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

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S49
negative in plasma (3.54 and 3.83 log10 IU/ml) and 6 gave the oppo-
site results (mean viral load: 2.73 log10 IU/ml). All positive (n = 36)
samples were analyzed. Linear regression analysis showed a good
correlation between the two methods: r = 0.75, p < 0.0001, (slope
of Deming regression 1.299 [CI 95%: 0.900–1.698] and y-intercept
0.49 [CI 95%: −1.908 to 0.9199]. The Bland-Altman representation
showed that the CMV-DNA quantitation inwhole blood gave higher
virus loads than did the CMV-DNA quantitation in plasma: the aver-
age deviation was −0.54 log10 IU/ml (SD = 0.60).
The influence of the blood compartment was also analyzed by
comparing the virus load kinetics for successive samples selected
fromfour immunosuppressedpatients (16 samples). Overall results
showed similar patterns with variation in the same direction.
Whole blood was the only compartment that tested positive in one
patient for very low virus loads (2.02–2.78 log log10 IU/ml).
Conclusion: The H-DIACMVQ kit® provides precise, repro-
ducible results and it satisfies quality requirements for routine
monitoring of DNA-CMV in plasma or whole blood samples.
Abstract no: 65
Presentation at ESCV 2016: Poster 56
Use of recombinant virus technology to produce
non-infectious, whole process controls for
emerging viruses such as Ebola, Chikungunya,
Dengue-2, Norovirus GII, MERS-CoV and Zika
C. Plachot∗, C. Huang, R. Vemula, J. Wu, H.J. Lee,
B. Anekella
SeraCare Life Sciences, United States
Background: Outbreaks of viral communicable disease and
appearance of new viral strains can represent public health emer-
gencies. As diagnostic laboratories and test developers design,
manufacture and validate diagnostic assays to prepare for these
threats, positive reference materials are needed. SeraCare has
developed AccuPlexTM recombinant virus technology to produce
whole process reference materials that mimic clinical samples.
They are mammalian virus products and are non-infectious. Accu-
Plex technology was used to develop quality controls for amplified
nucleic acid tests for the emerging viruses Ebola, Chikungunya,
Dengue-2, Norovirus GII, MERS-CoV and Zika as well as drug resis-
tant HIV-1. Here we demonstrate the performance of these quality
control materials and show that they have an extended stability at
2–8 ◦C and do not require freezer storage.
Methods: AccuPlexTM controls for RNA viruses employ engi-
neered Sindbis virus, and a portion of the Sindbis structural genes
are replaced with up to ∼4000 bp of the diagnostic targets of inter-
est. Where the diagnostic targets are well defined, those regions
were incorporated into one recombinant virus. For example, the
recombinant Chikungunya reference material contains portions of
the NSP1, NSP2, NSP4, Capsid, E3 and E1 genes, and is based on
the sequence of strain IND-06-Guj. The recombinant Dengue ref-
erence material contains portions of 3 UTR, NSP5, Capsid, and E1
genes from serotype 2. Recombinant Norovirus, Ebola, and MERS-
coV reference materials follow a similar design scheme.
However, when diagnostic targets are undefined, as is the case
for Zika, a different design scheme is required. The entire Zika
genome was divided into four segments and each segment was
used to generate an AccuPlex recombinant virus. The Zika reference
material therefore is a mixture of four distinct AccuPlex recom-
binant viruses. Dividing the pathogen’s genome among multiple
constructs ensures each recombinant virus is not functional. Addi-
tional safety features such as gene truncation, multiple stop codons
and frame shifts are also used and the products are heat treated for
viral inactivation.
Results: Recombinant AccuPlex viruses were diluted in defib-
rinated plasma or other commutable matrices and characterized
by Digital PCR using pathogen specific primers and probes. The
target concentration range of the reference materials is from
5E + 05 copies/mL for recombinant Ebola, to 5E + 06 copies/mL for
many of the other viruses. Functional testing of the reference mate-
rials on Altona RealStar RT-PCR Kits as well as Primer Design Ltd
GeneSigAdvanced kits showedpositive detection. The recombinant
viruses gave cycle threshold values (Ct) on these assays consistent
with a low positive control (Ct of 27–31.5). Accelerated stabil-
ity studies indicate that the product is stable at 4 ◦C for at least
two years. Real time stability data at Room Temperature has been
collected through 20 months and updated stability data will be
presented.
Conclusions: SeraCare has developed stable, well-characterized
whole process controls for pathogenic viruses. These reference
materials will enable laboratories to validate tests and train tech-
nicians to ensure preparedness for outbreaks. These products
demonstrate the utility of recombinant virus technology to pro-
duce non-infectious controls for select agents and viruses difficult
to source or propagate.
Abstract no: 67
Presentation at ESCV 2016: Poster 57
Characterisation and standardisation of Qnostic
products in the absence of higher order
standards
S. Kazi∗, A. Ricketts, F. Opdam
Qnostics, United Kingdom
Introduction: Viral load determination plays a critical role
in clinical diagnostics and a central role in monitoring patients’
response to treatment and disease progression. However, true
transferability of results remains elusive due to the lack of
inter-laboratory standardisation. Where available, International
Standards have helped to facilitate data comparison but where
there is no standard or Certified Reference Material available assay
variation obscuresmeaningful comparison of results at the technol-
ogy and laboratory level. The use of characterised control materials
with known performance characteristics would allow for objective
comparisons between laboratories and assays used. In this studywe
evaluated the use of digital PCR (dPCR) to quantify control materials
for four viral targets (Cytomegalovirus (CMV), Epstein-Barr Virus
(EBV), JC Virus (JCV) and BK Virus (BKV)), and established perfor-
mance across the top five available commercial assays in clinical
use for each. International Standards are available for CMV and
EBV but not for JCV and BKV. Digital PCR permits the characteri-
sation of control materials without the requirement of a standard
or certified reference material thereby allowing direct comparison
of results between laboratories.
Methods: Control materials for each of the 4 viral targets
(Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), JC Virus (JCV)
and BKVirus (BKV)) were prepared at a single titre in human plasma
at a concentration that fell within the linear range for most assays
in use. The controls were characterised internally using both an in-
house qPCR based method and digital PCR (BioRad QX200). Blind
panels were provided to laboratories participating in the study in
2015. Laboratories were asked to treat the materials as they would
a clinical sample and to return quantitative data along with infor-
mation on the assay workflow used to generate the results.