Comparative evaluation of four rapid diagnostic tests that detect human Trypanosoma cruzi-specific antibodies to support diagnosis of Chagas Disease in urban population of Argentina.

BACKGROUND: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is the most important endemic anthropozoonosis in Argentina. Since 2010, the World Health Organization has highlighted the urgent need to validate diagnostic systems that allow rapid detection of T. cruzi, infection in primary healthcare centers. Serological rapid diagnostic tests (RDTs) for T. cruzi, infection could be used to improve case management, as RDTs do not require specialized laboratories or highly trained staff to use them. We aimed to generate unbiased performance data of RDTs in Argentina, to evaluate their usefulness for improving T. cruzi, diagnosis rates. METHODS AND PRINCIPAL FINDINGS: This is a retrospective, laboratory-based, diagnostic evaluation study to estimate the clinical sensitivity/specificity of four commercially available RDTs for T. cruzi, using the Chagas disease diagnostic algorithm currently used in Argentina as the reference standard. In total, 400 serum samples were tested, 200 from individuals with chronic T. cruzi infection and 200 from individuals not infected with T. cruzi. All results were registered as the agreement of at least two operators who were blinded to the reference standard results. The sensitivity estimates ranged from 92.5-100% (95% confidence interval (CI) lower bound 87.9-98.2%); for specificity, the range was 76-96% (95% CI lower bound 69.5-92.3%). Most RDTs evaluated showed performances comparable with the reference standard method, showing almost perfect concordance (Kappa 0.76-0.92). CONCLUSIONS: Our study demonstrates that, under controlled laboratory conditions, commercially available RDTs for CD have a performance comparable to the Argentinian diagnostic algorithm, which is based on laboratory-based serological tests. For the next stage of our work, the RDTs will be evaluated in real-world settings.

ELISA F29 -A therapeutic efficacy biomarker in Chagas disease: Evaluation in pediatric patients treated with nifurtimox and followed for 4 years post-treatment.

BACKGROUND: Measurement of the success of antitrypanosomal treatment for Chagas disease is difficult, particularly in the chronic phase of the disease, because anti-Trypanosoma cruzi antibodies persist in serum for prolonged periods. We studied the effects of nifurtimox administered by two different treatment regimens on the T. cruzi calcium-binding flagellar protein F29 in children diagnosed with Chagas disease measured using an enzyme-linked immunosorbent assay (ELISA) technique (ELISA F29). METHODS AND PRINCIPAL FINDINGS: In a phase 3, randomized, double-blind, parallel-group, historically controlled study (ClinicalTrials.gov NCT02625974), blood samples obtained from children diagnosed with Chagas disease and treated with nifurtimox for either 60 days or 30 days were analyzed using an ELISA with an F29 recombinant protein as the antigen, as well as conventional serological tests (recombinant ELISA and indirect hemagglutination assay). In an exploratory approach, serological response to nifurtimox treatment was evaluated for 4 years post-treatment. In both treatment groups, the number of patients with negative ELISA F29 values increased over the period of observation. The incidence rate of negative seroconversion using ELISA F29 was 22.94% (95% CI: 19.65%, 26.63%) in the 60-day treatment group and 21.64% (95% CI: 17.21%, 26.86%) in the 30-day treatment group. In the subpopulation of patients who tested seropositive for F29 before nifurtimox treatment, 88 patients (67.7%) in the 60-day regimen and 39 patients (59.1%) in the 30-day regimen were F29 seronegative at 4 years post-treatment. All patients who had a positive ELISA F29 test at baseline and seroconverted to negative measured by conventional serology reached seronegativity in ELISA F29 earlier than in conventional serology. CONCLUSIONS: The results demonstrate a serological response to treatment with nifurtimox measured by the ELISA F29 test in children diagnosed with Chagas disease. The F29-based ELISA can be considered a potential early marker of response to antitrypanosomal therapy for Chagas disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT02625974.

Effectiveness of the repurposed drug isotretinoin in an experimental murine model of Chagas disease.

Benznidazole and nifurtimox are the drugs currently used for the treatment of Chagas disease, however its side effects may affect patient adherence. In the search for new alternative therapies, we previously identified isotretinoin (ISO), an FDA-approved drug widely used for the treatment of severe acne through a drug repurposing strategy. ISO shows a strong activity against Trypanosoma cruzi parasites in the nanomolar range, and its mechanism of action is through the inhibition of T. cruzi polyamine and amino acid transporters from the Amino Acid/Auxin Permeases (AAAP) family. In this work, a murine model of chronic Chagas disease (C57BL/6 J mice), intraperitoneally infected with T. cruzi Nicaragua isolate (DTU TcI), were treated with different oral administrations of ISO: daily doses of 5 mg/kg/day for 30 days and weekly doses of 10 mg/kg during 13 weeks. The efficacy of the treatments was evaluated by monitoring blood parasitemia by qPCR, anti-T. cruzi antibodies by ELISA, and cardiac abnormalities by electrocardiography. No parasites were detected in blood after any of the ISO treatments. The electrocardiographic study of the untreated chronic mice showed a significant decrease in heart rate, while in the treated mice this negative chronotropic effect was not observed. Atrioventricular nodal conduction time in untreated mice was significantly longer than in treated animals. Mice treated even with ISO 10 mg/kg dose every 7 days, showed a significant reduction in anti-T. cruzi IgG levels. In conclusion, the intermittent administration of ISO 10 mg/kg would improve myocardial compromise during the chronic stage.

Prospective multicenter evaluation of real time PCR Kit prototype for early diagnosis of congenital Chagas disease.

BACKGROUND: Current algorithm for Congenital Chagas Disease (cCD) diagnosis is unsatisfactory due to low sensitivity of the parasitological methods. Moreover, loss to follow-up precludes final serodiagnosis after nine months of life in many cases. A duplex TaqMan qPCR kit for Trypanosoma cruzi DNA amplification was prospectively evaluated in umbilical cord (UCB) and peripheral venous blood (PVB) of infants born to CD mothers at endemic and non-endemic sites of Argentina. METHODS: We enrolled and followed-up 370 infants; qPCR was compared to gold-standard cCD diagnosis following studies of diagnostic accuracy guidelines. FINDINGS: Fourteen infants (3·78%) had cCD. The qPCR sensitivity and specificity were higher in PVB (72·73%, 99·15% respectively) than in UCB (66·67%, 96·3%). Positive and negative predictive values were 80 and 98·73% and 50 and 98·11% for PVB and UCB, respectively. The Areas under the Curve (AUC) of ROC analysis for qPCR and micromethod (MM) were 0·81 and 0·67 in UCB and 0·86 and 0·68 in PVB, respectively. Parasitic loads ranged from 37·5 to 23,709 parasite equivalents/mL. Discrete typing Unit Tc V was identified in five cCD patients and in six other cCD cases no distinction among Tc II, Tc V or Tc VI was achieved. INTERPRETATION: This first prospective field study demonstrated that qPCR was more sensitive than MM for early cCD detection and more accurate in PVB than in UCB. Its use, as an auxiliary diagnostic tool to MM will provide more accurate records on cCD incidence. FUNDING: FITS SALUD 001-CHAGAS (FONARSEC, MINCyT, Argentina) to the Public-Private Consortium (INGEBI-CONICET, INP-ANLIS MALBRAN and Wiener Laboratories); ERANET-LAC-HD 328 to AGS and PICT 2015-0074 (FONCYT, MinCyT) to AGS and FA.

Serological reactivity against T. cruzi-derived antigens: Evaluation of their suitability for the assessment of response to treatment in chronic Chagas disease.

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects more than 6 million people worldwide. Following a mostly asymptomatic acute phase, the disease progresses to a long-lasting chronic phase throughout which life-threatening disorders to the heart and/or gastrointestinal tract will manifest in about 30% of those chronically infected. During the chronic phase, the parasitemia is low and intermittent, while a high level of anti-T. cruzi antibodies persist for years. These two features hamper post-chemotherapeutic follow-up of patients with the tools available. The lack of biomarkers for timely assessment of therapeutic response discourages a greater use of the two available anti-parasitic drugs, and complicates the evaluation of new drugs in clinical trials. Herein, we investigated in a blinded case-control study the serological reactivity over time of a group of parasite-derived antigens to potentially address follow up of T. cruzi chronically infected subjects after treatment. We tested PFR2, KMP11, HSP70, 3973, F29 and the InfYnity multiplexed antigenic array, by means of serological assays on a multi-national retrospective collection of samples. Some of the antigens exhibited promising results, underscoring the need for further studies to determine their potential role as treatment response biomarkers.

Cruzipain Sulfotopes-Specific Antibodies Generate Cardiac Tissue Abnormalities and Favor Trypanosoma cruzi Infection in the BALB/c Mice Model of Experimental Chagas Disease.

Trypanosoma cruzi cruzipain (Cz) bears a C-terminal domain (C-T) that contains sulfated epitopes "sulfotopes" (GlcNAc6S) on its unique N-glycosylation site. The effects of in vivo exposure to GlcNAc6S on heart tissue ultrastructure, immune responses, and along the outcome of infection by T. cruzi, were evaluated in a murine experimental model, BALB/c, using three independent strategies. First, mice were pre-exposed to C-T by immunization. C-T-immunized mice (C-TIM) showed IgG2a/IgG1 <1, induced the production of cytokines from Th2, Th17, and Th1 profiles with respect to those of dC-TIM, which only induced IL-10 respect to the control mice. Surprisingly, after sublethal challenge, both C-TIM and dC-TIM showed significantly higher parasitemia and mortality than the control group. Second, mice exposed to BSA-GlcNAc6S as immunogen (BSA-GlcNAc6SIM) showed: severe ultrastructural cardiac alterations while BSA-GlcNAcIM conserved the regular tissue architecture with slight myofibril changes; a strong highly specific humoral-immune-response reproducing the IgG-isotype-profile obtained with C-TIM; and a significant memory-T-cell-response demonstrating sulfotope-immunodominance with respect to BSA-GlcNAcIM. After sublethal challenge, BSA-GlcNAc6SIM showed exacerbated parasitemias, despite elevated IFN-γ levels were registered. In both cases, the abrogation of ultrastructural alterations when using desulfated immunogens supported the direct involvement of sulfotopes and/or indirect effect through their specific antibodies, in the induction of tissue damage. Finally, a third strategy using a passive transference of sulfotope-specific antibodies (IgG-GlcNAc6S) showed the detrimental activity of IgG-GlcNAc6S on mice cardiac tissue, and mice treated with IgG-GlcNAc6S after a sublethal dose of T. cruzi, surprisingly reached higher parasitemias than control groups. These findings confirmed the indirect role of the sulfotopes, via their IgG-GlcNAc6S, both in the immunopathogenicity as well as favoring T. cruzi infection.

In vitro studies and preclinical evaluation of benznidazole microparticles in the acute Trypanosoma cruzi murine model.

Chagas disease is a serious parasitic infection caused by Trypanosoma cruzi. Unfortunately, the current chemotherapeutic tools are not enough to combat the infection. The aim of this study was to evaluate the trypanocidal activity of benznidazole-loaded microparticles during the acute phase of Chagas infection in an experimental murine model. Microparticles were prepared by spray-drying using copolymers derived from esters of acrylic and methacrylic acids as carriers. Dissolution efficiency of the formulations was up to 3.80-fold greater than that of raw benznidazole. Stability assay showed no significant difference (P > 0.05) in the loading capacity of microparticles for 3 years. Cell cultures showed no visible morphological changes or destabilization of the cell membrane nor haemolysis was observed in defibrinated human blood after microparticles treatment. Mice with acute lethal infection survived 100% after 30 days of treatment with benznidazole microparticles (50 mg kg-1 day-1). Furthermore, no detectable parasite load measured by quantitative polymerase chain reaction and lower levels of T. cruzi-specific antibodies by enzyme-linked immunosorbent assay were found in those mice. A significant decrease in the inflammation of heart tissue after treatment with these microparticles was observed, in comparison with the inflammatory damage observed in both infected mice treated with raw benznidazole and untreated infected mice. Therefore, these polymeric formulations are an attractive approach to treat Chagas disease.

Effect of the Tc13Tul antigen from Trypanosoma cruzi on splenocytes from naïve mice.

Trypanosoma cruzi, the etiological agent of Chagas disease, releases factors, including antigens from the trans-sialidase (TS) superfamily, which modulate the host immune responses. Tc13 antigens belong to group IV of TSs and are characterized by C-terminal EPKSA repeats. Here, we studied the effect of the Tc13 antigen from the Tulahuén strain, Tc13Tul, on primary cultures of splenocytes from naïve BALB/c mice. Recombinant Tc13Tul increased the percentage of viable cells and induced B (CD19+) lymphocyte proliferation. Tc13Tul stimulation also induced secretion of non-specific IgM and interferon-γ (IFN-γ). The same effects were induced by Tc13Tul on splenocytes from naïve C3H/HeJ mice. In vivo administration of Tc13Tul to naïve BALB/c mice increased non-specific IgG in sera. In addition, in vitro cultured splenocytes from Tc13Tul-inoculated mice secreted a higher basal level of non-specific IgM than controls and the in vitro Tc13Tul stimulation of these cells showed an enhanced effect on IgM and IFN-γ secretion. Our results indicate that Tc13Tul may participate in the early immunity in T. cruzi infection by favouring immune system evasion through B-cell activation and non-specific Ig secretion. In contrast, as IFN-γ is an important factor involved in T. cruzi resistance, this may be considered a Tc13Tul effect in favour of the host.

Tamoxifen acts on Trypanosoma cruzi sphingolipid pathway triggering an apoptotic death process.

This study shows the effects of tamoxifen, a known estrogen receptor antagonist used in the treatment of breast cancer, on the sphingolipid pathway of Trypanosoma cruzi, searching for potential chemotherapeutic targets. A dose-dependent epimastigote growth inhibition at increasing concentration of tamoxifen was determined. In blood trypomastigotes, treatment with 10 µM showed 90% lysis, while 86% inhibition of intracellular amastigote development was obtained using 50 µM. Lipid extracts from treated and non-treated metabolically labelled epimastigotes evidenced by thin layer chromatography different levels of sphingolipids and MALDI-TOF mass spectrometry analysis assured the identity of the labelled species. Comparison by HPLC-ESI mass spectrometry of lipids, notably exhibited a dramatic increase in the level of ceramide in tamoxifen-treated parasites and a restrained increase of ceramide-1P and sphingosine, indicating that the drug is acting on the enzymes involved in the final breakdown of ceramide. The ultrastructural analysis of treated parasites revealed characteristic morphology of cells undergoing an apoptotic-like death process. Flow cytometry confirmed cell death by an apoptotic-like machinery indicating that tamoxifen triggers this process by acting on the parasitic sphingolipid pathway.

Experimental combination therapy using low doses of benznidazole and allopurinol in mouse models of Trypanosoma cruzi chronic infection.

This study evaluated the effectiveness of low doses of benznidazole (BNZ) on continuous administration (BNZc), combined with allopurinol (ALO), in C57BL/6J and C3H/HeN mice infected with Trypanosoma cruzi Nicaragua strain and T. cruzi Sylvio-X10/4 clone. TcN-C57BL/6J was also treated with intermittent doses of BNZ (BNZit). The drug therapy started 3 months post infection (pi) in the chronic phase of mice with heart disease progression, followed-up at 6 months pi. TcN-C57BL/6J treated with BNZc was also monitored up to 12 months pi by serology and electrocardiogram. These mice showed severe electrical abnormalities, which were not observed after BNZc or BNZit. ALO only showed positive interaction with the lowest dose of BNZ. A clear parasitic effect, with significant reductions in antibody titres and parasitic loads, was achieved in all models with low doses of BNZ, and a 25% reduction of the conventional dose showed more efficacy to inhibit the development of the pathology. However, BNZ 75 showed partial efficacy in the TcSylvio-X10/4-C3H/HeN model. In our experimental designs, C57BL/6J allowed to clearly define a chronic phase, and through reproducible efficacy indicators, it can be considered a good preclinical model.

Chagas cardiomyopathy associated with serological cure after trypanocidal treatment during childhood.

Chagas disease is a chronic parasitological disease, which could cause cardiac manifestations in approximately one-third of affected individuals. Benznidazole and nifurtimox are used to treat this parasitological infection caused by Trypanosoma cruzi. Conventionally, the criterion for cure is consistently negative serological tests after treatment. We report a case of a patient who was treated when she was 13 years old and achieved T. cruzi negative seroconversion but developed Chagas disease cardiomyopathy as an adult.

Chagas cardiomyopathy associated with serological cure after trypanocidal treatment during childhood

Abstract Chagas disease is a chronic parasitological disease, which could cause cardiac manifestations in approximately one-third of affected individuals. Benznidazole and nifurtimox are used to treat this parasitological infection caused by Trypanosoma cruzi. Conventionally, the criterion for cure is consistently negative serological tests after treatment. We report a case of a patient who was treated when she was 13 years old and achieved T. cruzi negative seroconversion but developed Chagas disease cardiomyopathy as an adult.

Elucidating the impact of low doses of nano-formulated benznidazole in acute experimental Chagas disease.

BACKGROUND: Chagas disease is a neglected parasitic infection caused by the protozoan Trypanosoma cruzi (T. cruzi) that affects more than 6 million people, mainly in Latin America. Benznidazole is still the drug of choice in many countries to treat it in spite of its dosage regimen and adverse side effects such as such as allergic dermatitis, peripheral neuropathy and anorexia. Thus, novel, safer, and more efficacious treatments for such neglected infection are urgently required. METHODOLOGY: In this study, the efficacy of orally administered low doses of benznidazole (BNZ) nanoparticles was evaluated during the acute phase in mice infected with T. cruzi Nicaragua (TcN) that were immunosuppressed during the chronic stage of the disease. Moreover, the production of T. cruzi-specific antibodies, cardiac tissue inflammation and reactive oxygen species generation by Vero cells treated with both BNZ nanoparticles (BNZ-nps) and raw BNZ (R-BNZ) were also evaluated. PRINCIPAL FINDINGS: T. cruzi infected mice treated with 10, 25 or 50 mg/kg/day of BNZ-nps survived until euthanasia (92 days post infection (dpi)), while only 15% of infected untreated mice survived until the end of the experiment. PCR analysis of blood samples taken after induction of immunosuppression showed that a dosage of 25 mg/kg/day rendered 40% of the mice PCR-negative. The histological analysis of heart tissue showed a significant decrease in inflammation after treatments with 25 and 50 mg/kg/day, while a similar inflammatory damage was observed in both infected mice treated with R-BNZ (50 mg/kg/day) and untreated mice. In addition, only BNZ-nps treated mice led to lower levels of T. cruzi-specific antibodies to 50-100%. Finally, mammalian Vero cells treated with BNZ-nps or R-BNZ lead to a significant increase in ROS production. CONCLUSIONS: Based on these findings, this research highlights the in-vitro/in-vivo efficacy of nanoformulated BNZ against T. cruzi acute infections in immunosuppressed and non-immunosuppressed mice and provides further evidence for the optimization of dosage regimens to treat Chagas disease.

Rapid detection of Trypanosoma cruzi by colorimetric loop-mediated isothermal amplification (LAMP): A potential novel tool for the detection of congenital Chagas infection.

Early diagnosis of congenital Trypanosomacruzi transmission in newborns is essential because babies show high indices of cure. Conventional diagnosis is based on microscopic examination and serology. Molecular diagnosis is a promising alternative to replace conventional diagnosis, although it is not well suited for adoption in laboratories with limited resources. Isothermal DNA amplification methods have the advantage of not requiring expensive equipment. The aim of this work was to apply loop-mediated isothermal amplification (LAMP) to detect congenital infection in babies colorimetrically. This assay was able to detect all T. cruzi discrete typing units and Leishmania braziliensis, but not other pathogens. The assay showed a limit of detection of 50 parasites/mL in spiked artificial samples. This assay was tested in 27 blood samples of babies born to T. cruzi-infected mothers and showed 100% of concordance with conventional diagnosis. This is the first study to detect T. cruzi in clinical samples by LAMP, showing that this assay would be useful in the detection of congenital T. cruzi infection. The advantages of this novel tool include the speed with which the assays can be completed, the no-need of trained personnel, and the fact that it can be performed without complex and expensive laboratory equipment.

Promising Efficacy of Benznidazole Nanoparticles in Acute Trypanosoma cruzi Murine Model: In-Vitro and In-Vivo Studies.

The aim of this study was to evaluate the effectiveness of benznidazole nanoparticles (BNZ-nps) on trypomastigote forms and on intracellular infection in mammalian cells and primary cardiac myocyte cells. Its effectiveness was also evaluated on acute Trypanosoma cruzi Nicaragua mice infection. Trypomastigotes from culture were treated with different concentrations of BNZ-nps to determine the drug concentration that lyses 50% of trypomastigotes (LC50). Infected mammalian cells were incubated with different concentrations of BNZ-nps to determine the percentage of amastigote inhibition. C3H/HeN mice with lethal acute infection were treated with 10, 25, and 50 mg/kg/day of BNZ-nps for 30 and 15 days to control the survival rate of animals. BNZ-nps having a mean particle size of 63.3 nm, a size distribution of 3.35, and a zeta potential of -18.30 were successfully prepared using poloxamer 188 as a stabilizer. BNZ-nps 25 and 50 µg/mL showed no significant differences in the percentage of inhibition of infected mammalian cells. Infected mice treated with BNZ-nps (50, 25, and 10 mg/kg/day) for 30 days and with BNZ-nps (50 and 25 mg/kg/day) for 15 days presented a 100% survival, whereas the animals treated with 10 mg/kg/day for 15 days of BNZ-nps showed a 70% survival rate. The results obtained demonstrate, for the first time, that benznidazole nanoparticles are a useful and attractive approach to treat Chagas disease in infected mice.

Involvement of sulfates from cruzipain, a major antigen of Trypanosoma cruzi, in the interaction with immunomodulatory molecule Siglec-E.

In order to investigate the involvement of sulfated groups in the Trypanosoma cruzi host-parasite relationship, we studied the interaction between the major cysteine proteinase of T. cruzi, cruzipain (Cz), a sulfate-containing sialylated molecule and the sialic acid-binding immunoglobulin like lectin-E (Siglec-E). To this aim, ELISA, indirect immunofluorescence assays and flow cytometry, using mouse Siglec-E-Fc fusion molecules and glycoproteins of parasites, were performed. Competition assays verified that the lectins, Maackia amurensis II (Mal II) and Siglec-E-Fc, compete for the same binding sites. Taking into account that Mal II binding remains unaltered by sulfation, we established this lectin as sialylation degree control. Proteins of an enriched microsomal fraction showed the highest binding to Siglec-E as compared with those from the other parasite subcellular fractions. ELISA assays and the affinity purification of Cz by a Siglec-E column confirmed the interaction between both molecules. The significant decrease in binding of Siglec-E-Fc to Cz and to its C-terminal domain (C-T) after desulfation of these molecules suggests that sulfates contribute to the interaction between Siglec-E-Fc and these glycoproteins. Competitive ELISA assays confirmed the involvement of sulfated epitopes in the affinity between Siglec-E and Cz, probably modified by natural protein environment. Interestingly, data from flow cytometry of untreated and chlorate-treated parasites suggested that sulfates are not primary receptors, but enhance the binding of Siglec-E to trypomastigotic forms. Altogether, our findings support the notion that sulfate-containing sialylated glycoproteins interact with Siglec-E, an ortholog protein of human Siglec-9, and might modulate the immune response of the host, favoring parasitemia and persistence of the parasite.

Predictive role of polymerase chain reaction in the early diagnosis of congenital Trypanosoma cruzi infection.

The efficacy of specific chemotherapy in congenital Chagas disease before the first year of life ranges between 90 and 100%. Between this age and 15 years of age, the efficacy decreases to around 60%. Therefore, early infection detection is a priority in vertical transmission. The aim of this work was to assess whether polymerase chain reaction (PCR) plays a predictive role in the diagnosis of congenital Chagas disease as compared to conventional parasitological and serological methods. To this end, we studied a total of 468 children born to Trypanosoma cruzi seroreactive mothers came from Argentina, Bolivia and Paraguay, who lived in the city of Buenos Aires and suburban areas (Argentina), a non-endemic area of this country. These children were assessed by PCR from 2004 to 2009 with the specific primers Tcz1 and Tcz2, and 121 and 122. PCR allowed detecting 49 T. cruzi-positive children. Eight of these 49 children were excluded from the analysis: six because they did not complete follow-up and two because the first control was performed after 12 months of age. Parasitological methods allowed detecting 25 positive children, 7 of whom had been earlier diagnosed by PCR (1.53±2.00 vs. 6.71±1.46 months; p=0.0002). Serological methods allowed detecting 16 positive children, 12 of whom had been earlier diagnosed by PCR (1.46±1.48 vs. 11.77±4.40 months; p<0.0001). None of the children negative by PCR was positive by serological or parasitological methods. This study shows that PCR allows early diagnosis in congenital Chagas disease. At present, an early positive PCR is not indicative for treatment. However, a positive PCR would alert the health system to search only those infected infants diagnosed by early PCR and thus generate greater efficiency in the diagnosis and treatment of congenital T. cruzi infection.

Effects of chlorate on the sulfation process of Trypanosoma cruzi glycoconjugates. Implication of parasite sulfates in cellular invasion.

Sulfation, a post-translational modification which plays a key role in various biological processes, is inhibited by competition with chlorate. In Trypanosoma cruzi, the agent of Chagas' disease, sulfated structures have been described as part of glycolipids and we have reported sulfated high-mannose type oligosaccharides in the C-T domain of the cruzipain (Cz) glycoprotein. However, sulfation pathways have not been described yet in this parasite. Herein, we studied the effect of chlorate treatment on T. cruzi with the aim to gain some knowledge about sulfation metabolism and the role of sulfated molecules in this parasite. In chlorate-treated epimastigotes, immunoblotting with anti-sulfates enriched Cz IgGs (AS-enriched IgGs) showed Cz undersulfation. Accordingly, a Cz mobility shift toward higher isoelectric points was observed in 2D-PAGE probed with anti-Cz antibodies. Ultrastructural membrane abnormalities and a significant decrease of dark lipid reservosomes were shown by electron microscopy and a significant decrease in sulfatide levels was confirmed by TLC/UV-MALDI-TOF-MS analysis. Altogether, these results suggest T. cruzi sulfation occurs via PAPS. Sulfated epitopes in trypomastigote and amastigote forms were evidenced using AS-enriched IgGs by immunoblotting. Their presence on trypomastigotes surface was demonstrated by flow cytometry and IF with Cz/dCz specific antibodies. Interestingly, the percentage of infected cardiac HL-1 cells decreased 40% when using chlorate-treated trypomastigotes, suggesting sulfates are involved in the invasion process. The same effect was observed when cells were pre-incubated with dCz, dC-T or an anti-high mannose receptor (HMR) antibody, suggesting Cz sulfates and HMR are also involved in the infection process by T. cruzi.

Solanesyl diphosphate synthase, an enzyme of the ubiquinone synthetic pathway, is required throughout the life cycle of Trypanosoma brucei.

Ubiquinone 9 (UQ9), the expected product of the long-chain solanesyl diphosphate synthase of Trypanosoma brucei (TbSPPS), has a central role in reoxidation of reducing equivalents in the mitochondrion of T. brucei. The ablation of TbSPPS gene expression by RNA interference increased the generation of reactive oxygen species and reduced cell growth and oxygen consumption. The addition of glycerol to the culture medium exacerbated the phenotype by blocking its endogenous generation and excretion. The participation of TbSPPS in UQ synthesis was further confirmed by growth rescue using UQ with 10 isoprenyl subunits (UQ10). Furthermore, the survival of infected mice was prolonged upon the downregulation of TbSPPS and/or the addition of glycerol to drinking water. TbSPPS is inhibited by 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid, and treatment with this compound was lethal for the cells. The findings that both UQ9 and ATP pools were severely depleted by the drug and that exogenous UQ10 was able to fully rescue growth of the inhibited parasites strongly suggest that TbSPPS and UQ synthesis are the main targets of the drug. These two strategies highlight the importance of TbSPPS for T. brucei, justifying further efforts to validate it as a new drug target.