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

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S37
based in vitro diagnostic test intended for qualitative detection of
Zika virus RNA in clinical specimens (K 2 EDTA plasma, serum and
urine) from symptomatic individuals and/or individuals who have
suspected exposure to ZIKV based on epidemiological criteria. The
assay workflow utilizes seamless automation from viral nucleic
acid extraction (up to 24 tests) to PCR setup, followed by detec-
tion on the ABI 7500 Fast Dx system. Thus, minimizing hands-on
time and human error while enabling maximum throughput with
high precision.
The Sentosa® SA ZIKV RT-PCR Test contains reagents for reverse
transcription and specific amplification of a 103 base pair (bp) frag-
ment of the NS4A gene within the open reading frame (ORF) of the
ZIKV. Additionally, the assay has a built-in positive control that acts
as an external control to monitor workflow operation, a negative
control to detectworkflowcontamination and an extraction control
to confirm validity of the extraction process.
Methods: ZIKV positive samples simulated by spiking ZIKV into
EDTA plasma, serum and urine were used for establishing the per-
formance characteristics of the assay. Sentosa ® SX101 is a liquid
handling instrument that automates the extraction of samples and
controls using the Sentosa ® SX Virus Total Nucleic Acid Kit and PCR
set-up using the Sentosa® SA ZIKV RT-PCR Test. ABI 7500 Fast Dx
was used for PCR detection of ZIKV.
Results: The analytical limit of detection (LoD) resulting in >95%
detection rate assessed by the workflow for Sentosa® SA ZIKV RT-
PCR Test was determined as 6 copies/ L of viral concentration in K2
EDTA plasma, serum and urine. The assay is reactive to both African
and Asian strains of ZIKV, MR-766 and PRVABC59. The oligonu-
cleotide sequences were aligned to 82 ZIKV sequences from NCBI
using BLAST command line and revealed 100% and >95% alignment
for the primers and probe, respectively. Furthermore, the assay
showed no cross-reactivity to closely related flaviviruses and other
viruses causing similar febrile illnesses based on wet-testing and in
silico analysis, which would predict no potential false positive RT-
PCR results. The workflow was stressed by extraction of a mixture
of high positive (10,000× LoD) and negative samples to determine
the chance of cross-contamination. Our data showed that the con-
tamination rate of the workflow is 0% within and between runs.
The precision of the workflow was assessed with consideration of
assay lots, operators, instruments and day-to-day variability. The
detection of controls and low positive samples (1.5× LoD) is highly
reproducible, achieving a CV of <5% and 100% agreement.
Conclusion: We have developed a robust, minimal hands-on,
high throughput and workflow-automated ZIKV NAT-based assay,
which can contribute significantly to the diagnosis of ZIKV-infected
patients.
Abstract no: 246
Presentation at ESCV 2016: Poster 33
Comparative performance of new Aptima HCV
Quant Dx assay with Abbott HCV Real-Time
assay
Anna Rosa Garbuglia ∗, Angela Bibbò,
Roberta Sciamanna, Marina Pisciotta,
Maria Rosaria Capobianchi
Laboratory of Virology, “Lazzaro Spallanzani”
National Institute for Infectious Diseases, IRCCS,
Rome, Italy
Background: It is estimated that chronic hepatitis C virus (HCV)
infection affects up to 170million people and there were 350,000
deaths due to HCV-related liver disease each year. The introduction
of newdirect-acting antiviral (DAA) therapies represents a relevant
promise to eradicate the HCV in patients who cannot be cured with
PEGilated interferon/ribavirin treatment. The use of newDAA poses
new challenges for clinicians, who must accurately interpret HCV-
RNA. Thus molecular HCV assay accuracy play a relevant role in the
correct management of patients under therapy.
Objective: In this study, we compare the concordance of
absolute quantification of ABBOTT HCV RealTime assay (ABBOTT
HCV-Abbott HCV, LOQ = 12 IU/ml) and Aptima HCV Quant Dx assay
(Aptima HCV-Aptima HCV, LOQ = 10 IU/ml) by using diluted HCV
WHO standard samples as well as by testing clinical samples of
chronically HCV-infected patients attending INMI L. Spallanzani
IRCCS Hospital.
Materials and methods: Serial dilutions were prepared from
5th WHO standard (NIBSC code 14/150, genotype 1a) to achieve
nominal concentration corresponding to 2000, 1000, 500, 250, 125,
50, 25,12.5, 6.25 IU/ml. Ten replicates (2000; 1000;500; 250; 125;
12.5; 6.25 IU/ml conc.) or 20 replicates (50; 25 IU/ml conc.) were
tested. % CV values based on long-transformed results were calcu-
lated for each dilution and both platform. In the second approach,
117 prospective and 178 retrospective clinical samples, were tested
by side-by side in the two assays.
Results: A good correlation were observed between the
expected and observed results obtained both with Aptima HCV
(r= 0.998) and Abbott HCV (r = 0.9011). Abbott HCV showed a lower
% CV in comparison to Aptima HCV at 2000 (0.84 vs 2.37); 125 (4.08
vs 7.15), at 12.5 IU/ml (5.30 vs 7.36), at 50 IU/ml (3.36 vs 6.36), and
at 6.25 IU/ml (14.26 vs 15.62). % CV variation was lower in Aptima
HCV at the following WHO standard concentration: 1000 IU/ml
(2.76 vs 2.97), 500 IU/ml (2.54 vs 2.71), 250 IU/ml (2.54 vs 2.71),
12.5 IU/ml (12.28 vs 12.76). At HCV RNA concentrations higher than
125 IU/ml, Abbott HCV tended to underestimate HCV RNA level.
Although detecting all replicates, Aptima HCV quantified 9/10 sam-
ples at 12.5 IU/ml and 6/10 samples at 6.25 IU/ml. Among clinical
samples, we observed 23 discrepant results (23/117, 19.7%). They
were retested with COBAS Ampliprep/COBAS TaqMan v.2 (Roche).
Six samples resulted “not-detected” with Aptima HCV and Roche
and detected >12 IU/ml or quantifiable by Abbott HCV. Two sam-
ples were detected <10 IU/ml by Aptima HCV and “not detected” by
Abbott HCV and Roche assays. The other samples showed variable
HCV RNA values in the 3 considered assays.
Conclusion: Overall evaluation and comparison of the Aptima
HCV and Abbott HCV commercially HCV RNA assays revealed
comparable precision, quantification level, and detection rates.
Consequently, HCV RNA monitoring during therapy, monitored
with both platform, can be considered comparable for treatment
decisions.