Using this background knowledge, the study aims at re- evaluating broadly applied clinical definitions of disease progres- sion: LTNP elite controllers (LTNP-EC), viremic controllers (LTNP-VC), viremic non controllers (LTNP-NC), chronic pro- gressors (P) and rapid progressors (RP) under the information provided by a selected number of viral and host genetic characteristics. Importantly, the study is not a de-novo genetic or viral study, rather the combined application of well established knowledge that should result in a clear readout in small sets of individuals. The analysis emphasizes, in particular, the character- istics of the least investigated group of individuals, the rapid progressors. Materials and Methods Ethics statement The study was approved by the Comite de Etica del Centro Sanitario Sandoval, C/Sandoval 7, Madrid 28010 and by the University of Lausanne, Faculty of Medicine Commission d’ Ethique de la Recherche Clinique rue du Bugnon 21, 1005 Lausanne. Study subjects We included 64 treatment na¨ıve individuals, from the Centro Sanitario Sandoval (IMSALUD, Madrid, Spain), and from the Swiss HIV Cohort Study (www.shcs.ch) that fulfilled criteria for the various clinical progression definitions (Table 1). In the absence of standardized definition of the various clinical progression profiles, we based strict definitions on published literature [1,2,13,14], and on their broad clinical use. LTNPs patients, including elite and viremic controllers, from Spain met the strictest definition of more than 10 years of infection and undetectable or low viremia. Participants gave informed genetic consent for the study (which was oral and general for different type of studies for participants with a long term follow up in the Centro Sanitario Sandoval), and the study and the consents was approved by the Committees of the two Centers. Seroconversion date was estimated from either a documented negative test, less than two years before the first documented positive test, or biological criteria of primary infection: incomplete western blot and/or Table 1. Definitions of the five clinical progression groups. HIV-1 Progression Definitions positive p24 Ag and/or high viremia (.1 million copies per milliliter of blood). Seroconversion date was defined as the mid- point between the two dates. These individuals have not been included in previous genetic studies, with the exception of 10 chronic progressors [3]. Host genetic characterization Host genetic variants were chosen on the basis of genome-wide association studies [3,4], or selected from the literature according to the quality of their supporting evidence (www.hiv-pharmaco- genomics.org). These included HLA-B alleles associated with protection or progressive disease, the HCP5 rs2395029 allele in linkage disequilibrium with HLA-B*5701, the HLA-C-35 (rs9264942) variant, ZNRD1 rs9261174 and HLA-A10 serogroup alleles in linkage disequilibrium, CCR5 D32 (rs333), CCR2 V64I (rs1799864), CCR5 haplotypes, and copy number variation of CCL3L1. CCR5 haplotypes were constructed according to the published nomenclature [15]; considering 8 polymorphisms in the CCR5/CCR2 promoter and coding region (rs2856758, rs2734648, rs1799987, rs1799988, rs1800023, rs1800024, rs333, rs1799864); CCR5_P1 haplotype is carried by HHE, HHG*1 and HHF*1 haplotypes. HLA typing was done by sequencing, and SNP (single nucleotide polymorphism) analysis was done by TaqMan. Additive unweighted genetic scores were used to compile genetic informa- tion [16]. In this model the impact of each allele is assumed to be the same except for the sign (i.e. alleles with a protective effect were added, and risk alleles were subtracted. A simplified score was applied: CCR5 D32 [score 0,1], CCR2 V64I [0,1,2], CCR5 P1 homozygous [0,22], HLA-C–35 [0,1,2], and protective HLA-B+ [0,1,2] and detrimental HLA-B- alleles [0,21,22]). This simplified procedure was also applied to estimate allelic frequencies for groups (e.g. protective HLA B alleles). Viral load and DNA quantification, PCR amplification and nucleotide sequencing Plasma HIV-1 RNA viral load (VL) was quantified with the Branched DNA Siemens versant HIV RNA 3.0 assay (bDNA), or the Roche Amplicor with a detection limit of 75 and 50 copies/ml respectively. Viral set point was defined as the average of viral load LTNP-EC LTNP-VC LTNP-NC P RP N Asymptomatic HIV Infection over 10 year after seroconversion N Plasma HIV RNA levels without ART that are below the level of detection for the respective assay (e.g., ,75 copies/mL by bDNA or ,50 by ultrasensitive PCR). N Isolated episodes of viremia up to 1000 copies/mL as long as they are not consecutive and represent the minority of all available determinations. N Longitudinal HIV RNA that includes a minimum of 3 determinations, in the absence of antiretroviral agents, which span at least a 12-month period. N Asymptomatic HIV Infection over 10 year after seroconversion. N Plasma HIV RNA levels without ART that are equal or below 2000 copies/mL. N Isolated episodes of viremia above 2000 copies/mL as long as such episodes represent the minority of all available determinations. N Longitudinal HIV RNA that includes a minimum of 3 determinations, in the absence of ART, which span at least a 12-month period. N Asymptomatic HIV Infection over 10 year after seroconversion N Plasma HIV RNA levels above 2.000 copies/mL without ART, in more than 50% of the samples. N Symptomatic infection or initiation of ART within 10 years after seroconversion N Longitudinal HIV RNA that includes a minimum of 3 determinations, in the absence of ART, with a viral set point above 2000 copies/mL N $2 CD4 T cell measurements below 350/mm3 within 3 years after seroconversion, with no value $350 afterwards in the absence of ART. N And/or, ART initiated within 3 years after seroconversion, and at least one preceding CD4 , 350/mm3. N And/or, AIDS or AIDS-related Death within 3 years after seroconversion and at least one preceding CD4,350/mm3. LTNP-EC: long term non-progressor, elite controllers; LTNP-VC: long term non-progressor, viremic controllers; LTNP-NC: long term non-progressor, viremic non controllers; P: chronic progressors, RP: rapid progressors, ART: antiretroviral therapy. Clinical groups summarize different definitions from the literature [1,2,13,14]. doi:10.1371/journal.pone.0011079.t001 PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11079