Monitoring the emergence of resistance mutations in patients under salvage therapy with Raltegravir in Rio de Janeiro, Brazil: a six month follow-up Caroline Passaes 1, Monick Guimarães 1, Sandra Wagner 2, Valdiléa Veloso 2, Beatriz Grinsztejn 2, Mariza Morgado Laboratório de AIDS e Imunologia Molecular – IOC – FIOCRUZ, Rio de Janeiro, Brazil. 2 - Instituto de Pesquisa Clínica Evandro Chagas – IPEC – FIOCRUZ, Rio de Janeiro, Brazil. Brazilian Ministry of Health Oswaldo Cruz Foundation Oswaldo Cruz Institute

Background Raltegravir (RAL) was approved for salvage therapy in Brazil in Since Raltegravir was approved, several efforts have been made in order to describe the emergence of resistance mutations in patients taking HAART under a Raltegravir-containing regimen. N155H + L74M, E92Q, T97A, V151I, and/or G163R; Q148K/R/H + E138K/A and/or G140S/A; Y143R/C + L74A/I, E92Q, T97A, I203M, and/or S230R. Resistance pathways:

Question How long will it take to the integrase resistance mutations emerge in multi- experienced patients under RAL-containing regimens?

Methods Samples: The Evandro Chagas Clinical Research Institute (IPEC) is the clinical Unit of the Oswaldo Cruz Foundation (FIOCRUZ) that is responsible for the treatment and follow-up of more than 2000 HIV positive patients in the Rio de Janeiro State, Brazil. Prospective and longitudinal study. Study design: Baseline Start treatment with RAL 2 months 12 months 4 months 6 months 9 months CD4 (FACS Calibur Cytometer) Viral load (bDNA) Genotyping of HIV-1 integrase (home brew method developed in our laboratory)

Methods Samples: Four patients receiving a Raltegravir-containing regimen (HAART) at IPEC, Rio de Janeiro, Brazil 6 months follow-up. All patients have more than 10 years of infection and ARV use. All of them have used at least 3 classes of ARVs (NRTI, NNRTI and PI).

Results HIV+ since 1995 Previous therapeutic scheme: 3TC/DDI/TDF/LPV/r Actual therapeutic scheme: 3TC/TDF/DRV/r/Raltegravir Subtype B HIV+ since 1993 Previous therapeutic scheme: 3TC/TDF/DRV/r/T20 Actual therapeutic scheme: 3TC/TDF/DRV/r/Raltegravir Subtype F Sustained Virologic Response

Results HIV+ since 1986 Previous therapeutic scheme: AZT/3TC/TDF/DRV/r/T20 Actual therapeutic scheme: AZT/3TC/ TDF/DRV/r/Raltegravir Subtype B HIV+ since 1991 Previous therapeutic scheme: 3TC/TDF/DRV/r/ETR Actual therapeutic scheme: 3TC/TDF/fAPV/r/Raltegravir Subtype B Virologic Failure HS Q148H/G140S

Questions Is it possible to detect the integrase resistance mutations in the proviral compartment before its detection in the plasma samples? Can this approach be used as a predictor of the emergence of integrase resistance mutations?

Methods Samples: Provirus: Amplification by PCR (limit dilution) Baseline Start treatment with RAL 2 months 12 months 4 months 6 months 9 months 10 clones for each visit of all patients 150 sequences analyzed

Results Major resistance mutations were detected in proviral clones only in that sample presenting major resistance mutations in the plasma. However, these mutations were detected in the provirus after their emergence in the plasma. Some minor resistance mutations were observed in the DNA analysis: L74M; Q146H; Q95K; E138Q; M154I; G163R; S230N and R263K. G140S Q148H

Conclusions In spite of being a good option for the clinical management of patients presenting highly resistance to multiple antiretroviral drugs, in our study 2 out of 4 individuals showed early virologic failure to a RAL-containing regimen. For one of these cases integrase resistance mutations could be detected. Similar cases of early failure to RAL have been recently described (Baldanti et al., J Med Virol, 2010; da Silva et al., J Antimicrob Chemother, 2010; Tommasi et al., Scand J Infect Dis, 2010). The DNA provirus analysis did not antecipate the emergence of integrase resistance mutations. Close follow-up of multi-drug experienced patients should be warranted.

Acknowledgments AIDS 2010 Organizers International Scholarship Program/Sidaction Laboratory of AIDS and Molecular Immunology team – IOC/FIOCRUZ Evandro Chagas Clinical Research Institute team – IPEC/FIOCRUZ All patients enrolled in the study PAPES V/FIOCRUZ, CNPq and FAPERJ grants