A-275 Evaluation of Dried Blood Spots for Serological Detection of Chagas Disease

Abstract Background Chagas Disease (CD) is a vector-borne parasitic infection of Trypanosoma cruzi, endemic to the Americas. Sequela of this infection include cardiomyopathy and gastrointestinal dysmotility disorders. Approximately 6 million people are chronically infected worldwide, of which 300,000 reside in the United States (US). Community-based screening has been useful in facilitating diagnosis and linkage to care, as well as supporting an understanding of the prevalence of disease. However, such settings are often not amenable for phlebotomy and processing necessary to generate specimen for serology. Dried blood spots (DBS) are an attractive alternative specimen type because they require less specialized collection, transport, and storage methods that can facilitate screening more at-risk individuals. In this study, we evaluated DBS sampling using an established T.cruzi serology assay to define test performance. Methods Sample selection: A panel of plasma samples was selected from a set of 775 confirmed CD positives and negatives identified by the American Red Cross (ARC) by blood donor testing. Confirmed positives were stratified based on their reactivity (Optical density 450nm [OD450]). 54 positive specimens were randomly selected by quartiles of reactivity: 18 ‘high positives’, 18 ‘intermediate positives’ and 18 ‘low positives’. 36 confirmed negatives were selected at random. In total, 90 samples were randomized into a 96 well plate format. DBS preparation: ARC plasma samples were reconstituted with packed red blood cells to a hematocrit of 4.6 x 10^6/L to develop a contrived whole blood specimen. Controls from the Chagas Detect Fast ELISA kit (InBios International) were prepared in the same way. 50uL of each contrived whole blood sample and control was pipetted onto 903 Protein Saver Cards (Whatman). Blood spots were allowed to air dry and then 6mm punches were excised and distributed into a 96 well plate. T. cruzi serology: Testing was performed using the Chagas Detect Fast ELISA. DBS punches were eluted in 100uL of InBios sample diluent to an estimated serum dilution of 1:20 and incubated overnight. DBS were tested following the manufacture protocol, with the DBS eluate used in lieu of diluted plasma volume. Neat plasma samples from the same specimens were tested in tandem. Test performance was calculated as positive and negative percent agreement between neat plasma and DBS specimens. Results DBS demonstrated reduced absorbance on the ELISA relative to neat plasma samples (Fig 1). The mean difference in OD450 between plasma and DBS across the linear range (plasma OD450<3.73) was -0.54. However, for all 90 samples, the qualitative result for DBS was concordant with neat serum: 100% (54/54) positive percent agreement and 100% (36/36) negative percent agreement. If neat plasma control cutoffs were applied to DBS specimens, two seropositive specimens turned indeterminate, demonstrating the need for positive and negative controls to be prepared as DBS samples. Conclusion DBS represent an inexpensive and practical alternative to traditional blood draws for screening Chagas Disease. In our study, DBS demonstrated lower reactivity compared to plasma, but the qualitative test result was equivalent to plasma, supporting the use of the Chagas Detect Fast ELISA for DBS testing.