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Background People who inject drugs (PWID) are the most important risk group for incident Hepatitis C virus (HCV) infection. In PWID in Europe HCV genotype 3a is highly prevalent. Unfortunately, many of the recently developed directly acting antiviral drugs against HCV (DAAs) are suboptimal for treatment of this genotype. Detection of resistance-associated variants (RAV) in genotype 3a may help to optimize treatment decisions, however, robust protocols for amplification and sequencing of HCV NS5A as an important target for treatment of genotype 3a are currently lacking. Objectives The aim of this study was to establish a protocol for sequencing of HCV NS5A in genotype 3a and to determine the frequency of RAVs in treatment-naive PWID living in Germany. Study design The full NS5A region was amplified and sequenced from 110 HCV genotype 3a infected PWID using an in-house PCR protocol. Results With the established protocol the complete NS5A region was successfully amplified and sequenced from 110 out of 112 (98.2%) genotype 3a infected PWID. Phylogenetic analysis of sequences from PWID together with unrelated genotype 3a sequences from a public database showed a scattered distribution without geographic clustering. Viral polymorphisms A30K and Y93H known to confer resistance in a GT3a replication model were present in 8 subjects (7.2%). Conclusions A protocol for amplification of nearly all GT3a samples was successfully established. Substitutions conferring resistance to NS5A inhibitors were detected in a few treatment-naive PWID.

Background: In 1997 a new human virus, TTV, was identified. The clinical significance of the TTV infection, however, remains unknown. Objective: Establishment of the prevalence of TTV DNA in different population groups in Germany and the assessment of the possible clinical significance of TTV infection. Study design: Detection of the TTV DNA by PCR in blood donors, patients with end-stage liver disease, and multiple transfused patients with haemotological disorders. Results: TTV DNA was detected in 16 of 122 (13.1%) volunteer blood donors, in 34 of 77 (44.2%) patients with end-stage liver disease, and in 21 of 38 (55.3%) multiple transfused patients. There was no difference in the prevalence of the TTV DNA in end-stage liver disease patients with regard to sex, age, presence of HCV and HBV infection markers, and etiology of liver disease. Phylogenetic analysis of the amplified DNA fragments from 12 randomly selected TTV infected subjects demonstrated that in Germany at least two putative TTV genotypes and four subtypes are circulating. Conclusions: (i) TTV is widely spread in German population; (ii) one of the possible ways of its transmission is blood transfusion; (iii) TTV infection most probably does not generally lead to the development of the end-stage liver disease.