HIV Genotypischer Resistenzalgorithmus Deutschland Multicenter comparison of genotypic tropism testing: results from viral RNA and proviral DNA M. Obermeier 5/2012
Aims of the study Genotypic tropism testing from viral RNA is widely accepted as routine diagnostic method. It‘s usage is implemented by local and European guidelines. Although there is a huge interest in performing tropism testing in patients with low or undetectable viral load, data about tropism testing from proviral DNA ist still limited To better assess the usage of tropism testing from proviral DNA, we conducted a multicenter study to compare V3 loop sequence pairs from viral RNA and proviral DNA in routine diagnostic samples M. Obermeier 5/2012
HIV in Germany The HI V-GRADE laboratory network Labor Lademannbogen Rolf Kaiser 69500 HIV-pos. Hamburg Univ Cologne ~ 20% female Patrick Braun 2700 Infections/year Lab Fenner Lab Dr. Knechten Hamburg 700 deaths/year Aachen < 2% of newborns HIV-pos Martin Däumer Martin Obermeier Lab Dr. Thiele Lab Dr. Berg Kaiserslautern Berlin Alexander Thielen Hauke Walter MPI Informatik Univ Erlangen Eva Wolf Martin Stürmer Lab Jäger Univ. Frankfurt Munich Josef Eberle Univ Munich Ref.: RKI M. Obermeier 5/2012
Specification of the samples Sequencing from viral RNA and proviral DNA was performed in 210 samples Viral load range was between <50 copies/ml and 8.1 million cop./ml HIV1-Subtype was in most cases B (71%) M. Obermeier 5/2012
Subtype distribution of samples Subtype was determined using COMET tool (http://comet.retrovirology.lu/) M. Obermeier 5/2012
Coreceptor-tropism classification 2 Classification systems were used Geno2pheno [coreceptor] (http://www.geno2pheno.org) • Cut-offs for coreceptor tropism classification were used as implemented in the german guidelines • FPR <12.5% -> CXCR4 • FPR >20% -> CCR5 • FPR 12.5% – 20% -> equivocal WebPSSM ( http://indra.mullins.microbiol.washington.edu/) • Cut-offs as described in: Jensen, M.A. u. a. Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences. J. Virol 77 , 13376-13388 (2003). • FPR > -2.88 -> CXCR4 • FPR < -6.96 -> CCR5 • FPR -2.88 - -6.96 -> equivocal • Ambiguous positions in Sequences were resolved with using the highest potential score of any possible sequences M. Obermeier 5/2012
Comparison using geno2pheno (N=210) 86% M. Obermeier 5/2012
Comparison using WebPSSM (N=191) 82% M. Obermeier 5/2012
Range of FPR values for geno2pheno (all samples N=210) M. Obermeier 5/2012
Range of FPR values for geno2pheno (divergent samples N=29) M. Obermeier 5/2012
Range of scores for WebPSSM (all samples N=191) M. Obermeier 5/2012
Range of scores for WebPSSM (divergent samples N=33) p=0.04 { M. Obermeier 5/2012
Range of scores for WebPSSM (divergent samples N=33) PSSM score R5 viral proviral M. Obermeier 5/2012
Viral vs proviral geno2pheno WebPSSM M. Obermeier 5/2012
Conclusions: Genotypic tropism testing from viral RNA and proviral DNA shows a high level of concordance In routine diagnostics tropism testing can be performed from proviral DNA alone and can replace tropism testing from viral RNA to simplify routine diagnostic procedures Genotypic tropism testing from proviral DNA shows at least a trend towards an higher CXCR4 classification If performing genotypic tropism testing from proviral DNA cut-offs should be lowered compared to cut-offs in viral RNA M. Obermeier 5/2012
HIV-1 coreceptor tropism HIV-GRADE study – correlation to HIV-1 subtype M. Obermeier 5/2012
Aims Differences of coreceptor tropism for HIV-1 subtypes? Identification of polymorphisms responsible for subtype dependent coreceptor tropism differences M. Obermeier 5/2012
Methods V3-RT-PCR from plasma or proviral V3-DNA-PCR from EDTA-blood samples Sequencing of PCR amplicons HIV-1 subtyping by COMET tool Coreceptor tropism prediction by geno2pheno (FPR>10% => R5) CHi 2 test of absolute values and expected values expected values were calculated by the normalized mean of each subtype-specific R5-X4 ratio M. Obermeier 5/2012
Results – subtype distribution M. Obermeier 5/2012
Results – subtype tropism M. Obermeier 5/2012
Results – statistical analyses CHi 2 0.001 0.015 0.610 0.171 0.010 0.919 0.022 M. Obermeier 5/2012
Summary Difference of percentages for subtypes detected Normalized overall mean: 70.5% R5 vs. 29.5% X4 Higher rate of X4 tropism prediction for subtypes CRF01_AE and D Higher rate of R5 tropism prediction for subytpes A1 and G No differences for all other subtypes (B, C, F) M. Obermeier 5/2012
Discussion Higher rate of X4 prediction for subtype D as described before confirmed For subtype C R5 prediction rate similar to subtype B Contradictory publications available Limitations: no further laboratory parameters and no clinical data available Short sequence length may influence subtyping performance (use of additional subtyping tools) M. Obermeier 5/2012
HIV-GRADE association Thomas Berg, Medizinisches Labor Dr. Berg, Berlin Patrick Braun, PZB, Aachen Martin Däumer, Institut für Virologie, Köln Josef Eberle, Pettenkofer-Institut, München Robert Ehret, PZB Aachen Rolf Kaiser, Institut für Virologie, Köln Klaus Korn, NRZ für Retroviren, Erlangen Claudia Kücherer, Robert Koch Institut, Berlin Harm Müller,Labor Fenner, Hamburg Christian Noah, Labor Lademannbogen, Hamburg Martin Stürmer, Institut für Medizinische Virologie, Frankfurt Alexander Thielen, Max Planck Institut Saarbrücken Hauke Walter, NRZ für Retroviren, Erlangen
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