APHL 2019 POSTER ABSTRACTS
Testing Anti-Zika Virus NS1 IgA Additionally to IgM
Increases Sensitivity in Acutely Infected Patients
from Regions Endemic for Flaviviruses
K. Steinhagen, N. Muigg, O. Sendscheid and W. Schlumberger,
EUROIMMUN
Introduction: Specific IgM response to Zika virus (ZIKV) can be low
or absent in patients with acute ZIKV infection and a history of
other infections with related flaviviruses, e. g. dengue virus (DENV),
presenting with an early high IgG titer. In these ZIKV cases, IgA against
ZIKV non-structural protein 1 (NS1) was observed in the acute phase,
suggesting anti-ZIKV IgA as alternative acute marker in secondary
infections. In this study, we investigated the diagnostic benefit of an
ELISA for combined detection of anti-ZIKV NS1 IgA and IgM.
Anti-ZIKV NS1 antibodies were determined in each sample using
a commercial NS1-based Anti-Zika virus ELISA IgM (Euroimmun
AG, Germany) and a corresponding ELISA (Euroimmun), applying a
combination of anti-human IgA/IgM conjugated with peroxidase.
Results: In panel 1, 29% (9/31) of samples were positive for anti-
ZIKV NS1 IgM, whereas 100% were positive for combined specific
IgA and IgM. In panel 2, none of the sera reacted in the Anti-Zika
virus ELISA IgM, two samples were reactive in the Anti-Zika virus
IgAM ELISA (5.0%).
Discussion: As patients with acute ZIKV infection from flavivirus
endemic regions may not develop NS1-specific antibodies class IgM,
additional testing of anti-ZIKV NS1 IgA is required.
Presenter: Maite Sabalza, EUROIMMUN US, Inc.,
[email protected]
Dried Blood Spots Represent Well-suited Specimens
for Detection of Anti-Trypanosoma cruzi Antibodies
A. Franke 1 , F. Lindhorst 2 , O. Sendscheid 2 , K. Steinhagen 2 ; 1 University
Luebeck, 2 EUROIMMUN
Introduction: The parasitic kinetoplast Trypanosoma cruzi can be
found in warm rural areas. In Brazil, the estimated seroprevalence
of T. cruzi is 1-6%. CDC estimates that more than 300,000 persons
with a Trypanosoma cruzi infection live in the United States.
Transmission occurs by hematophagous bugs and infection causes
Chagas disease, which can be fatal if untreated. Diagnosis is usually
performed by microscopic detection in blood smears (early during
infection), by xenodiagnostics or by detection of specific antibodies.
Blood spotting onto filter paper is an easy and well-established
method for collecting specimens used in serology. After drying,
analytes such as antibodies can be extracted from these Dried
Blood Spots (DBS) and subsequently be used for the diagnosis
of different diseases. For efficient analysis by ELISA, DBS have to
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LAB MATTERS Summer 2019
Methods: To validate DBS as proper specimens, 12 paired samples
covering a wide range of the calibration curve were analyzed in
triplicate. As DBS lose reactivity over time, the extraction volume
was adapted such that freshly produced DBS show 120% recovery
compared to the respective serum (diluted 1:101). Reproducibility
was examined in 10 measurements at 5 days including sample
duplicates. DBS were stored in a closed bag with desiccant
at -20°C, 4°C, RT or 37°C for 4 weeks. Measurements were
performed after 1, 2 and 4 weeks. To test for stability during
transport, DBS were stored at 30°C in a closed bag with desiccant
or in an open bag for 3 weeks, and measured weekly. In addition,
we examined DBS obtained from 440 pregnant women from Brazil.
Results: The detection of anti-T. cruzi IgG from DBS showed high
reproducibility as well as 100% sensitivity and specificity compared
to serum analysis. Storage of DBS over 4 weeks at -20°C, 4°C or
RT resulted in a temperature-independent loss of reactivity of 20%
on average. DBS stored at 37°C showed 78% recovery compared
to serum after 4 weeks. Among 440 DBS from pregnant Brazilian
women, 5 positive or equivocal results could be confirmed in
duplicates resulting in a seroprevalence of 1.1%.
Discussion: DBS fulfill all conditions for efficient serological
diagnostics. Due to their high-temperature stability and simplicity of
collection, DBS can be used in rural areas with less infrastructure
or in serological studies: blood is collected on filter paper by the
patient and then sent to a laboratory for analysis without the
need for permanent cooling. It has been shown that also other
immunoglobulin isotypes can be extracted and subsequently
detected from DBS. Automated punching systems for DBS facilitate
the entire process.
Presenter: Oliver Sendscheid, EUROIMMUN US, Inc.,
[email protected]
Development of Anti-Powassan ELISA for the Detection of
Specific Antibodies Based on Different Antigenic Substrates
O. Klemens 1 , J. Boethfuer 1 , S. Wong 2 , L. Binnenkade 1 , O.
Sendscheid 1 , K. Steinhagen 1 ; 1 EUROIMMUN, 2 New York State
Department of Health
Introduction: The tick-borne Powassan virus (POWV) is a member of
the family Flaviviridae. POWV infections in humans were reported
in the US, Canada and Russia with a considerable increase in
recent years, with only 27 cases in the second half of the 20th
century but 98 cases within the last ten years (CDC statistics).
Since other tick-borne diseases transmitted by the same vector
occur at high incidences (e.g., Lyme disease), an underestimation
of POWV infections is suspected. POWV infections present with a
wide spectrum of symptoms including severe neurological signs like
encephalitis. Besides imaging techniques to diagnose neurological
manifestations, diagnostic methods comprise direct virus detection
or the detection of specific antibodies against POWV. No serological
assays are commercially available. We have developed different
ELISAs for the detection of antibodies against POWV to enable
sensitive screening of suspected cases and a more accurate
prevalence estimation of this rare but life-threatening disease.
PublicHealthLabs
@APHL
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Methods: The following human serum panels were included in this
study: [1] A sensitivity panel (panel 1) comprising acute serum
samples (day 8-16 post symptom onset) of 31 residents from the
Dominican Republic (2015), where ZIKV and DENV are endemic.
Patients had been tested positive for ZIKV RNA and anti-DENV IgG
during the viremic phase (≤ day 5). [2] A specificity panel (panel 2)
consisting of serum samples (day 3-7 post symptom onset) of 40
Vietnamese patients, hospitalized with DENV hemorrhagic fever
according to the World Health Organization case definition grade
I and tested positive for DENV nucleic acid and anti-DENV IgG.
Vietnam (2015) is endemic for DENV but not for ZIKV.
meet several requirements, including comparability to serum (gold
standard), reproducibility of extraction, stability during storage and
transport. In this study, we analyzed the extraction of anti-T. cruzi
IgG from DBS and the detection of these antibodies using the Anti-
Trypanosoma cruzi IgG ELISA (Euroimmun AG, Germany).