Multicenter comparison of genotypic tropism testing: results from viral RNA and proviral DNA

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1 HIV Genotypischer Resistenzalgorithmus Deutschland Multicenter comparison of genotypic tropism testing: results from viral RNA and proviral DNA

2 Financial disclosure Study was financially supported by ViiV Healthcare Germany

3 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

4 Patrick Braun Lab Dr. Knechten Aachen Martin Däumer Lab Dr. Thiele Kaiserslautern Alexander Thielen MPI Informatik Rolf Kaiser Univ Cologne HIV in Germany The HIV-GRADE laboratory network Labor Lademannbogen HIV-pos. Hamburg ~20% female 2700 Infections/year Lab Fenner 700 deaths/year Hamburg <2% of newborns HIV-pos Martin Obermeier Lab Dr. Berg Berlin Hauke Walter Univ Erlangen Martin Stürmer Univ. Frankfurt Eva Wolf Lab Jäger Munich Ref.: RKI Josef Eberle Univ Munich

5 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%)

6 Subtype distribution of samples Subtype was determined using COMET tool (

7 Coreceptor-tropism classification 2 Classification systems were used Geno2pheno [coreceptor] ( 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 ( 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, (2003). FPR > > CXCR4 FPR < > CCR5 FPR > equivocal Ambiguous positions in Sequences were resolved with using the highest potential score of any possible sequences

8 Comparison using geno2pheno (N=210) geno2pheno viral CCR5 CXCR4 equivocal proviral CCR CXCR equivocal %

9 Comparison using WebPSSM (N=191) WebPSSM viral CCR5 CXCR4 equivocal proviral CCR CXCR equivocal %

10 Range of FPR values for geno2pheno (all samples N=210) R5

11 Range of FPR values for geno2pheno (divergent samples N=29) R5

12 Range of scores for WebPSSM (all samples N=191) R5

13 Range of scores for WebPSSM (divergent samples N=33) p=0.04 R5

14 Range of scores for WebPSSM (divergent samples N=33) R5 PSSM score viral proviral

15 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

16 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

Deep Sequencing Detects V3 loop Forms Present in Functional X4 Viruses Growing in MT 2 assays

Deep Sequencing Detects V3 loop Forms Present in Functional X4 Viruses Growing in MT 2 assays Christian Pou 1, Rocío Bellido 1, Francisco M. Codoñer 1, Alexander Thielen 3, Cecilia Cabrera 1, Judith Dalmau