Author(s):
Videira e Castro, Sandra 
; Sousa, Carina ; Pádua, Elizabeth ; Esteves, Aida ; Parreira, Ricardo ; Piedade, João
Date: 2013
Persistent ID: http://hdl.handle.net/10400.18/1588
Origin: Repositório Científico do Instituto Nacional de Saúde
Subject(s): HIV-1; Injecting Drug Users; Antiretroviral Drug Resistance; Enzymatic Inhibitors; Maroviroc; Infecções Sexualmente Transmissíveis
Description
Genotypic resistance profiles to antiretroviral drugs in HIV-1 circulating among injecting drug users in the Greater Lisbon
Sandra Videira e Castro1; Carina Sousa1; Elizabeth Pádua3; Aida Esteves1,2; Ricardo Parreira1,2; João Piedade1,2
1Grupo de Virologia, UEI de Microbiologia Médica; 2Unidade de Parasitologia e Microbiologia Médicas (UPMM)
Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa
Rua da Junqueira nº100, 1349-008 Lisboa, Portugal
3Laboratório de Referência da Sida, Instituto Nacional de Saúde Dr. Ricardo Jorge
Avenida Padre Cruz, 1649-016 Lisboa, Portugal
Background and Objectives
The introduction of highly active antiretroviral therapy has significantly improved the ability to maintain human immunodeficiency virus type 1 (HIV-1) replication to very low levels for an extended period of time, providing a better quality of life to infected patients.
The acquisition and transmission of HIV-1 drug resistance mutations is a major public health concern, so the search for better antiretroviral (ARV) therapeutic agents is a constant challenge. In recent years, in an effort to target other functions at the virus replication cycle beyond those carried out by the viral enzymes protease (PR), reverse transcriptase (RT) and integrase (IN), some novel agents have emerged, e.g. CCR5 coreceptor antagonists, as maraviroc (MVC), the first directed to a cellular target. Nevertheless, the discovery and licensing of new ARVs is followed by descriptions of the rapid emergence of resistant and multidrug-resistant viral variants (Hartman and Buckheit, 2012). Therefore, resistance information is useful in the clinical setting, as it can guide the choice of ARVs in therapy, also providing a repository of data on the extent of circulation of resistant viral strains (Vandamme et al., 2011).
Monitoring of HIV-1 resistance to ARVs can be performed with genotypic tests. These assays are based on the direct nucleotide sequencing of target genes and the identification of mutations associated to resistance (and of other genetic polymorphisms) is made through the use of algorithms that allow the reporting of associations of mutation patterns with different levels of susceptibility to ARVs. Thus, the aims of this study were:
• to identify PR, RT and IN mutations associated to different levels of resistance to currently licensed enzymatic inhibitors, in HIV-1 strains circulating among injecting drug users (IDUs) from the Greater Lisbon;
• to describe, in the same population, natural genetic polymorphisms putatively associated with reduced susceptibility to MVC.
Results and Discussion
RNA extracted from plasma taken from 61 HIV-1 seropositive IDUs, collected from 1998 to 2009, was amplified by RT-nested PCR using the Titan One Tube RT-PCR kit (Roche Applied Science, Germany), followed by the illustra™ puReTaq Ready-To-Go PCR Beads (GE Healthcare, England) according to the manufacturers’ instructions. HIV-1 DNA sequences for PR (n=41), RT (n=41), IN (n=41) of pol gene and C2V3C3-gp120 (n=35) of env gene were generated by sequencing, in both directions, of PCR products or plasmid clones. PR, RT, IN and C2V3C3 nucleotide sequences were edited with BioEdit v.7.0.9.0 (Hall, 1999) to originate fragments of 297, 598, 813 and 516 bp, respectively.
The analysis of mutations associated with decreased susceptibility to currently licensed ARV enzymatic inhibitors was performed using the HIVdb program, implemented through the Genotypic Resistance Interpretation Algorithm from the Stanford University HIV Drug Resistance Database (at http://hivdb.stanford.edu).
The analysis for PR, RT, IN coding regions revealed a total of 15 resistance associated mutations, present in 50.0% (22/44) of the subjects, with a distribution of 1-3 mutations/sequence (4 minor mutations in PR; 3 high-level and one low-level resistance mutations and 2 polymorphisms in RT; 5 mutations in IN, one major and 4 minor). Half of the subjects infected with these strains (11/22) were drug-naïve, showing transmission of resistance mutations between infected individuals. However, only 26.7% (4/15) of these mutations confer a putative phenotypic profile of resistance and just to one class of inhibitors (RT or IN), being found in 9.1% (4/44) of the subjects.
Conversely, the mutations associated with resistance to MVC were analyzed after translation of the C2V3C3 nucleotide sequences and subsequent comparison to a subtype B consensus. Based on published data, we observed a total of 12 different single mutations, as well as two mutational patterns, in the V3 loop, present in all the sequences studied, at variable numbers. The presence of the patterns 11S+26V and 20F+25D+26V, in 3 sequences, is of particular interest, since these patterns have unequivocally been associated with MVC resistance in vivo. The presence of these mutations in MVC-naïve patients is certainly the result of the high variability of theV3 region. Their impact at MVC treatment baseline is still unknown, but may have important clinical implications.
Conclusions
Overall, a very high frequency of genetic polymorphisms was found, both for pol (PR, RT, IN) and env (C2V3C3) coding regions. Although the majority of the polymorphisms described has only a marginal importance on ARV resistance, they may play a role on viral fitness and/or the evolution of resistance, once drug pressure is applied. On the other hand, even though a resistance profile for MVC is not defined yet, the presence of these mutations in MVC-naïve populations may have a significant impact in their clinical management in the future, especially considering the introduction of this drug in salvage therapy. Finally, the presence of resistance associated mutations in drug-naïve individuals shows that the transmission of these mutations is ongoing among IDUs in the Greater Lisbon area.
The monitoring of ARV resistance is a fundamental issue to the control of the HIV/AIDS epidemic. The results reported will surely be of value for ARV therapy implementation and monitoring of infection in this IDU population, having also an impact on the clinical management of HIV-1 drug-experienced individuals, particularly in those who have experienced previous virologic failures.
References
Hartman, Tracy and Buckheit Jr., Robert. The continuing evolution of HIV-1 therapy: identification and development of novel antiretroviral agents targeting viral and cellular targets. Molecular Biology International, v.2012, 401965 (2012).
Vandamme, Anne-Mieke, et al. European recommendations for the clinical use of HIV drug resistance testing: 2011 update. AIDS Reviews, v.13, p.77-108. (2011).
Hall, Thomas. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, v.41, p.95-98. (1999).
