Accomplishing the genotype-specific serodiagnosis of single and dual Trypanosoma cruzi infections by flow cytometry Chagas-Flow ATE-IgG2a

Abstract

The methods currently available for genotype-specific diagnosis of T. cruzi infection still present relevant limitations, especially to identify mixed infection. In the present investigation, we have evaluated the performance of Chagas-Flow ATE-IgG2a test for early and late differential diagnosis of single and dual genotype-specific T. cruzi infections. Serum samples from Swiss mice at early and late stages of T. cruzi infection were assayed in parallel batches for genotype-specific diagnosis of single (TcI, TcVI or TcII) and dual (TcI+TcVI, TcVI+TcII or TcII+TcI) infections. The intrinsic reactivity to TcI, TcVI and TcII target antigens, including amastigote (AI/AVI/AII), trypomastigote-(TI/TVI/TII) and epimastigote (EI/EVI/EII), at specific reverse of serum dilutions (500 to 64,000), was employed to provide reliable decision-trees for “early” vs “late”, “single vs “dual” and “genotype-specific” serology. The results demonstrated that selective set of attributes “EII 500/EI 2,000/AII 500” were able to provide high-quality accuracy (81%) to segregate early and late stages of T. cruzi infection. The sets “TI 2,000/AI 1,000/EII 1,000” and “TI 8,000/AII 32,000” presented expressive scores to discriminate single from dual T. cruzi infections at early (85%) and late stages (84%), respectively. Moreover, the attributes “TI 4,000/TVI 500/TII 1,000”, “TI 16,000/EI 2,000/EII 2,000/AI 500/TVI 500” showed good performance for genotype-specific diagnosis at early stage of single (72%) and dual (80%) T. cruzi infections, respectively. In addition, the attributes “TI 4,000/AII 1,000/EVI 1,000”, “TI 64,000/AVI 500/AI 2,000/AII 1,000/EII 4,000” showed moderate performance for genotype-specific diagnosis at late stage of single (69%) and dual (76%) T. cruzi infections, respectively. The sets of decision-trees were assembled to construct a sequential algorithm with expressive accuracy (81%) for serological diagnosis of T. cruzi infection. These findings engender new perspectives for the application of Chagas-Flow ATE-IgG2a method for genotype-specific diagnosis in humans, with relevant contributions for epidemiological surveys as well as clinical and post-therapeutic monitoring of Chagas disease.

Human Chagas-Flow ATE-IgG1 for advanced universal and Trypanosoma cruzi Discrete Typing Units-specific serodiagnosis of Chagas disease

“…This phenomenon may reflects the phylogenetic origin of T. cruzi DTUs 5 indicating TcI and TcII as ancestors DTUs with polarized biological features 8,9,[60][61][62][63][64] and TcVI as a hybrid DTU presenting intermediate profile 8,60,61 . Corroborating the previous findings from Alessio et al 30 for experimental T. cruzi infection, the present work also identified TcI antigen as the root attribute in the decision tree algorithms proposed for DTUspecific diagnosis of human infection.…”

“…Battacharyya et al 27 demonstrated that TSSA lineage serology lacks specificity to detect TcI DTU. More recently, the Chagas-Flow ATE-IgG2a has been presented as an innovative method with outstanding performance for universal and DTU-specific diagnosis of experimental T. cruzi infection 29,30 . Moreover, the Human Chagas-Flow ATE-IgG1 methodology has been developed and standardized showing elevated performance for universal diagnosis and post-therapeutic cure assessment in Chagas disease 35 .…”

“…The amastigote, trypomastigote and epimastigote of the TcI, TcVI and TcII T. cruzi DTUs were obtained as described previously by Alessio et al 30 . Briefly, live trypomastigotes (T) and amastigotes (A) forms were harvested from supernatant of desynchronized L929 cell line cultures in vitro after 4–6 days and 8–15 days post-infection, respectively.…”

“…Recently, a flow cytometry-based test has been proposed as a strategy for DTU-specific serotyping. The Chagas-Flow ATE-IgG2a methodology has been standardized for the DTU-specific diagnosis of experimental T. cruzi infection displaying high performance to discriminate the hosts infected with distinct T. cruzi DTUs 29,30 . The present study show the Human Chagas-Flow ATE-IgG1 as a promising technique for advanced universal and DTU-specific serodiagnosis of Chagas disease.…”

A lineage-specific rapid diagnostic test (Chagas Sero K-SeT) identifies Brazilian Trypanosoma cruzi II/V/VI reservoir hosts among diverse mammalian orders

“…Mixed infections, such as TcI and TcII, may occur in single mammals, and are not currently detectable by lineage-specific serology; nevertheless, due to sustained infection antibodies to TcII should not be lost. The Chagas Sero K-SeT RDT described here does not make redundant T. cruzi genotyping, or advanced analytical techniques such as flow cytometry [47]; however, by detecting lineage-specific host IgG, it can provide epidemiological information when direct parasite genotyping is hampered or subject to confounding biases.…”