AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 24, Number 3, 2008 © Mary Ann Liebert, Inc. DOI: 10.1089/aid.2007.0135 Emergence of an NNRTI Resistance Mutation Y181C in an HIV-Infected NNRTI-Naive Patient EMMANOUIL MAGIORKINIS, 1 DIMITRIOS PARASKEVIS, 1 HELEN SAMBATAKOU, 2 PANAGIOTIS GARGALIANOS, 3 CATERINA HAIDA, 1 ALEXANDROS VASSILAKIS, 1 and ANGELOS HATZAKIS 1 ABSTRACT The purpose of our study was to examine the emergence of the Y181C resistance mutation in an NNRTI-naive subject (index patient) at different time points. Phylogenetic trees in protease (PR) and partial reverse tran- scriptase (RT) regions were inferred by the maximum likelihood (ML) method. The Y181C mutation was de- tected for the first time when the patient was receiving d4T  ddI  LPV/r; the previous drug combination was 3TC  AZT  IDV. The particular mutation (Y181C) was not present at any time point during the treat- ment period with 3TC  AZT  IDV. Moreover, there was no evidence of resistance mutations in RT before the initiation of antiretroviral therapy. Phylogenetic analysis including sequences from the index patient and his spouse sampled at different time points, as well as control sequences belonging to the same HIV-1 subtype, revealed that there is no evidence of coinfection or reinfection with Y181C resistance strains, while the virus for both subjects was classified as subtype CRF14_BG. Overall, our findings suggest that the Y181C resistance mutation may be selected, not only by NNRTIs, but also by d4T. This may be of particular significance in de- veloping countries where treatment with Triomune, a fixed combination of d4T, ddI, and nevirapine, is com- mon. The genetic barrier against resistance of this combination may be lower than previously thought. 413 H IV-1 DRUG RESISTANCE constitutes one of the major causes of antiretroviral therapy failure. Resistance is mediated by mutations emerging under the selective pressure of antiretrovi- ral drugs in the targeted genomic regions (reverse transcriptase, protease, etc.). The introduction of highly active antiretroviral therapy (HAART) led to the selection of multidrug-resistant isolates carrying complex mutational profiles. Resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs) is mediated by single mutations such as K103N and Y181C, 1 which confer broad class cross-resistance. Mutations at codons 100, 106, 108, 188, and 190 also confer different lev- els of resistance to this class. NNRTIs block viral cDNA elon- gation at a secondary or allosteric site instead of at the active sites utilized by the NRTIs. Our objective was to investigate the emergence of the Y181C mutation in an NNRTI-naive patient failing a d4T + ddI-based treatment, identified as a consequence of routine genotypic re- sistance testing. Genotypic resistance testing was performed in six samples collected from the index patient at different time points and in two samples of his treatment-naive spouse. HIV-RNA testing was performed by using the Versant HIV-1 RNA 3.0 assay (bDNA) (Diagnostics Division, Bayer Corp., Tarrytown, NY). Genotypic resistance testing was performed using the TrueGene HIV-1 Genotyping Test (Siemens Medical Solutions Diagnos- tics, NY) or ViroSeq (Celera Diagnostics/Abbott Laboratories, IL), according to the manufacturer’s recommendations. Virtual phenotype testing was performed using the VircoType HIV-1 (Virco, Mechelen, Belgium). HIV-1 subtype classification was performed by phylogenetic analysis using all previously char- acterized subtypes and CRFs (http://hiv-lanl.gov). In particular, phylogenetic trees were inferred by the maximum likelihood (ML) method, using the best fitted nucleotide substitution model (general time reversible including -distributed rates het- erogeneity among sites), as implemented in PAUP*. 4 To ex- clude the possibility of coinfection or reinfection of a resistant 1 Department of Hygiene and Epidemiology, University of Athens Medical School, GR-11527 Athens, Greece. 2 Hippokrateion General Hospital, Athens, Greece. 3 G. Genimatas General Hospital, Athens, Greece.