Comparing in vivo bioluminescence imaging and the Multi-Cruzi immunoassay platform to develop improved Chagas disease diagnostic procedures and biomarkers for monitoring parasitological cure

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and is a serious public health problem throughout Latin America. With 6 million people infected, there is a major international effort to develop new drugs. In the chronic phase of the disease, the parasite burden is extremely low, infections are highly focal at a tissue/organ level, and bloodstream parasites are only intermittently detectable. As a result, clinical trials are constrained by difficulties associated with determining parasitological cure. Even highly sensitive PCR methodologies can be unreliable, with a tendency to produce "false-cure" readouts. Improved diagnostic techniques and biomarkers for cure are therefore an important medical need. METHODOLOGY/PRINCIPAL FINDINGS: Using an experimental mouse model, we have combined a multiplex assay system and highly sensitive bioluminescence imaging to evaluate serological procedures for diagnosis of T. cruzi infections and confirmation of parasitological cure. We identified a set of three antigens that in the context of the multiplex serology system, provide a rapid, reactive and highly accurate read-out of both acute and chronic T. cruzi infection. In addition, we describe specific antibody responses where down-regulation can be correlated with benznidazole-mediated parasite reduction and others where upregulation is associated with persistent infection. One specific antibody (IBAG39) highly correlated with the bioluminescence flux and represents a promising therapy monitoring biomarker in mice. CONCLUSIONS/SIGNIFICANCE: Robust, high-throughput methodologies for monitoring the efficacy of anti-T. cruzi drug treatment are urgently required. Using our experimental systems, we have identified markers of infection or parasite reduction that merit assessing in a clinical setting for the longitudinal monitoring of drug-treated patients

Target product profile for a test for the early assessment of treatment efficacy in Chagas disease patients: An expert consensus.

Six to 7 million people are estimated to be infected by Trypanosoma cruzi, the parasite causing Chagas disease. Thirty to 40% of them, i.e., 1.8 to 2.4 million people, will suffer cardiac disorders and/or digestive clinical manifestations if they are not treated early during the course of the infection [1, 2]. However, only a small fraction of patients are properly diagnosed and treated [3]. Current clinical guidelines recommend treating T. cruzi–infected people if they are asymptomatic or present early symptoms of the disease (Table 1) [4, 5]. Benznidazole (BNZ) and nifurtimox (NFX) are the first-line antiparasitic treatments currently available, both with long administration regimens (60 days) that can produce adverse side effects [6–8]. Despite the fact they are not 100% effective in patients with chronic disease [9–12], they are the only drugs currently registered, and the benefits of their administration have been confirmed in several clinical studies. Currently, clinical trials with new compounds, using alternative regimens that aim to maintain efficacy whilst reducing toxicity, are ongoing and could lead to new therapeutic opportunities and/or policy change

Evaluation of INNO-LIA Syphilis Assay as a Confirmatory Test for Syphilis

We evaluated the sensitivity and specificity of a new confirmatory test for treponemal antibodies, INNO-LIA Syphilis (Innogenetics NV, Ghent, Belgium), on a large number of sera from a clinical laboratory. This multiparameter line immunoassay (LIA) uses recombinant and synthetic polypeptide antigens derived from Treponema pallidum proteins. In a single-blinded cross-sectional retrospective study, 289 seronegative sera, 219 seropositive sera, and 23 sera with an indeterminate serological status for syphilis were analyzed. All sera were tested by the T. pallidum hemagglutination assay (TPHA), the immunoglobulin (IgG)-fluorescent T. pallidum absorption assay (IgG-FTA-ABS), and the Venereal Disease Research Laboratory (VDRL) test. In addition, some seropositive samples were analyzed by the 19S-IgM-FTA-ABS test, an enzyme immunoassay (IgM-EIA), and the MarDx immunoblotting assay. Based on a consensus diagnosis derived from conventional serology, all of the sera were classified as positive, negative, or indeterminate, and the results were compared with the findings of the INNO-LIA Syphilis assay. The sensitivity and specificity of the LIA were 100% (219 of 219) and 99.3% (286 of 288), respectively. Compared to TPHA and IgG-FTA-ABS, the new test gave a significantly higher number (P = 0.021 and P < 0.0001, respectively) of correct results than either of the other two tests. The multiparameter INNO-LIA Syphilis assay is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques

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

BACKGROUND:Chagas 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 FINDINGS:We 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/SIGNIFICANCE:The 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

Trypanosoma-Cruzi Cross-Reactive Antibodies Longitudinal Follow-Up: A Prospective Observational Study in Hematopoietic Stem Cell Transplantation

Antibodies named TcCRA "Trypanosoma cruzi Cross Reactive Antibodies" were detected in 47% of blood donors from French population unexposed to the parasite. In order to evaluate the passive or active transmissibility of TcCRA and further characterize its role and etiology, we have conducted a study in a cohort of 47 patients who underwent allogeneic Hematopoietic Stem Cell Transplantations (allo-HSCT). Donors and recipients were tested for TcCRA prior to transplantation. Recipients were further tested during follow-up after transplantation. Demographical, clinical and biological data were collected. Our primary end-point was to assess the risk of TcCRA acquisition after transplantation. During this initial analysis, we observed no seroconversion in patients receiving cells from TcCRA negative donors (n = 23) but detected seroconversion in 4 out of 24 patients who received hematopoietic stem cells from positive donors. Here, we are discussing possible scenarios to explain TcCRA-immune status in recipient after transplantation.status: publishe