Development of a Novel Multiplex Immunoassay Multi-cruzi for the Serological Confirmation of Chagas Disease

Abstract

BackgroundChagas disease is due to the parasite Trypanosoma cruzi, a protist disseminated by a Triatome vector. This disease is endemic to Latin America and considered by WHO as one of the 17 world’s neglected diseases. In Europe and in North America, imported cases are also detected, due to migration of population outside of the endemic region. Diagnosis of T. cruzi infection is usually made indirectly by the detection of specific antibodies to T. cruzi antigens. Following initial diagnostic evaluation or screening test (qualifying or discarding blood donation), a confirmation test is performed for samples initially reactive. The test presented in this study aims at the confirmation/refutation of the infectious status of human blood samples and will permit taking appropriate clinical measures.Methodology/Principal FindingsWe designed a novel array of twelve antigens and printed these antigens onto 96-well plates. We tested 248 positive samples T. cruzi, 94 unscreened blood donors’ samples from non-endemic area, 49 seronegative blood donors, 7 false-positive and 3 doubtful samples. The observed reactivities were analyzed to propose a decision-tree algorithm that correctly classifies all the samples, with the potential to discriminate false-positive results and sticky samples. We observed that antibodies levels (Sum of all antigens) was significantly higher for PCR positive than for PCR negative samples in all studied groups with Multi-cruzi.Conclusion/SignificanceThe results described in this study indicate that the Multi-cruzi improves the serological confirmation of Chagas disease. Moreover the “sum of all antigens” detected by Multi-cruzi could reflect parasitemia level in patients–like PCR signals does—and could serve as an indicator of parasite clearance in longitudinal follow-ups. Validation of this assay is still required on an independent large collection of well characterized samples including typical false-reactive samples such as Leishmaniasis.

A novel antibody surrogate biomarker to monitor parasite persistence in Trypanosoma cruzi-infected patients

“…The assay presented in this study is an extended version of the MultiCruzi confirmatory assay previously described [ 26 ]. We used the sciFLEXARRAYER system (SCIENION, Germany) to print fifteen T .…”

“…The aim of the study we present in this manuscript was to characterize serological profiles of T . cruzi -infected patients on an extended version of the MultiCruzi assay [ 26 ]. By measuring diversity of antibodies we sought to identify surrogate antibody biomarker(s) that could be sensitive and specific for monitoring parasite persistence regardless of PCR results.…”

Chagas Disease Diagnostic Applications

“…New generation tests displaying potentially improved accuracy such as the Chemoluminescent Microparticle ImmunoAssay (CMIA) and its improved version, Architect Chagas (both from Abbott Laboratories, Wiesbaden, Germany) (Abras et al, 2016; Praast et al, 2011), or the Multi-cruzi test (InfYnity Biomarkers, Lyon, France) (Granjon et al, 2016) have been recently developed. Confirming the above mentioned trend, both use a large panel of T. cruzi antigens belonging to the 'parental repertoire' -those discovered in the 80's-, which in the case of the Multi-cruzi test is supplemented with TSSA (Trypomastigote Small Surface Antigen (Di Noia et al, 2002)).…”

“…Confirming the above mentioned trend, both use a large panel of T. cruzi antigens belonging to the 'parental repertoire' -those discovered in the 80's-, which in the case of the Multi-cruzi test is supplemented with TSSA (Trypomastigote Small Surface Antigen (Di Noia et al, 2002)). Despite the virtual lack of antigen innovation, these tests incorporate a large degree of automation and highly sensitive detection systems (Abras et al, 2016) or major technical improvements, such as a multiplexed printing method inside ELISA microplates (Granjon et al, 2016). …”

Next-generation ELISA diagnostic assay for Chagas Disease based on the combination of short peptidic epitopes

“…cruzi immunodominant antigens [ 14 ]. However, several studies showed that the use of single antigens in an assay did not confer the sensitivity required for a diagnostic test [ 14 , 15 ], which prompted the development of tests based on combinations of antigens[ 16 , 17 ], some of which were evaluated in multicenter trials and are commercially available [ 18 – 20 ]. Synthetic peptides are advantageous for diagnostic applications because they are: i) well defined (ease of quality control), ii) easily produced in large amounts, ii) highly pure and often cost-saving if compared to the production of natural or recombinant antigens in vitro [ 21 ]; and iv) also chemically stable (can be stored lyophilized or dessicated and tend to be stable for several years).…”