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Acute HCV infection was defined by a positive HCV RNA test preceded by a negative HCV RNA test in patients without evidence of past HCV infection.
Patients were either prospectively identified during acute infection or retrospectively by determining HCV RNA in anti-HCV-positive patients in stored sera from earlier time points.
The presence of new infections with a different genotype (genotype switch), or with a different strain from the same genotype (clade switch), was assessed by systematically sequencing the virus present during the entire viremic period.
The study population consisted of 85 HIV-infected MSM attending the HIV treatment clinic of the Academic Medical Center (AMC) in Amsterdam, the Netherlands, who acquired a primary HCV infection sexually between 19.
For the detection of new infections with the same genotype (i.e.
clade typing), a 590-bp fragment from the envelope, spanning positions 1295–1885 relative to the H77 strain, which includes the hyper-variable region 1 (designated ‘E2/HVR1’), was amplified and directly sequenced.
If a genotype switch was observed between the first and the last RNA-positive time points, samples taken between these two time points were genotyped to determine the interval of genotype switch.
For visual inspection of sequences, maximum likelihood trees were constructed for each genotype under a Hasegawa–Kishino–Yano evolutionary model with invariant sites and a gamma distribution of among-site rate heterogeneity (HKY I G) as implemented in Molecular Evolution Genetic Analysis version 5 .
Trees were unrooted and bootstrap values were determined from 1000 bootstrap resamplings of the original data.
The majority of patients were participants of the MSM Observational Study of Acute Infection with hepatitis C (MOSAIC) – a prospective cohort study on acute hepatitis C infection in HIV-infected MSM The presence of HCV RNA was assessed by either transcription-mediated amplification (VERSANT HCV RNA Qualitative Assay; Siemens Healthcare Diagnostics Inc., Tarrytown, New York, USA) or branched-chain DNA (VERSANT HCV RNA 3.0 Assay, Siemens).
For genotyping, a 389-base pair (bp) fragment spanning positions 8616–8275 relative to the H77-strain (AF009606) of the NS5B region was amplified and sequenced as described by Murphy .