Sequencing Analysis in the Field of HIV Variability and Drug Resistance

Sequence analysis plays a crucial role in routine clinical practice, especially in the field of HIV-1 drug resistance. Resistance testing in HIV-1 infected patients is now recommended to guide the choice of antiretroviral therapy in clinical practice. In this regard, genotypic testing is the most common method used for detecting drug-resistant strains of HIV. It measures indirectly the drug resistance by detecting mutations in HIV genome known to be associated with a reduced susceptibility to antiviral drugs either in the laboratory and/or in clinical situations (http://www.iasusa.org/resistance_mutations; http://hivdb.stanford.edu). Various tools are now available to assist the provider in interpreting genotypic test results. Many clinicians currently use on a routine basis the genetic information on HIV-genotype to make treatment decisions for HIV-infected patients (both drug-naive or drug-experienced). Sequences obtained by genotypic assays can also be used to evaluate the HIV-1 genetic variability. Indeed, it should be considered that antiretroviral drug design, resistance research, and interpretation systems have been largely based on HIV-1 subtype B, which has historically been the most prevalent in North America, Western Europe, and Australia. However, subtype B viruses account for only about 12% of the worldwide HIV-1 infections. An increasing number of individuals who are infected with non-B subtype strains are now receiving antiretroviral therapy because of rollout programs in resource-limited world and because of increasing migration to the developed world, particularly to European countries. Therefore, HIV-1 subtype determination is essential to appreciate the effects of HIV-1 genetics on the characteristics of drug susceptibility and drug resistance. Finally, genotypic assays can be used for surveillance purposes both in developed and resource-constrained countries. Regarding these last settings, a careful evaluation of HIV drug resistance is needed since the number of antiretroviral drug-experienced patients is rapidly increasing. In light of previous considerations, in the present chapter book we reported the results obtained in two already published studies (Fokam et al., 2011; Ceccarelli et al., 2012). The HIV-1 genetic diversity and the prevalence of resistance among Cameroonian individuals were assessed trough the analysis of HIV-1 sequences. Sequences from 239 adults and 92 children (including both drug-naive and drug-experienced patients) were performed and analyzed. In particular, drug resistance mutations in HIV-1 pol region (corresponding to the entire protease and the first 280/300 amino acids of the reverse transcription open reading frame) were characterized; phylogenetic analysis was performed for subtype determination. Among 40 children failing first-line antiretroviral therapy, treatment response was also evaluated at weeks 24 and 48 after treatment, on the basis of genotypic results. The phylogenetic analysis revealed a great genetic diversity of HIV-1 viral strains circulating in Cameroon. The totality of the analyzed population was infected by non-B subtypes. In particular, CRF02_AG was the most common viral subtype (about 60% both in children and adults). Among drug-naive patients drug resistance was rather low (8.2% in adults and 4.9% in children), while a very high prevalence of resistance was observed in patients failing therapy (about 80% in adults and 90% in children). The high level of drug resistance observed in the analyzed cohort of treated patients is alarming, since it occurred as a result of few years of treatment. Adherence to therapy, adequate physicians’ education and the appropriate use of genotypic resistance testing are the critical points of intervention to improve patient’s care. For example, in the analysis performed in children starting a second-line regimen after failure, treatment change driven by genotypic resistance test was successful for most of them, highlighting the importance of choosing a therapy according to the genetic characteristics of the virus. Overall, these data well describe the role of HIV-1 genotypic resistance test in clinical practice. This testing has revolutionized the care of HIV infected patients and significantly advanced HIV research. Making use of this assay, physicians can closely monitor the emergence of drug resistance mutations and optimize the management of patients infected with drug resistant HIV. To date, important efforts are addressed to the improvement of these tests, in term of sensitivity, cost-effectiveness and detection of drug resistance in non-B subtypes. The introduction of new techniques for next generation sequencing (detecting also minority species representing 1% of viral population) and of new affordable algorithms to evaluate the importance of resistance patterns for non-subtype B strains is crucial to achieve this goal.