Journal of Clinical Virology - Volume 82 - Supplement 1

S44

Abstracts / Journal of Clinical Virology 82S (2016) S1–S142

Results:

IDS iSYs VCA IgM: Sens. during PrI = 97.5%

(95%CI:92.6–99.4).

Spe.

during AI

and PastI = 97,4%

(95%CI:92.6–99). Spec with interfering sera from other viral

primary infections =88.1% (95%CI:79.2–93.5).

IDS iSYS VCA IgG: Sens. during PrI and PastI = 71.2%

(95%CI:62.3–78.7) and 98.2 (95%IC 95.4–99.3) respectively.

Spe. during AI = 94.4% (95% CI:92.2–97.6)

IDS iSYS EBNA IgG: Sens. during PastI = 97.3 (95%CI 93.6–98.4).

Spe. during AI and PrI = 98.9 (95%CI:93.9–99.8) and 90.1% (95%CI:

83.4–94.4) respectively.

Agreement for the determination of EBV status between IDS iSYS

system and reference test = 84. 4 (95%CI:80.6–87.6).

Conclusion:

The IDS- iSYS is a real “Walk-Away System”, easy to

use, fast and secure and appears to offer new reliable commercial

immunoassays for the determination of EBV serological status.

http://dx.doi.org/10.1016/j.jcv.2016.08.085

Abstract no: 335

Presentation at ESCV 2016: Poster 46

Evaluation of process control “Mengo Virus”

using 3 RNA extraction kits and 2 different

types of methods of one-step real-time RT-PCR

in Donax sp (Palabritas)

Pablo Londone-Bailon

Instituto Tecnologico de la Produccion, Peru

There are many problems to extract viral genetic material that

is contaminating bivalve molluscs, this is due to bivalve molluscs,

specifically the hepatopancreas, have many inhibitors to PCR, for

that reason extraction methods should consider a virus process

control used to measure the efficiency of extraction. In the mar-

ket there are many commercial kits for extracting the nucleic acid

of the virus and to performOne-step real-time RT-PCR, but most are

not tested on bivalve mollusks, for this reason, the aim to evaluate

the efficiency of extraction process control (Mengo virus) using 3

different RNA extraction kits and 2 treatments of One-Step real time

RT-PCR. They were used to study 30 samples of hepatopancreas of

Donax sp.

(Palabritas) to which was added 10 l of Mengo virus at a

concentration of 1.6

particles/ l, and processing of the sam-

ple according to the ISO/TS 15216-2:2013, then RNA was extracted

of each sample with the kits: 1. BioMerieux NucliSENS

system

(bioMerieux SA, France), 2. PureLink

RNA Mini Kit (Ambion-

Life Technologies

, USA) and 3. Hugh Pure RNA Tissue Kit (Roche

SA, Germany). Once extracted RNA was performed one-step real-

time RT-PCR using the following treatments: 1. the UltraSense

One-step qRT-PCR (Invitrogen, USA) kit according to the ISO/TS

15216-2:2013 was used and 2. Kit Mengovirus@ceeramTools

(CEERAM, France) according to the manufacturer’s specifications

was used. It turned out that the measuring efficiency of the extrac-

tion process control (Mengo virus) the best method of extraction

was BioMerieux NucliSENS

system with an efficiency 10 times

greater than the second; and with respect to matters related to Kits

One-step real-time RT-PCR it can be concluded that treatment 1

kit UltraSense One-step qRT-PCR has an efficiency of 32% over the

Mengovirus@ceeramTools

Kit.

http://dx.doi.org/10.1016/j.jcv.2016.08.086

Abstract no: 337

Presentation at ESCV 2016: Poster 47

Ion Torrent next generation sequencing for

accurate genotyping and detection of resistance

associated variants in HCV and HIV

Elian Rakhmanaliev

1 ,

∗

, Pramila Ariyaratne

Charlie Lee

, Pornpimon Nimitsuntiwong

Chortip Wathtphan

, Ekawat Passomsub

Kok Siong Poon

, Cui Wen Chua

, Mui Joo Khoo

Zhang Rui

, Wen Huang

, Evelyn S. Koay

Wasun Chantratita

, Gerd Michel

Vela Research Ltd., Singapore

Department of Pathology, Faculty of Medicine,

Ramathibodi Hospital Mahidol University, Bangkok,

Thailand

Molecular Diagnosis Centre, Department of

Laboratory Medicine, National University Hospital,

Singapore

Background:

Detection of resistance-associated mutations is

well established in HIV ART (as DRMs) and is increasingly used

in HCV patients selected for treatment (as RAVs) with direct

acting antiviral agents (DAAs). Both for DAA treatment and con-

ventional interferon-based therapy accurate determination of HCV

genotypes (GTs) is essential. Sanger sequencing has recognized lim-

itations in sensitivity and turn around time. NGS provides excellent

accuracy, speed and sensitivity enabling detection of rare mutants,

HCV subtypes as well as mixed infections.

Objectives:

To develop improved detection of clinically rele-

vant viral mutations using ion torrent based NGS in an automated

workflow.

Materials and methods

: We have used NGS in combination

with workflow automation on a newly developed platform based

on the emotion 5075 system (Eppendorf, Germany) consisting of a

continuous robotic process starting with sample extraction and RT-

PCR followed by automated library preparation, Ion Torrent deep

sequencing and direct online data analysis to determine HCV geno-

types and RAVs as well as DRMs in HIV. We have employed target

sequences from the HCV NS3, NS5A and NS5B regions. For HIV

sequences in reverse transcriptase, protease and integrase were

selected for NGS.

Results

: We are reporting results from an evaluation study con-

ducted on >200 HCV sera comparing HCV genotyping with line

probe assay. Two cases of mixed GT infections were detected. Con-

firmation of discrepant results between NGS and line probing by

Sanger sequencing indicated 100% accurate GTs by NGS whereas in

several cases line probe results would have led to selection of sub-

optimal therapy regimens. In an HIV pilot study (

= 112 patients),

comparing NGS results to TruGene sequencing the

Sentosa

SQ HIV

Genotyping Assay detected 100% (199/199) of all mutations in

the protease gene and more that 98% mutations (427/435) in the

reverse transcriptase gene.

Conclusions:

Given the crucial role of accurate sequencing anal-

ysis in HCV and HIV treatment management, workflow automated

NGS appears as a highly reliable tool for differentiating HCV GTs

and RAVs, which can help to prevent diagnostic errors potentially

leading to suboptimal treatment.

Considering the pivotal role of DRMs in HIV patients under

HAART the

Sentosa

SQ HIV Genotyping workflow appears as a valu-

able new tool for detecting clinically relevant HIV variants. Given

its high sensitivity compared to Sanger based systems and the