Evaluation of InBios Chagas Detect Fast, a Novel Enzyme-Linked Immunosorbent Assay for the Detection of Anti-Trypanosoma cruzi Antibodies

LETTER

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, affects an estimated 6 million people worldwide, predominantly in Latin America (1). Up to 30% of cases will manifest as cardiac disease, including conduction disorders and heart failure, and/or gastrointestinal motility disorders, with high resulting morbidity and mortality (2, 3). Diagnosis of chronic T. cruzi infection requires concordant positive results by two distinct serological tests. No single assay has demonstrated sensitivity and specificity adequate enough to be used alone (4, 5).

We evaluated the Chagas Detect Fast (CDF; InBios International, Seattle, WA) enzyme-linked immunosorbent assay (ELISA) in 775 CD plasma samples identified by blood donor testing by the American Red Cross (ARC) (seronegative, n = 285; seropositive, n = 490) (6). ARC results were determined according to published testing protocols (7). Region of birth data were available for 199 confirmed positive samples (Mexico, n = 73; Central America, n = 69; South America, n = 57).

Prior to testing, aliquots were randomized to 96-well plates and stored at −20°C. Samples had undergone 3 freeze-thaw cycles at the time of testing. The Chagas Detect Fast (CDF) ELISA was performed in accordance with the package insert. Plates were washed using the ELx50 auto strip washer (BioTek, Winooski, VT). Optical density at 450 nm (OD₄₅₀) values were read using the Victor X4 multilabel plate reader (PerkinElmer, Waltham, MA, USA). To establish an optimal OD₄₅₀ cutoff, data from a training set consisting of a 20% random selection of the samples (n = 155; seronegative, n = 52; seropositive, n = 103) were used. We calculated the maximum Youden J statistic and applied a Fluss correction to determine the point at which sensitivity and specificity were maximized in the training set (8). An OD₄₅₀ value of 0.35 (indeterminate cutoff ±10%) was established and used to calculate the signal to cutoff ratio (S/CO) for the test set, which comprised the remaining 80% of ARC samples (n = 620). A comparison of the reactivity of ARC seropositive specimens with region of origin data was performed using Mann-Whitney test between groups. Data analysis was performed using STATA 14.2 and GraphPad Prism 9.0.

This study was approved by the ARC institutional review board and Human Research Protection Program at the University of California, San Francisco (UCSF).

The CDF results agreed with ARC status in 98.7% of positive samples (383/388; 95% confidence interval [CI], 98.46% to 98.97%) and 98.3% of negative samples (228/232; 95% CI, 97.85% to 98.7%). No statistically significant difference was found in reactivity (S/CO) between samples from donors born in Mexico, Central America, and South America (P > 0.05) (Fig. 1).

FIG 1.

Comparison of reactivity between seropositive donors born in different regions of Latin America. The median S/CO for donors born in Mexico was 8.79 (interquartile range [IQR], 7.78 to 8.95), 8.83 for those born in Central America (IQR, 8.67 to 8.94), and 8.85 (IQR, 8.74 to 9.15) for those born in South America. No significant difference in the reactivity of seropositive specimens was observed between regions. Bar and whiskers represent median with interquartile range.

Previously, we reported the test performance of U.S. Food and Drug Administrative (FDA)-cleared and next-generation CD serology tests (6, 9). This evaluation adds data for the novel CDF ELISA (research use only) demonstrating high negative and positive agreement (>98%) with results from U.S. blood donor testing algorithms (7). Reactivity differences by region of birth are less pronounced for this assay than for previously evaluated kits. Our data suggest that the CDF ELISA could provide an attractive additional option for CD testing in the United States if validated as a laboratory developed test or pursued for FDA clearance.