Relapse or reinfection after failing hepatitis C direct acting antiviral treatment: Unravelled by phylogenetic analysis

Abstract

Despite high response rates associated to hepatitis C virus (HCV) treatment, no protective immunity is acquired, allowing for reinfection and continued infectiousness. Distinguishing between relapse and reinfection is crucial for patient counselling and to choose the most appropriate retreatment. Here, refined phylogenetic analysis using multiple genes served to assess genotype and reinfection for 53 patients for whom the virus was sampled before start of therapy and at time of sustained virological response evaluation at week 12. At baseline, genotypes were determined as HCV1a (41.5%), HCV1b (24.5%), HCV4 (18.9%) and HCV3a (15.1%), while six cases revealed to be discordantly assigned by phylogeny and commercial assays. Overall, 60.4% was co-infected with HIV. The large majority was classified as people who inject drugs (78.6%), often co-infected with HIV. Transmission was sexual in seven cases, of which five in HIV-positive men-who-have-sex-with-men. Overall, relapse was defined for 44 patients, while no conclusion was drawn for four patients. Five patients were reinfected with a different HCV strain, of which three with a different genotype, showing that phylogeny is needed not only to determine the genotype, but also to distinguish between relapse and intra-subtype reinfection. Of note, phylogenies are more reliable when longer fragments of the viral genome are being sequenced.

Fig 1. Maximum-likelihood tree of the concatenated alignment covering genetic regions NS5A and NS5B.

Both the entire phylogenetic tree (left) and the HCV1b clade in detail (right) are visualized. Patient 18, coloured in green, is a clear example of reinfection with a different HCV strain, since although both viruses at baseline and at time of SVR12 evaluation are classified as HCV1b, they cluster in a different clade in the tree, with a bootstrap support of 100% for the segregation of the different clades. For patient 4, coloured in blue, both strains cluster together with a high bootstrap support (100%), suggesting that this patient experienced a virological relapse. Bootstrap replicates are only visualized for patients who experienced a relapse, all indicated in blue. Patients for which only a baseline or SVR12 evaluation sequence is included in the NS5A-NS5B alignment, are coloured in red. The sequence at time of SVR12 evaluation and baseline for patients 7 and 49 respectively, cluster outside the large HCV1b clade, which might be due to a potential event of recombination. However, detailed analyses with TreePuzzle and RDP4 for all 53 patients, could not support a recombination event. The in-depth Simplot analysis for patients 7 and 49 showed that both strains were classified as HCV1a. Despite the absence of evidence of recombination, we did not draw any conclusions concerning the occurrence of reinfection or relapse for patient 7. The bar at the bottom represents the number of nucleotide substitutions per site.

Fig 2. Maximum-likelihood tree of the concatenated alignment covering genetic regions NS3 and NS5A.

Both the entire phylogenetic tree and the HCV4 (4a and 4d) clade in detail are visualized. Clusters of patients coloured in blue, are suggested to have experienced a virological relapse, supported by bootstrap values >70%. However, for patient 6, indicated in red, only a bootstrap value of 38 was obtained, resulting in lack of evidence to distinguish between a relapse and reinfection. For patient 39, the fragment NS3-NS5A was only sequenced for the sample at time of SVR12 evaluation. The bar at the bottom represents the number of nucleotide substitutions per site.