Chagas disease affects an estimated 300,000 individuals in the United States. Diagnosis in the chronic phase requires positive results from two different IgG serological tests. Three enzyme-linked immunosorbent assays (ELISAs) (Hemagen, Ortho, and Wiener) and one rapid test (InBios) are FDA cleared, but comparative data in U.S. populations are sparse. We evaluated 500 seropositive and 300 seronegative blood donor plasma samples. Country of birth was known for 255 seropositive specimens, which were grouped into regions as follows: Mexico (n = 94), Central America (n = 88), and South America (n = 73).
Specimens were tested by the four FDA-cleared IgG serological assays. Test performance was evaluated by two comparators and latent class analysis. InBios had the highest sensitivity (97.4% to 99.3%) but the lowest specificity (87.5% to 92.3%). Hemagen had the lowest sensitivity (88.0% to 92.0%) but high specificity (99.0% to 100.0%). The level of sensitivity was intermediate for Ortho (92.4% to 96.5%) and Wiener (94.0% to 97.1%); both had high specificity (98.8% to 100.0% and 96.7% to 99.3%, respectively).
The levels of antibody reactivity and clinical sensitivity were lowest in donors from Mexico, intermediate in those from Central America, and highest in those from South America. Our findings provide an initial evidence base to improve laboratory diagnosis of Chagas disease in the United States. The best current testing algorithm would employ a high-sensitivity screening test followed by Abbott a high-specificity confirmatory test.
Chagas disease is the most important tropical disease in the Americas. The attributable disease burden in the region, based on disability-adjusted life years lost, is nearly 8 times greater than that due to malaria and 20% higher than that for dengue. An estimated 6 million people, predominantly in Mexico, Central, and South America, are currently infected with Trypanosoma cruzi . Chronic infection persists lifelong in the absence of treatment, with tissue tropism for cardiac myocytes and the enteric nervous system . Over time, 20% to 30% of infected individuals develop cardiac or gastrointestinal disease.
Widespread enzootic transmission cycles involving wildlife and sylvatic triatomine vectors occur in the United States, but autochthonous T. cruzi transmission to humans appears to be very rare. Locally acquired infections are greatly outnumbered by the estimated 300,000 infected immigrants from Latin America residing in the United States. The U.S. Food and Drug Administration (FDA) approved benznidazole, the first-line Chagas disease treatment, in 2017, increasing the need for reliable diagnostic testing for both individual and public health needs in the United States.
In the chronic phase, confirmed diagnosis requires positive results by two serological tests for IgG antibodies to T. cruzi, preferably based on different antigens.
Currently, four serological assays, namely, the Ortho T. cruzi enzyme-linked immunosorbent assay (ELISA) (Ortho Clinical Diagnostics, Raritan, NJ), Hemagen Chagas’ kit ELISA (Hemagen Diagnostics, Inc., Columbia, MD), Wiener Chagatest Recombinante v.3.0 ELISA (Wiener Laboratories, Rosario, Argentina), and InBios Chagas Detect Plus (CDP) rapid test (InBios International, Inc, Seattle, WA), are cleared by the FDA for diagnostic use . The Ortho and Hemagen ELISAs are based on native parasite proteins. The other two assays are based on recombinant proteins. The Wiener ELISA uses trypomastigote-shed acute-phase antigens (SAPA) and recombinant epimastigote antigens 1, 2, 13, 30, and 36 .
The InBios test is based on the recombinant multiepitope fusion antigen ITC8.2 . All four assays report high sensitivity and specificity in their FDA 510(k) clearance applications (reported percent sensitivity/specificity: Ortho, 98.9/99.99; Hemagen, 100/98.7; Wiener, 99.3/98.7; InBios, 95 to 100/87 to 98). However, comparative performance data are lacking for at-risk populations in the United States, as well as for those in Mexico and Central America, the predominant regions of origin of U.S. immigrants. Emerging evidence suggests variation in test sensitivity by geographic location and a high rate of discordance between serological test results, particularly in Mexico. Comprehensive studies are needed to provide the basis for development of reliable testing algorithms. In this study, we compared the performances of the four FDA-cleared serological tests in specimens from U.S. blood donors to provide the first systematic evidence to improve laboratory diagnosis of Chagas disease in the United States.
This study was approved by the American Red Cross (ARC) institutional review board and was deemed exempt from review by the Human Research Protection Program at the University of California, San Francisco (UCSF).
Sample selection and preparation.
We evaluated archived plasma samples from 800 blood donations (BDs) collected by the ARC between September 2006 and June 2018. Specimen selection was based on confirmed T. cruzi infection status in ARC BD testing algorithms at the time of blood donation. ARC provided a list of 1,091 seropositive specimens, defined by repeat reactive results generated by an FDA-licensed screening test (Ortho ELISA or Abbott PRISM [Abbott Laboratories, Abbott Park, IL]) followed by confirmed-positive results generated by a supplemental test (radioimmunoprecipitation assay [RIPA], performed by Quest Diagnostics [Chantilly, VA], or Abbott enzyme strip assay [ESA]).
We prioritized selection of BD-positive specimens with country-of-birth data; the remainder of the 500 BD-positive specimens were selected at random. A random sample of 300 specimens was compiled from a list of 3,938 seronegative blood donations, frequency matched by region of donation to the BD-positive specimen set. No country-of-birth data were available for seronegative specimens.
Donated plasma units from each donation were frozen at –20°C within 24 h of collection. Plasma units retrieved from ARC collections used for research purposes were thawed in a temperature-controlled water bath, divided aliquots and placed into multiple tubes, and refrozen. Aliquots tested by Hemagen, Wiener, and InBios assays at UCSF were thawed and refrozen only once. For the current analysis, the Ortho ELISA was rerun on all 800 specimens in 2019. Aliquots used for current Ortho testing were thawed and refrozen twice.
Ortho ELISA testing for this study was conducted at Innovative Blood Resources, Minneapolis, MN, using the fully automated Ortho Summit system. The Ortho ELISA has FDA approval for blood donation screening and clearance for diagnostic purposes but is not yet marketed for the latter use. For the Ortho ELISA, signal-to-cutoff (S/CO) ratios of 1.00 or greater are considered representative of reactive results; in the blood donation screening algorithm, all reactive units are retested two more times. A blood donation is considered repeat reactive if at least 2 of 3 sample results have an S/CO ratio greater than 1.00.
Hemagen ELISA, Wiener ELISA, and InBios rapid tests were conducted at UCSF. Plasma samples were thawed at 4˚C and spun at 2,300 relative centrifugal force for 10 min to pellet any precipitate.
Samples were divided into aliquots and placed at randomly assigned positions in 96-deep-well plates to blind readers performing the InBios rapid test. Plasma aliquots of 10 μl (Hemagen and Wiener) or 5 μl (InBios) were tested and interpreted in accordance with package inserts using the kit reagents, a ELx405 Select microplate washer (BioTek, Winooski, VT), and a SpectraMax Plus 384 microplate reader (Molecular Devices, San Jose, CA).
The InBios package insert defines any visible test line as representative of a positive result. For quantification of the results this assay, a set of 7 quality control samples was used to construct a semiquantitative scale ranging from 0 (negative) to 6 (strongly positive) (see Fig. S1 in the supplemental material). InBios test results were scored by two independent readers blind to other assay results. The only deviation from package insert protocols was the use of plasma for Hemagen tests; the manufacturer recommends use of serum only.