Journal of Clinical Virology - Volume 82 - Supplement 1 - September 2016

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S15
between October to December 2015 was tested for both anti-HEV
IgM and HEV RNA. They were evaluated by EIA (recomWell HEV
IgM, version 2015, Mikrogen) and in case of anti-HEV IgM posi-
tive or equivocal results the samples were retested by immunodot
assay (recom Line HEV IgM, version 2015, Mikrogen). Nucleic acid
was extracted fromeach plasma sample (400 l) using Nucleic Acid
Isolation Kit I (MagNA Pure Compact system, Roche Diagnostics)
and HEV RNA was detected using an in house real-time RT-PCR
(Jothikumar and colleagues, 2006) and two commercial real-time
RT-PCR kits (ampliCube HEV 2.0, Mikrogen and RealStar HEV 1.0,
Altona).
Results: An overall HEV IgG seroprevalence in Portuguese popu-
lation of 16% was found with seropositivity increasing significantly
with age (p < 0.05). Differences between regions were also observed
ranging from 8% to 28%. From the total of 2115 blood donors,
7 (0.33%) were found positive for anti-HEV IgM. HEV RNA was
detected in 19 (0.90%) blood donors by the three real-time RT-PCR
used. These plasma samples were negative for both anti-HEV IgM
and IgG. HEV RNA concentration of plasma samples ranged from
2.0 × 104 to 8.4 ×101 IU/ml when calculated by a WHO/IS strain
standard curve (r2 = 0.998).
Conclusions: This study provides insight in the exposure of the
Portuguese general population to HEV and generates information
on risk profiles regarding demographic data (age and region of res-
idence). The prevalence of HEV silent infection in Portuguese blood
donors was 1.2% based on the presence of IgM and HEV RNA. This
high prevalence could be the due to the high volume of plasma used
for acid nucleic extraction and the screening of unpooled samples.
Abstract no: 322
Presentation at ESCV 2016: Oral 27
Assessment of the Illumina MiSeq massively
parallel sequencing platform for simultaneous
analysis of Hepatitis C virus resistance to all
direct-acting antivirals combination regimes
E. Martró ∗, V. Saludes , K. Salvatierra ,
G. Rech , L. Sumoy , A. Artacho ,
R.M. Morillas , M. Berenguer ,
F.X. López-Labrador
1 Microbiology Service, Germans Trias i Pujol
University Hospital, Germans Trias i Pujol Health
Sciences Research Institute (IGTP), Badalona, Spain
2 Consorcio de Investigación Biomédica en
Epidemiología y Salud Pública (CIBERESP), Instituto
de Salud Car, Spain
3 Virology Laboratory, Genomics and Health Area.
Fundación para el Fomento de la Investigación
Sanitaria y Biomédica de la Comunitat Valenciana
(FISABIO), Valencia, Spain
4 Bioinformatics and Genomics, Institute of
Predictive and Personalized Medicine of Cancer
(IMPPC), Can Ruti Campus, Badalona, Spain
5 Institut d’Investigació en Ciències de la Salut
Germans Trias i Pujol (IGTP), Can Ruti Campus,
Badalona, Spain
6 Genomics and Health Area, Fundación para el
Fomento de la Investigación Sanitaria y Biomédica de
la Comunitat Valenciana (FISABIO), Valencia, Spain
7 Liver Unit, Hospital Universitari Germans Trias i
Pujol, Badalona, Spain
8 Consorcio de Investigación Biomédica en
Enfermedades Hepáticas y Digestivas (CIBEREHD),
Instituto de Salud Carlos III, Madrid, Spain
9 Digestive Medicine, Liver transplantation Unit,
Hospital Universitari La Fe, Valencia, Spain
10 Consorcio de Investigación Biomédica de
Epidemiología y Salud Pública (CIBERESP), Spain
Background: Interferon-free direct-acting antivirals (DAA)
combinations regimens are highly effective for the treatment of
chronic hepatitis C virus (HCV) infection. However, DAA regimes are
often specific to a particular HCV genotype, and the re-treatment of
HCVpatientswith failure toDAAs remains challenging, as a result of
the emergence of resistance associated variants (RAVs). Resistance
testing prior to treatment was not previously recommended. How-
ever, new consensus guidelines are now incorporating resistance
testing for the management of DAA failures. From the available
next-generation sequencing (NGS) platforms, the Roche-454 (FLX
or Junior) are being discontinued in 2016, and there is a need for
new HCV resistance NGS assays.
Methods: We evaluated the Illumina MiSeq platform for the
simultaneous sequencing of the three HCV genes target of the cur-
rently approved and phase III DAA combinations: NS3(protease),
NS5A and NS5B. In a pilot, validation study, the three HCV genes
from 45 HCV-genotype 1b infected patients (DAA naïve) were
amplified by RT-PCR. The different amplicons corresponding to the
same patient were pooled equimolarly before library preparation.
Patient-specific indexed paired-end libraries were obtained with
the Nextera XT DNA Sample Preparation Kit and the Nextera Index
Kit. After normalization, all libraries were pooled and sequenced
in parallel using the MiSeq Reagent Kit v2 (300 cycle) in a MiSeq
system. To determine the error rates of the process, a plasmid with
a cloned HCV-NS3 protease was also processed in the run.