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

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Abstracts / Journal of Clinical Virology 82S (2016) S1–S142 S43
in 10 nasopharyngeal swabs obtained from two immunocompro-
mised patients (n1 = 7 and n2 = 3) with extended A(H1N1)pdm09
viral shedding.
Results: High correlations (using Spearman correlation test)
were observed between qPCR and NGS, (r2 = 0.9187, p < 0.01), and
between ddPCR and NGS (r2 = 0.9132, p < 0.01). ddPCR demon-
strated higher performances than qPCR, using NGS assay as a gold
standard. ddPCR was able to detect 1.5–2% of oseltamivir-resistant
viruses in a WT IV population and 3–4% of WT IV in an oseltamivir-
resistant population (variation coefficient <10%). In the clinical
specimens of the first patient, the oseltamivir-resistant IV popula-
tion reach 56% 14 days after the beginning of oseltamivir treatment.
The treatment was then stopped during 22 days and resistant
IV population dropped to be below quantification level by qPCR
and ddPCR (<1.5%) Oseltamivir treatment was re-introduced and
oseltamivir-resistant IV population rocketed up to 96% in 5 days
before lethal complications. For the second patient, oseltamivir-
resistant IV populations reached 22%, 9 days after treatment onset,
on the last specimen collected before death.
Discussion: Use of new molecular tools can improve accu-
racy and speed of diagnosis especially for infectious diseases
when therapeutic resistance may lead to severe complications. Our
study showed that ddPCR is cheaper, less time-consuming and
demonstrated higher performances than classical qPCR to estimate
oseltamivir-resistant IV subpopulation percentages. This technique
could be used to monitor the emergence of H275Y-NA mutation in
immunocompromised patients. An early detection of oseltamivir-
resistant population may improve the therapeutic management
and decrease the rate of therapeutic failure.
Abstract no: 314
Presentation at ESCV 2016: Poster 44
A new combination of old techniques for HIV
disease progression – qRT-PCR and ONEp-PCR
Quirina dos Santos Costa ∗,
José Miguel Azevedo Pereira
Maria Manuel Lopes , Margarida Rocheta
1 Faculdade de Farmácia da Universidade de Lisboa,
Portugal
2 Faculdade de Agronomia da Universidade de
Lisboa, Portugal
Background: Human immunodeficiency virus type 2 (HIV-2)
have unique properties as a human pathogenic agent: is less effi-
cient on developing pathologic manifestations, the infection is
generally defined as less virulent and HIV-2 infected individuals
have a lower viral burden, accomplishing to a lower transmission
rate.
Primary and chimeric HIV-2 viruses have been shown to use
alternative coreceptors beyond the main CCR5 or CXCR4 to enter
host cells. These features seem to be related to a flexible envelope
oligomeric structure. HIV coreceptor usage, cDNA integration, and
HIV pathogenesis are mechanisms with poorly understood dynam-
ics.
Sensitive methods are needed for quantifying gene expression
and copy number in HIV infected cells. Combining qRT-PCR with
ONEp -PCR (one primer-PCR), which is a relatively simple cus-
tomized technique that can be used to investigate fingerprinting,
polymorphisms, genomic instability in HIV infected cells, and has
the potential to reveal associated markers, is a renewable resource
for numerous studies in various fields of modern biology and
medicine.
Findings: In this work it is shown that primary HIV-2 R5 and
ROD/envR5 or ROD/env-R5/-X4 chimeric viruses have differential
behaviour related to copy number integration and expression.
Conclusions: HIV-2 env-V1V2 region is responsible to trigger
different signal pathways leading to ccr5 and env expression, and
envcopy number, in infected human T-lymphocytes genomic DNA.
These results also point out the potential usefulness of combin-
ing qRT-PCR and ONEp-PCR to detect changes in HIV proviral DNA
within the infected cell’s genome, as a new tool for HIV disease
progression.
Abstract no: 328
Presentation at ESCV 2016: Poster 45
Performance of the IDS-iSYS “walk away”
immunoassay system for the determination of
Epstein–Barr Virus (EBV) serological status
M. Baccard Longere ∗, J. Lupo , J. Tolenaere ,
C. delmas , P. Morand
1 Laboratoire de Virologie CHU Grenoble, France
2 Laboratoire de Virologie CHU Grenoble, Institut de
Biologie Structurale Universite Grenoble Alpes,
France
3 Immunodiagnostic Systems, Paris, France
4 Laboratoirede Virologie CHU Grenoble, Institut de
Biologie Structurale Universite Grenoble Alpes,
France
Background:The combination of EBV specific serological mark-
ers is the best strategy to assess the immune status against EBV
infection. Usually the simultaneous detection of IgM and IgG anti-
bodies against the viral capsid antigen (VCA) and against the EBV
nuclear 1Ag (EBNA1) is sufficient to differentiate between: absence
of infection (AI), past infection (PastI) and primary infection (PrI).
Here we report the performance of a new commercial automated
immunoassay system, IDS-iSYS, for the detection of VCA IgM, VCA
IgG and EBNA IgG on a large panel of samples.
Material and methods: A panel of 435 sera (426 patients)
was retrospectively selected from our university hospital routine
screening EBV serology.
The immunoassay routinely used in our laboratory for the
detection of IgG and IgM antibodies against EBV (Enzygnost®
Immunoassay, Siemens Healthcare Diagnostics;) and IgG antibod-
ies against EBNA (BioMedicalDiagnostics) were used as “reference
tests” to classify the 435 samples as: (i) seronegative (EBV IgG/IgM
and EBNA IgG negative n = 90); (ii) past infection (EBV IgM neg, EBV
IgG pos, EBNA IgG pos, n = 108); primary infection (EBV IgMpos and
EBV IgG pos or neg, EBNA IgG neg n: 117).
Additionally, 22 sera (22 patients) with an “isolated EBV IgG”
status and 15 sera (7 patients) with all the three markers detected
as positive (EBV IgM/IgG pos and EBNA IgG pos) were re-classified
as primary infection or past infectionwith further analysis (indirect
immunofluorescence assay, heterophile antibodies detection) and
tested with the IDS-iSYS system. Eighty-three serologically proven
primary infections caused by other viruses (cytomegalovirus, hep-
atitis viruses, parvovirus. . .) were also used to test the IDS-iSYS IgM
VCA cross reactivity.
Sensitivity (Sens.) and specificity (Spe.) of the three IDS-iSYS
parameters were compared to the reference tests. The agreement
between IDS iSYS EBV status obtained with the three iSYS markers
and the expected EBV status obtained with the referent test was
established in accordance with the criteria of interpretation of the
manufacturers.