

Molecular epidemiology of hepatitis C virus strains from Romania.

BACKGROUND AND AIMS: A high seroprevalence of Hepatitis C Virus (HCV) infection has been reported in Romania, with limited data on the viral subtypes' distribution. In order to detect any changes in the genetic composition of the epidemic, a survey on the recent profile of circulating HCV genotypes was conducted. METHODS: 241 hepatitis C infected patients with active viral replication diagnosed between September 2004 - October 2008 were included in a retrospective study. Genotyping using commercial Line Probe Assay (Innogenetics) was confirmed by sequencing of Core PCR products followed by phylogenetic analysis. RESULTS: HCV subtype 1b was found in 92.6% of the samples, subtype 1a in 5.4 % of the samples, subtype 4a in 1.2%, and subtype 3a in 0.8% of the samples. Chronic hepatitis C infections with subtype 1b were found in women aged 40-60 years old with a history of blood transfusions received during surgical/obstetrical interventions. No geographical clustering was evident for HCV 1b sequences. The new emerging non-1b genotypes were detected mainly in younger patients with a history of intravenous drug use. The genetic distances among the HCV 1a strains are very homogeneous and small, with a high sequence identity with other European strains, suggesting the recent entrance of this subtype in Romania from singular or limited sources of infection. CONCLUSION: The introduction of new HCV genotypes in Romania stimulates a continuous epidemiological surveillance, suggesting shifts in the transmission pathways and risk factors, with the possible emergence of recombinant strains in patients with multiple infections.

HEPATITIS C VIRUS GENOTYPES IN INJECTING DRUG USERS FROM ROMANIA.

Due to the increasing number of infections related to injecting drug use, both the pattern of hepatitis C virus (HCV) transmission, and the circulating genotypes in Europe have changed. As there are little available data in this respect for Romania, the aim of our study was a preliminary analysis of the distribution of HCV genotypes circulating among injecting drug users (IDUs). Of the 45 IDUs evaluated (86.7% men, mean age - 27.6±3.7 years, mean age at first drug use - 17.5±3.9 years), 88.9% presented anti-HCV antibodies, with higher rates in those with an injecting history of more than 10 years; 57.8% of the subjects had detectable HCV viral load. Only 6.7% had markers of chronic hepatitis B infection, and none had anti-HIV antibodies. While HCV subtype 1b is still prevalent (in 50% of the viraemic subjects), other subtypes begin to emerge, especially in younger patients (1a - in 23.1%, 4 - in 11.5%, 3a - in 7.7% of the cases). These data indicate the possibility of major shifts in the distribution of the dominant subtype, underlining the need for close surveillance of HCV infections in IDUs, who can act as a bridging group toward the general population.