Dogs are considered the main mammal reservoir of Trypanosoma cruzi in domiciliary environments. Consequently, accurate detection of T. cruzi infection in canine populations is epidemiologically relevant. Here, we analysed the utility of the T. cruzi recombinant antigens FRA, SAPA, CP1, Ag1 and a SAPA/TSSA VI mixture, in an ELISA format. We used a positive control group of sera obtained from 38 dogs from the Chaco region in Argentina with positive homogenate-ELISA reaction, all of them also positive by xenodiagnosis and/or PCR. The negative group included 19 dogs from a nonendemic area. Sensitivity, specificity, area under the curve (AUC) of the receiver operating charactheristic (ROC) curve and Kappa index were obtained to compare the diagnostic efficiency of the tests. The SAPA/TSSA VI had the highest performance, with a sensitivity of 94.7% and an AUC ROC of 0.99 that indicates high accuracy. Among individual antigens, SAPA-ELISA yielded the highest sensitivity (86.8%) and AUC ROC (0.96), whereas FRA-ELISA was the least efficient test (sensitivity = 36.8%; AUC ROC = 0.53). Our results showed that the use of SAPA/TSSA VI in ELISAs could be a useful tool to study dogs naturally infected with T. cruzi in endemic areas.

In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans.

The threadworm, Strongyloides stercoralis, is endemic in tropical and subtropical areas. Data on the prevalence and distribution of infection with this parasite species is scarce in many critical regions. We conducted a seroprevalence study of S. stercoralis infection in 13 locations in the Gran Chaco and Yungas regions of Argentina and Bolivia during the period 2010–2016. A total of 2803 human serum samples were analyzed by ELISA-NIE which has a sensitivity of 75% and specificity of 95%. Results showed that 551 (19.6%) of those samples were positive. The adjusted prevalence was 20.9%, (95% confidence interval (CI) 19.4–22.4%). The distribution of cases was similar between females and males with an increase of prevalence with age. The prevalence in the different locations ranged from 7.75% in Pampa del Indio to 44.55% in Santa Victoria Este in the triple border between Argentina, Bolivia, and Paraguay in the Chaco region. Our results show that S. stercoralis is highly prevalent in the Chaco and Yungas regions, which should prompt prospective surveys to confirm our findings and the design and deployment of control measures.

Trypanosoma cruzi, as other kinetoplastids, has a complex mechanism of editing of mitochondrial mRNAs that requires guide RNAs (gRNAs) coded in DNA minicircles in the kinetoplast. There are many variations on this mechanism among species. mRNA editing and gRNA repertoires are almost unknown in T. cruzi. Here, gRNAs were inferred based on deep-sequenced minicircle hypervariable regions (mHVRs) and editing cascades were rebuilt in strains belonging to the six main T. cruzi lineages. Inferred gRNAs were clustered according to their sequence similarity to constitute gRNA classes. Extreme diversity of gRNA classes was observed, which implied highly divergent gRNA repertoires among different lineages, even within some lineages. In addition, a variable gRNA class redundancy (i.e., different gRNA classes editing the same mRNA region) was detected among strains. Some strains had upon four times more gRNA classes than others. Such variations in redundancy affected gRNA classes of all mRNAs in a concerted way, i.e., there are correlated variations in the number of gRNAs classes editing each mRNA. Interestingly, cascades were incomplete for components of the respiratory complex I in several strains. Finally, gRNA classes of different strains may potentially edit mitochondrial mRNAs from other lineages in the same way as they edit their own mitochondrial mRNAs, which is a prerequisite for biparental inheritance of minicircle in hybrids. We propose that genetic exchange and biparental inheritance of minicircles combined with minicircle drift due to (partial) random segregation of minicircles during kDNA replication is a suitable hypothesis to explain the divergences among strains and the high levels of gRNA redundancy in some strains. In addition, our results support that the complex I may not be required in some stages in the life cycle as previously shown and that linkage (in the same minicircle) of gRNAs that edit different mRNAs may prevent gRNA class lost in such stage.

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BackgroundGenetic exchange in Trypanosoma cruzi is controversial not only in relation to its frequency, but also to its mechanism. Parasexual genetic exchange has been proposed based on laboratory hybrids, but population genomics strongly suggests meiosis in T. cruzi. In addition, mitochondrial introgression has been reported several times in natural isolates although its mechanism is not fully understood yet. Moreover, hybrid T. cruzi DTUs (TcV and TcVI) have inherited at least part of the kinetoplastic DNA (kDNA = mitochondrial DNA) from both parents. Methodology/Principal findingsIn order to address such topics, we sequenced and analyzed fourteen nuclear DNA fragments and three kDNA maxicircle genes in three TcI stocks which are natural clones potentially involved in events of genetic exchange. We also deep-sequenced (a total of 6,146,686 paired-end reads) the minicircle hypervariable region (mHVR) of the kDNA in such three strains. In addition, we analyzed the DNA content by flow cytometry to address cell ploidy. We observed that most polymorphic sites in nuclear loci showed a hybrid pattern in one cloned strain and the other two cloned strains were compatible as parental strains (or nearly related to the true parents). The three clones had almost the same ploidy and the DNA content was similar to the reference strain Sylvio (a nearly diploid strain). Despite maxicircle genes evolve faster than nuclear housekeeping ones, we detected no polymorphisms in the sequence of three maxicircle genes showing mito-nuclear discordance. Lastly, the hybrid stock shared 66% of its mHVR clusters with one putative parent and 47% with the other one; in contrast, the putative parental stocks shared less than 30% of the mHVR clusters between them.

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