In Vitro and in Vivo Study of a Photostable Quinone Compound with Enhanced Therapeutic Efficacy against Chagas Disease.

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi poses a significant health challenge in rural areas of Latin America. The current pharmacological options exhibit notable side effects, demand prolonged administration, and display limited efficacy. Consequently, there is an urgent need to develop drugs that are safe and clinically effective. Previously, we identified a quinone compound (designated as compound 2) with potent antiprotozoal activity, based on the chemical structure of komaroviquinone, a natural product renowned for its antitrypanosomal effects. However, compound 2 was demonstrated considerably unstable to light. In this study, we elucidated the structure of the light-induced degradation products of compound 2 and probed the correlation between the quinone ring's substituents and its susceptibility to light. Our findings led to the discovery of quinones with significantly enhanced light stability, some of which exhibiting antitrypanosomal activity. The most promising compound was evaluated for drug efficacy in a mouse model of Chagas disease, revealing where a notable reduction in blood parasitemia.

Anti-trypanosomal Lignans Isolated from Salvadoran Peperomia pseudopereskiifolia.

A search for anti-trypanosomal natural compounds from plants collected in El Salvador, a country particularly endemic for Chagas disease, resulted in the isolation of five lignan-type compounds (1-5) from Peperomia pseudopereskiifolia. The lignan derivatives 1, 2, and 4 are new. Their absolute configuration was determined by chemical derivatization. Compounds 1, 5, 6, and 8 exhibited anti-trypanosomal activity against the amastigote form of T. cruzi comparable to that of the existing drug benznidazole.

Integrated Computational Approaches for Drug Design Targeting Cruzipain.

Cruzipain inhibitors are required after medications to treat Chagas disease because of the need for safer, more effective treatments. Trypanosoma cruzi is the source of cruzipain, a crucial cysteine protease that has driven interest in using computational methods to create more effective inhibitors. We employed a 3D-QSAR model, using a dataset of 36 known inhibitors, and a pharmacophore model to identify potential inhibitors for cruzipain. We also built a deep learning model using the Deep purpose library, trained on 204 active compounds, and validated it with a specific test set. During a comprehensive screening of the Drug Bank database of 8533 molecules, pharmacophore and deep learning models identified 1012 and 340 drug-like molecules, respectively. These molecules were further evaluated through molecular docking, followed by induced-fit docking. Ultimately, molecular dynamics simulation was performed for the final potent inhibitors that exhibited strong binding interactions. These results present four novel cruzipain inhibitors that can inhibit the cruzipain protein of T. cruzi.

The Hidden Hand of Asymptomatic Infection Hinders Control of Neglected Tropical Diseases: A Modeling Analysis.

BACKGROUND: Neglected tropical diseases are responsible for considerable morbidity and mortality in low-income populations. International efforts have reduced their global burden, but transmission is persistent and case-finding-based interventions rarely target asymptomatic individuals. METHODS: We develop a generic mathematical modeling framework for analyzing the dynamics of visceral leishmaniasis in the Indian sub-continent (VL), gambiense sleeping sickness (gHAT), and Chagas disease and use it to assess the possible contribution of asymptomatics who later develop disease (pre-symptomatics) and those who do not (non-symptomatics) to the maintenance of infection. Plausible interventions, including active screening, vector control, and reduced time to detection, are simulated for the three diseases. RESULTS: We found that the high asymptomatic contribution to transmission for Chagas and gHAT and the apparently high basic reproductive number of VL may undermine long-term control. However, the ability to treat some asymptomatics for Chagas and gHAT should make them more controllable, albeit over relatively long time periods due to the slow dynamics of these diseases. For VL, the toxicity of available therapeutics means the asymptomatic population cannot currently be treated, but combining treatment of symptomatics and vector control could yield a quick reduction in transmission. CONCLUSIONS: Despite the uncertainty in natural history, it appears there is already a relatively good toolbox of interventions to eliminate gHAT, and it is likely that Chagas will need improvements to diagnostics and their use to better target pre-symptomatics. The situation for VL is less clear, and model predictions could be improved by additional empirical data. However, interventions may have to improve to successfully eliminate this disease.

Post-therapeutic cure criterion in chronic Chagas disease using Trypanosoma cruzi chimeric proteins.

Chagas disease (CD) is a neglected disease caused by Trypanosoma cruzi Chagas, 1909. Causative treatment can be achieved with two drugs: benznidazole or Nifurtimox. There are some gaps that hinder progress in eradicating the disease. There is no test that can efficiently assess cure control after treatment. Currently, the decline in anti-T. cruzi antibody titres is assessed with conventional serological tests, which can take years. However, the search for new markers of cure must continue to fill this gap. The present study aimed to evaluate the decline in serological titres using chimeric proteins after treatment with benznidazole in chronic patients diagnosed with CD. It was a prospective cross-sectional cohort study between 2000 and 2004 of T. cruzi-positive participants from the Añatuya region (Argentina) treated with benznidazole. Serum samples from ten patients were collected before treatment (day zero) and after the end of treatment (2, 3, 6, 12, 24 and 36 months). For the detection of anti-T. cruzi antibodies, an indirect ELISA was performed using two chimeric recombinant proteins (IBMP-8.1 and IBMP-8.4) as antigens. The changes in reactivity index within the groups before and after treatment were evaluated using the Friedman test. All participants experienced a decrease in serological titres after treatment with benznidazole, especially IBMP-8.1. However, due to the small number of samples and the short follow-up period, it is premature to conclude that this molecule serves as a criterion for sustained cure. Further studies are needed to validate tests based on these or other biomarkers to demonstrate parasitological cure.

Acrylonitrile derivatives: In vitro activity and mechanism of cell death induction against Trypanosoma cruzi and Leishmania amazonensis.

Leishmaniasis and Chagas disease are parasitic infections that affect millions of people worldwide, producing thousands of deaths per year. The current treatments against these pathologies are not totally effective and produce some side effects in the patients. Acrylonitrile derivatives are a group of compounds that have shown activity against these two diseases. In this work, four novels synthetic acrylonitriles were evaluated against the intracellular form and extracellular forms of L. amazonensis and T. cruzi. The compounds 2 and 3 demonstrate to have good selectivity indexes against both parasites, specifically the compound 3 against the amastigote form (SI = 6 against L. amazonensis and SI = 7.4 against T. cruzi). In addition, the parasites treated with these two compounds demonstrate to produce a programmed cell death, since they were positive for the events studied related to this type of death, including chromatin condensation, accumulation of reactive oxygen species and alteration of the mitochondrial membrane potential. In conclusion, this work confirms that acrylonitriles is a source of possible new compounds against kinetoplastids, however, more studies are needed to corroborate this activity.

Synthesis of new trypanocidal agents from the hybridisation of metronidazole and eugenol analogues.

Nitroimidazole compounds are well-known bioactive substances, and the structural activity relationship has been reported whereby the position of the nitro group within the imidazole ring has a large influence on the activity. This study focuses on synthesising new trypanocidal agents from the hybridisation of metronidazole with different natural phenols (eugenol, dihydroeugenol and guaiacol). Two different coupling methodologies have been explored in order to analyse the influence of the connector on bioactivity: i) classic direct esterification (AD compounds) and ii) "click" chemistry using a triazole connector (AC compounds). The in vitro trypanocidal tests show good results for both AC and AD hybrid compounds against both epimastigote and trypomastigote forms of T. cruzi. In silico studies showed positive data for most of the synthesised compounds and, in general present low toxicological risks. The AC compounds present lower ClogP (lipophilicity) values than those found for the AD series and higher TPSA (topological polar surface area) values, suggesting lower lipophilicity may be related to the presence of the triazole connector. The AD series compounds have higher Drug Score values than the AC series derivatives, suggesting better general properties for a pharmacological action.

TNF blockers alone and associated with Benznidazole impact in vitro cytokine dynamics in chronic Chagas disease.

Studies involving the immune response in Chagas disease suggest an imbalance in the immune response of symptomatic patients, with an inflammatory profile dominating in Chagas heart disease, mainly by tumour necrosis factor (TNF). TNF is considered a key cytokine in immunopathology in chronic carriers in several processes during the immune response. Our work aimed to evaluate regulatory (interleukin [IL]-4 and IL-10) and inflammatory (TNF, interferon-gamma [IFN-γ], IL-2 and IL-6) cytokines in peripheral blood mononuclear cells culture supernatants. of affected patients with undetermined clinical forms-IND (n = 13) mild heart form-CARD1 (n = 13) and severe cardiac form-CARD2 (n = 16), treated in vitro with two TNF blockers, Adalimumab (ADA) and Etanercept (ETA) alone or in association with Benznidazole (BZ). The results indicate that ADA was more competent in blocking TNF (compared to ETA) in all groups but with much lower levels in the CARD2 group. ETA statistically decreased TNF levels only in the CARD2 group. IFN-γ increased in the CARD2 group after treatment with ETA relative to ADA. IL-4 had its levels decreased when treated by both drugs. IL-2 was detected in cells from CARD2 carriers compared to the NEG group after treatment with both drugs. The association with BZ decreased levels of IL-2/TNF and increased IL-4. These data reinforce the participation of TNF in severe Chagas heart disease and bring perspectives on using these blockers in the immunological treatment of Chagas disease since the use of BZ is extremely limited in these patients.

A promising pipeline of preclinical drug candidates for leishmaniasis and chronic Chagas' disease.

The drug discovery pipeline for leishmaniasis and trypanosomiasis has been filling with novel chemical entities with known mechanisms of action. González et al. and Braillard et al. report a cytochrome bc1 complex inhibitor as another promising preclinical candidate for visceral leishmaniasis (VL) and, in combination with benznidazole, for chronic Chagas' disease (CCD).

Multiple myeloma and Chagas disease: qPCR as a marker for preemptive antiparasitic therapy: a case reports series and review.

Multiple myeloma (MM) associated with Chagas disease is rarely described. This disease and its therapy suppress T cell and macrophage functions and increase regulatory T cell function, allowing the increase of parasitemia and the risk of Chagas Disease Reactivation (CDR). We aimed to analyze the role of conventional (cPCR) and quantitative Polymerase Chain Reaction (qPCR) for prospective monitoring of T. cruzi parasitemia, searching for markers of preemptive antiparasitic therapy in MM patients with Chagas disease. Moreover, we investigated the incidence and management of hematological diseases and CDR both inside and outside the transplant setting in the MEDLINE database. We found 293 studies and included 31 of them. Around 1.9-2.0% of patients with Chagas disease were reported in patients undergoing Stem Cell Transplantation. One case of CDR was described in eight cases of MM and Chagas disease. We monitored nine MM and Chagas disease patients, seven under Autologous Stem Cell Transplantation (ASCT), during 44.56±32.10 months (mean±SD) using parasitological methods, cPCR, and qPCR. From these patients, three had parasitemia. In the first, up to 256 par Eq/mL were detected, starting from 28 months after ASCT. The second patient dropped out and died soon after the detection of 161.0 par Eq/mL. The third patient had a positive blood culture. Benznidazole induced fast negativity in two cases; followed by notably lower levels in one of them. Increased T. cruzi parasitemia was related to the severity of the underlying disease. We recommend parasitemia monitoring by qPCR for early introduction of preemptive antiparasitic therapy to avoid CDR.

Quantitative PCR as a marker for preemptive therapy and its role in therapeutic control in Trypanosoma cruzi/HIV coinfection.

BACKGROUND: Trypanosoma cruzi and HIV coinfection can evolve with depression of cellular immunity and increased parasitemia. We applied quantitative PCR (qPCR) as a marker for preemptive antiparasitic treatment to avoid fatal Chagas disease reactivation and analyzed the outcome of treated cases. METHODOLOGY: This mixed cross-sectional and longitudinal study included 171 Chagas disease patients, 60 coinfected with HIV. Of these 60 patients, ten showed Chagas disease reactivation, confirmed by parasites identified in the blood, cerebrospinal fluid, or tissues, 12 exhibited high parasitemia without reactivation, and 38 had low parasitemia and no reactivation. RESULTS: We showed, for the first time, the success of the timely introduction of benznidazole in the non-reactivated group with high levels of parasitemia detected by qPCR and the absence of parasites in reactivated cases with at least 58 days of benznidazole. All HIV+ patients with or without reactivation had a 4.0-5.1 higher chance of having parasitemia than HIV seronegative cases. A positive correlation was found between parasites and viral loads. Remarkably, treated T. cruzi/HIV-coinfected patients had 77.3% conversion from positive to negative parasitemia compared to 19.1% of untreated patients. Additionally, untreated patients showed ~13.6 times higher Odds Ratio of having positive parasitemia in the follow-up period compared with treated patients. Treated and untreated patients showed no differences regarding the evolution of Chagas disease. The main factors associated with all-cause mortality were higher parasitemia, lower CD4 counts/µL, higher viral load, and absence of antiretroviral therapy. CONCLUSION: We recommend qPCR prospective monitoring of T. cruzi parasitemia in HIV+ coinfected patients and point out the value of pre-emptive therapy for those with high parasitemia. In parallel, early antiretroviral therapy introduction is advisable, aiming at viral load control, immune response restoration, and increasing survival. We also suggest an early antiparasitic treatment for all coinfected patients, followed by effectiveness analysis alongside antiretroviral therapy.

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 µM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 µM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

Design and Synthesis of Novel 3-Nitro-1H-1,2,4-triazole-1,2,3-triazole-1,4-disubstituted Analogs as Promising Antitrypanosomatid Agents: Evaluation of In Vitro Activity against Chagas Disease and Leishmaniasis.

A series of 28 compounds, 3-nitro-1H-1,2,4-triazole, were synthesized by click-chemistry with diverse substitution patterns using medicinal chemistry approaches, such as bioisosterism, Craig-plot, and the Topliss set with excellent yields. Overall, the analogs demonstrated relevant in vitro antitrypanosomatid activity. Analog 15g (R1 = 4-OCF3-Ph, IC50 = 0.09 µM, SI = >555.5) exhibited an outstanding antichagasic activity (Trypanosoma cruzi, Tulahuen LacZ strain) 68-fold more active than benznidazole (BZN, IC50 = 6.15 µM, SI = >8.13) with relevant selectivity index, and suitable LipE = 5.31. 15g was considered an appropriate substrate for the type I nitro reductases (TcNTR I), contributing to a likely potential mechanism of action for antichagasic activity. Finally, 15g showed nonmutagenic potential against Salmonella typhimurium strains (TA98, TA100, and TA102). Therefore, 3-nitro-1H-1,2,4-triazole 15g is a promising antitrypanosomatid candidate for in vivo studies.

Repositioning FDA-Approved Drug Against Chagas Disease and Cutaneous Leishmaniosis by Structure-Based Virtual Screening.

BACKGROUND: Chagas disease and cutaneous leishmaniasis, two parasitic diseases caused by Trypanosoma cruzi (T. cruzi) and Leishmania mexicana (L. mexicana), respectively, have a major global impact. Current pharmacological treatments for these diseases are limited and can cause severe side effects; thus, there is a need for new antiprotozoal drugs. METHODS: Using molecular docking, this work describes a structure-based virtual screening of an FDA-approved drug library against Trypanosoma cruzi and Leishmania mexicana glycolytic enzyme triosephosphate isomerase (TIM), which is highly conserved in these parasites. The selected compounds with potential dual inhibitory activity were tested in vitro to confirm their biological activity. RESULTS: The study showed that five compounds: nilotinib, chlorhexidine, protriptyline, cyproheptadine, and montelukast, were more active against T. cruzi, than the reference drugs, nifurtimox and benznidazole while chlorhexidine and protriptyline were the most active against L. mexicana. CONCLUSIONS: The analysis of these compounds and their structural characteristics may provide the basis for the development of new antiprotozoal agents.

Schistosoma mansoni co-infection modulates Chagas disease development but does not impair the effect of benznidazole-based chemotherapy.

The adequate management of parasite co-infections represents a challenge that has not yet been overcome, especially considering that the pathological outcomes and responses to treatment are poorly understood. Thus, this study aimed to evaluate the impact of Schistosoma mansoni infection on the efficacy of benznidazole (BZN)-based chemotherapy in Trypanosoma cruzi co-infected mice. BALB/c mice were maintained uninfected or co-infected with S. mansoni and T. cruzi, and were untreated or treated with BZN. Body weight, mortality, parasitemia, cardiac parasitism, circulating cytokines (Th1/Th2/Th17); as well as heart, liver and intestine microstructure were analyzed. The parasitemia peak was five times higher and myocarditis was more severe in co-infected than T. cruzi-infected mice. After reaching peak, parasitemia was effectively controlled in co-infected animals. BZN successfully controlled parasitemia in both co-infected and T. cruzi-infected mice and improved body mass, cardiac parasitism, myocarditis and survival in co-infected mice. Co-infection dampened the typical cytokine response to either parasite, and BZN reduced anti-inflammatory cytokines in co-infected mice. Despite BZN normalizing splenomegaly and liver cellular infiltration, it exacerbated hepatomegaly in co-infected mice. Co-infection or BZN exerted no effect on hepatic granulomas, but increased pulmonary and intestinal granulomas. Marked granulomatous inflammation was identified in the small intestine of all schistosomiasis groups. Taken together, our findings indicate that BZN retains its therapeutic efficacy against T. cruzi infection even in the presence of S. mansoni co-infection, but with organ-specific repercussions, especially in the liver.

Efficacy of short-course treatment for prevention of congenital transmission of Chagas disease: A retrospective cohort study.

BACKGROUND: In regions with controlled vector transmission of T. cruzi, congenital transmission is the most frequent route of infection. Treatment with benznidazole (BZ) or nifurtimox (NF) for 60 days in girls and women of childbearing age showed to be effective in preventing mother to child transmission of this disease. Reports on short-course treatment (≤30 days) are scarce. METHODS: Retrospective cohort study. Offspring of women with Chagas disease who received short-course treatment (≤30 days) with BZ or NF, attended between 2003 and 2022, were evaluated. Parasitemia (microhaematocrit and/or PCR) was performed at <8 months of age, and serology (ELISA and IHA) at ≥8 months to rule out congenital infection. RESULTS: A total of 27 women receiving ≤30 days of treatment and their children were included in this study. NF was prescribed in 17/27 (63%) women, and BZ in 10/27 (37%). The mean duration of treatment was 29.2 days. None of the women experienced serious adverse events during treatment, and no laboratory abnormalities were observed. Forty infants born to these 27 treated women were included. All newborns were full term, with appropriate weight for their gestational age. No perinatal infectious diseases or complications were observed. DISCUSSION: Several studies have shown that treatment of infected girls and women of childbearing age for 60 days is an effective practice to prevent transplacental transmission of T. cruzi. Our study demonstrated that short-duration treatment (≤30 days) is effective and beneficial in preventing transplacental transmission of Chagas disease.

Efficacy and safety of fexinidazole for treatment of chronic indeterminate Chagas disease (FEXI-12): a multicentre, randomised, double-blind, phase 2 trial.

BACKGROUND: More than six million people worldwide, particularly in vulnerable communities in Latin America, are infected with Trypanosoma cruzi, the causative agent of Chagas disease. Only a small portion have access to diagnosis and treatment. Both drugs used to treat this chronic, neglected infection, benznidazole and nifurtimox, were developed more than 50 years ago, and adverse drug reactions during treatment pose a major barrier, causing 20% of patients to discontinue therapy. Fexinidazole proved efficacious in an earlier, interrupted clinical trial, but the doses evaluated were not well tolerated. The present study evaluated fexinidazole at lower doses and for shorter treatment durations. METHODS: In this randomised, double-blind, phase 2 trial, we included adult patients (18-60 years old) with confirmed T cruzi infection by serology and PCR and without signs of organ involvement. We evaluated three regimens of fexinidazole-600 mg once daily for 10 days (6·0 g total dose), 1200 mg daily for 3 days (3·6 g), and 600 mg daily for 3 days followed by 1200 mg daily for 4 days (6·6 g)-and compared them with a historical placebo control group (n=47). The primary endpoint was sustained negative results by PCR at end of treatment and on each visit up to four months of follow-up. This study is registered with ClinicalTrials.gov, NCT03587766, and EudraCT, 2016-004905-15. FINDINGS: Between Oct 16, 2017, and Aug 7, 2018, we enrolled 45 patients (n=15 for each group), of whom 43 completed the study. Eight (19%) of 43 fexinidazole-treated patients reached the primary endpoint, compared with six (13%) of 46 in the historical control group. Mean parasite load decreased sharply following treatment but rebounded beginning 10 weeks after treatment. Five participants had seven grade 3 adverse events: carpal tunnel, sciatica, device infection, pneumonia, staphylococcal infection, and joint and device dislocation. Two participants discontinued treatment due to adverse events unrelated to fexinidazole. INTERPRETATION: The fexinidazole regimens in this study had an acceptable safety profile but did not prove effective against T cruzi infection. Development of fexinidazole monotherapy for treating T cruzi infection has been stopped. FUNDING: The Drugs for Neglected Diseases initiative.

Efficacy of three benznidazole dosing strategies for adults living with chronic Chagas disease (MULTIBENZ): an international, randomised, double-blind, phase 2b trial.

BACKGROUND: Treatment with benznidazole for chronic Chagas disease is associated with low cure rates and substantial toxicity. We aimed to compare the parasitological efficacy and safety of 3 different benznidazole regimens in adult patients with chronic Chagas disease. METHODS: The MULTIBENZ trial was an international, randomised, double-blind, phase 2b trial performed in Argentina, Brazil, Colombia, and Spain. We included participants aged 18 years and older diagnosed with Chagas disease with two different serological tests and detectable T cruzi DNA by qPCR in blood. Previously treated people, pregnant women, and people with severe cardiac forms were excluded. Participants were randomly assigned 1:1:1, using a balanced block randomisation scheme stratified by country, to receive benznidazole at three different doses: 300 mg/day for 60 days (control group), 150 mg/day for 60 days (low dose group), or 400 mg/day for 15 days (short treatment group). The primary outcome was the proportion of patients with a sustained parasitological negativity by qPCR during a follow-up period of 12 months. The primary safety outcome was the proportion of people who permanently discontinued the treatment. Both primary efficacy analysis and primary safety analysis were done in the intention-to-treat population. The trial is registered with EudraCT, 2016-003789-21, and ClinicalTrials.gov, NCT03191162, and is completed. FINDINGS: From April 20, 2017, to Sept 20, 2020, 245 people were enrolled, and 234 were randomly assigned: 78 to the control group, 77 to the low dose group, and 79 to the short treatment group. Sustained parasitological negativity was observed in 42 (54%) of 78 participants in the control group, 47 (61%) of 77 in the low dose group, and 46 (58%) of 79 in the short treatment group. Odds ratios were 1·41 (95% CI 0·69-2·88; p=0·34) when comparing the low dose and control groups and 1·23 (0·61-2·50; p=0·55) when comparing short treatment and control groups. 177 participants (76%) had an adverse event: 62 (79%) in the control group, 56 (73%) in the low dose group, and 59 (77%) in the short treatment group. However, discontinuations were less frequent in the short treatment group compared with the control group (2 [2%] vs 11 [14%]; OR 0·20, 95% CI 0·04-0·95; p=0·044). INTERPRETATION: Participants had a similar parasitological responses. However, reducing the usual treatment from 8 weeks to 2 weeks might maintain the same response while facilitating adherence and increasing treatment coverage. These findings should be confirmed in a phase 3 clinical trial. FUNDING: European Community's 7th Framework Programme.

An α-Gal antigenic surrogate as a biomarker of treatment evaluation in Trypanosoma cruzi-infected children. A retrospective cohort study.

BACKGROUND: Proper evaluation of therapeutic responses in Chagas disease is hampered by the prolonged persistence of antibodies to Trypanosoma cruzi measured by conventional serological tests and by the lack of sensitivity of parasitological tests. Previous studies indicated that tGPI-mucins, an α-Gal (α-d-Galp(1→3)-ß-d-Galp(1→4)-d-GlcNAc)-rich fraction obtained from T. cruzi trypomastigotes surface coat, elicit a strong and protective antibody response in infected individuals, which disappears soon after successful treatment. The cost and technical difficulties associated with tGPI-mucins preparation, however, preclude its routine implementation in clinical settings. METHODS/PRINCIPLE FINDINGS: We herein developed a neoglycoprotein consisting of a BSA scaffold decorated with several units of a synthetic α-Gal antigenic surrogate (α-d-Galp(1→3)-ß-d-Galp(1→4)-ß-d-Glcp). Serological responses to this reagent, termed NGP-Tri, were monitored by means of an in-house enzyme-linked immunosorbent assay (α-Gal-ELISA) in a cohort of 82 T. cruzi-infected and Benznidazole- or Nifurtimox-treated children (3 days to 16 years-old). This cohort was split into 3 groups based on the age of patients at the time of treatment initiation: Group 1 comprised 24 babies (3 days to 5 months-old; median = 26 days-old), Group 2 comprised 31 children (7 months to 3 years-old; median = 1.0-year-old) and Group 3 comprised 26 patients (3 to 16 years-old; median = 8.4 years-old). A second, control cohort (Group 4) included 39 non-infected infants (3 days to 5 months-old; median = 31 days-old) born to T. cruzi-infected mothers. Despite its suboptimal seroprevalence (58.4%), α-Gal-ELISA yielded shorter median time values of negativization (23 months [IC 95% 7 to 36 months] vs 60 months [IC 95% 15 to 83 months]; p = 0.0016) and higher rate of patient negative seroconversion (89.2% vs 43.2%, p < 0.005) as compared to conventional serological methods. The same effect was verified for every Group, when analyzed separately. Most remarkably, 14 out of 24 (58.3%) patients from Group 3 achieved negative seroconversion for α-Gal-ELISA while none of them were able to negativize for conventional serology. Detailed analysis of patients showing unconventional serological responses suggested that, in addition to providing a novel tool to shorten follow-up periods after chemotherapy, the α-Gal-ELISA may assist in other diagnostic needs in pediatric Chagas disease. CONCLUSIONS/SIGNIFICANCE: The tools evaluated here provide the cornerstone for the development of an efficacious, reliable, and straightforward post-therapeutic marker for pediatric Chagas disease.