Serologic Testing for Trypanosoma cruzi: Comparison of Radioimmunoprecipitation Assay with Commercially Available Indirect Immunofluorescence Assay, Indirect Hemagglutination Assay, and Enzyme-Linked Immunosorbent Assay Kits

The radioimmunoprecipitation assay (RIPA) has been used as a confirmatory test in several ongoing and published studies of Trypanosoma cruzi in blood donors in the United States. Despite its use as a confirmatory test, few studies are available comparing RIPA to commercially available serologic test methods. Thus, we compared RIPA with two indirect hemagglutination assays (Biolab Diagnostica SA, São Paulo, Brazil; Hemagen Diagnostics, Inc., Waltham, Mass.) and four different enzyme-linked immunosorbent assays (Abbott Laboratories, Abbott Park, Ill.; Embrabio, São Paulo, Brazil; Organon Teknika, São Paulo, Brazil; and Gull Laboratories, Salt Lake City, Utah) using a panel of 220 serum specimens from Brazilian blood donors with a range of T. cruzi antibody titers as determined by indirect immunofluorescence assay (IFA). A titer of 1:20 was used as the baseline for seropositivity. All IFA-negative serum specimens (n = 19) were nonreactive on all tests. At a titer of 1:20 (n = 9), reactivity rates varied considerably among the tests, with only the RIPA and the Organon and Gull assays identifying reactive specimens. For specimens at a 1:40 titer (n = 35), most assays identified at least 32 of 35 (91%) specimens as reactive, but the Biolab assay only identified 24 (69%). At higher titers (1:80, n = 56; 1:160, n = 101) the assays were comparable, with the exception of the Biolab assay, demonstrating rates of agreement with IFA of ≥98%. Overall, when compared with several other test formats, RIPA demonstrated equivalent or superior rates of agreement with IFA-positive specimens across all titers examined. In particular, at titers of >1:40, the RIPA compared favorably with other test methods currently in use, supporting its application as a confirmatory test, particularly in a research setting

Safety and efficacy assessment of two new leprosy skin test antigens: randomized double blind clinical study.

New tools are required for the diagnosis of pre-symptomatic leprosy towards further reduction of disease burden and its associated reactions. To address this need, two new skin test antigens were developed to assess safety and efficacy in human trials.A Phase I safety trial was first conducted in a non-endemic region for leprosy (U.S.A.). Healthy non-exposed subjects (n = 10) received three titrated doses (2.5 µg, 1.0 µg and 0.1 µg) of MLSA-LAM (n = 5) or MLCwA (n = 5) and control antigens [Rees MLSA (1.0 µg) and saline]. A randomized double blind Phase II safety and efficacy trial followed in an endemic region for leprosy (Nepal), but involved only the 1.0 µg (high dose) and 0.1 µg (low dose) of each antigen; Tuberculin PPD served as a control antigen. This Phase II safety and efficacy trial consisted of three Stages: Stage A and B studies were an expansion of Phase I involving 10 and 90 subjects respectively, and Stage C was then conducted in two parts (high dose and low dose), each enrolling 80 participants: 20 borderline lepromatous/lepromatous (BL/LL) leprosy patients, 20 borderline tuberculoid/tuberculoid (BT/TT) leprosy patients, 20 household contacts of leprosy patients (HC), and 20 tuberculosis (TB) patients. The primary outcome measure for the skin test was delayed type hypersensitivity induration.In the small Phase I safety trial, reactions were primarily against the 2.5 µg dose of both antigens and Rees control antigen, which were then excluded from subsequent studies. In the Phase II, Stage A/B ramped-up safety study, 26% of subjects (13 of 50) showed induration against the high dose of each antigen, and 4% (2 of 50) reacted to the low dose of MLSA-LAM. Phase II, Stage C safety and initial efficacy trial showed that both antigens at the low dose exhibited low sensitivity at 20% and 25% in BT/TT leprosy patients, but high specificity at 100% and 95% compared to TB patients. The high dose of both antigens showed lower specificity (70% and 60%) and sensitivity (10% and 15%). BL/LL leprosy patients were anergic to the leprosy antigens.MLSA-LAM and MLCwA at both high (1.0 µg) and low (0.1 µg) doses were found to be safe for use in humans without known exposure to leprosy and in target populations. At a sensitivity rate of 20-25% these antigens are not suitable as a skin test for the detection of the early stages of leprosy infection; however, the degree of specificity is impressive given the presence of cross-reactive antigens in these complex native M. leprae preparations.ClinicalTrials.gov NCT01920750 (Phase I), NCT00128193 (Phase II)

Diagnostic test statistics – Skin test.

<p>Diagnostic test statistics were calculated for each test method. Sensitivity (Se) is the likelihood to detect the presence of disease [Total Positive (TP)/TP + False Negative (FN)]. Specificity (Sp) is the likelihood to detect absence of disease [(Total Negative (TN)/TN + False Positive (FP)]. PPD served as an antigen control. Statistics for detecting tuberculosis: Sensitivity is (36/40) 90%, specificity is (41/100) 41%.</p

Dot plot of induration measurements.

<p>Induration results are provided across five subject groups, including BL/LL leprosy patients (<i>n</i> = 19 in low and <i>n</i> = 20 in high dose group), BT/TT leprosy patients (<i>n</i> = 20), HC (<i>n</i> = 20), TB (<i>n</i> = 20), and ECs (<i>n</i> = 50). Low and high dose groups were combined to show PPD responses: BL/LL leprosy patients (<i>n</i> = 39) and all other groups (<i>n</i> = 40). Mean and standard deviation are shown.</p

Number of positive responders and mean induration compared to ECs.

a<p>To allow calculations, the EC percent positive has been changed to 1.0.</p

Diagnostic test statistics – WB-IGRA^a.

a<p>Preliminary unpublished results.</p

Distribution frequency of induration.

<p>Frequency distribution graphs were used to establish cut off points for each skin test antigen at each dosage tested: A) MLSA-LAM low dose, B) MLCwA low dose, C) MLSA-LAM high dose, D) MLCwA high dose. Frequency of induration reaction (mm) of EC and TB groups were graphed against BT/TT and BL/LL leprosy groups. The anti-mode between the control and leprosy patient group represents the cut off for each antigen and antigen dose.</p

CONSORT flow diagram, Phase II, Stage C-1 Trial.

<p>CONSORT flow diagram, Phase II, Stage C-1 Trial.</p

Phase II, Stage A/B – DTH induration by subject.

<p>Phase II, Stage A/B graph depicting DTH indurations elicited by leprosy skin test antigens at the high dose (1.0 µg) and low dose (0.1 µg), and PPD at 5 TU: A) MLCwA, and B) MLSA-LAM. The first five subjects on both graphs represent subjects from Stage A, and the remaining 45 subjects on both graphs represent subjects from Stage B.</p