Lab Matters Summer 2019 - Page 74

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., maite.sabalza@euroimmun.us 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 72 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., oliver.sendscheid@euroimmun.us 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 APHL.org 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).