The use of rapid diagnostic tests for chronic Chagas disease: An expert meeting report.

Chagas disease, caused by Trypanosoma cruzi, affects millions of people globally and is associated with significant underdiagnosis and undertreatment. Current diagnostic algorithms face challenges in remote regions. We aimed to review the potential of rapid diagnostic tests (RDTs) for screening or diagnosing chronic Chagas disease in endemic areas. An expert panel representing scientific and academic institutions from the Americas convened with the aim of discussing the use of RDTs. The study employed the nominal group technique, gathering insights from diverse experts during a 3-day meeting. Panel discussions covered RDT application, research protocols, and regulatory mechanisms. The results indicate that RDTs play a crucial role in surveillance and screening, although limitations in sensitivity and specificity exist. The expert group recommends standardized protocols, emphasizes the importance of cost-effectiveness assessments, and highlights the need to consider geographic validation. Despite these challenges, RDTs present a promising avenue for improving Chagas disease diagnosis in resource-limited settings. Future research and a collaborative approach are deemed essential for effective implementation.

The expression of immune response genes in patients with chronic Chagas disease is shifted toward the levels observed in healthy subjects as a result of treatment with Benznidazole.

Introduction: Chagas disease, caused by the Trypanosoma cruzi parasite infection, is a potentially life-threatening neglected tropical disease with a worldwide distribution. During the chronic phase of the disease, there exists a fragile balance between the host immune response and parasite replication that keeps patients in a clinically-silent asymptomatic stage for years or even decades. However, in 40% of patients, the disease progresses to clinical manifestations mainly affecting and compromising the cardiac system. Treatment is recommended in the chronic phase, although there are no early markers of its effectiveness. The aim of this study is to identify differential expression changes in genes involved in the immune response in antigen-restimulated PBMC from chronic patients with Chagas disease due to benznidazole treatment. Methods: Thus, high-throughput real-time qPCR analysis has been performed to simultaneously determine global changes in the expression of 106 genes involved in the immune response in asymptomatic (IND) and early cardiac manifestations (CCC I) Chagas disease patients pre- and post-treatment with benznidazole. Results and discussion: The results revealed that 7 out of the 106 analyzed genes were differentially expressed (4 up- and 3 downregulated) after treatment in IND patients and 15 out of 106 (3 up- and 12 downregulated) after treatment of early cardiac Chagas disease patients. Particularly in CCC I patients, regulation of the expression level of some of these genes towards a level similar to that of healthy subjects suggests a beneficial effect of treatment and supports recommendation of benznidazole administration to early cardiac Chagas disease patients. The data obtained also demonstrated that both in asymptomatic patients and in early cardiac chronic patients, after treatment with benznidazole there is a negative regulation of the proinflammatory and cytotoxic responses triggered as a consequence of T. cruzi infection and the persistence of the parasite. This downregulation of the immune response likely prevents marked tissue damage and healing in early cardiac patients, suggesting its positive effect in controlling the pathology.

Synthesis and Anti-Trypanosoma cruzi Activity of New Pyrazole-Thiadiazole Scaffolds.

Chagas disease, a silent but widespread disease that mainly affects a socioeconomically vulnerable population, lacks innovative safe drug therapy. The available drugs, benznidazole and nifurtimox, are more than fifty years old, have limited efficacy, and carry harmful side effects, highlighting the need for new therapeutics. This study presents two new series of pyrazole-thiadiazole compounds evaluated for trypanocidal activity using cellular models predictive of efficacy. Derivatives 1c (2,4-diCl) and 2k (4-NO2) were the most active against intracellular amastigotes. Derivative 1c also showed activity against trypomastigotes, with the detachment of the flagellum from the parasite body being a predominant effect at the ultrastructural level. Analogs have favorable physicochemical parameters and are predicted to be orally available. Drug efficacy was also evaluated in 3D cardiac microtissue, an important target tissue of Trypanosoma cruzi, with derivative 2k showing potent antiparasitic activity and a significant reduction in parasite load. Although 2k potentially reduced parasite load in the washout assay, it did not prevent parasite recrudescence. Drug combination analysis revealed an additive profile, which may lead to favorable clinical outcomes. Our data demonstrate the antiparasitic activity of pyrazole-thiadiazole derivatives and support the development of these compounds using new optimization strategies.

Tumor-Suppressive Cross-Linking of Anti-T. cruzi Antibodies in Acute Lymphoblastic Leukemia.

Parasites have been associated with possible anticancer activity, including Trypanosoma cruzi, which has been linked to inhibiting the growth of solid tumors. To better understand this antitumor effect, we investigated the association of anti-T. cruzi antibodies with B cells of the acute lymphoblastic leukemia (ALL) SUPB15 cell line. The antibodies were generated in rabbits. IgGs were purified by affinity chromatography. Two procedures (flow cytometry (CF) and Western blot(WB)) were employed to recognize anti-T. cruzi antibodies on SUPB15 cells. We also used CF to determine whether the anti-T. cruzi antibodies could suppress SUPB15 cells. The anti-T. cruzi antibodies recognized 35.5% of the surface antigens of SUPB15. The complement-dependent cytotoxicity (CDC) results demonstrate the cross-suppression of anti-T. cruzi antibodies on up to 8.4% of SUPB15 cells. For the WB analysis, a band at 100 kDa with high intensity was sequenced using mass spectrometry, identifying the protein as nucleolin. This protein may play a role in the antitumor effect on T. cruzi. The anti-T. cruzi antibodies represent promising polyclonal antibodies that have the effect of tumor-suppressive cross-linking on cancer cells, which should be further investigated.

Aminopyridines in the development of drug candidates against protozoan neglected tropical diseases.

Neglected tropical diseases (NTDs) pose a major threat in tropical zones for impoverished populations. Difficulty of access, adverse effects or low efficacy limit the use of current therapeutic options. Therefore, development of new drugs against NTDs is a necessity. Compounds containing an aminopyridine (AP) moiety are of great interest for the design of new anti-NTD drugs due to their intrinsic properties compared with their closest chemical structures. Currently, over 40 compounds with an AP moiety are on the market, but none is used against NTDs despite active research on APs. The aim of this review is to present the medicinal chemistry work carried out with these scaffolds, against protozoan NTDs: Trypanosoma cruzi, Trypanosoma brucei or Leishmania spp.

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The effects of inflammation on connexin 43 in chronic Chagas disease cardiomyopathy.

Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC. Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43S368 and Cx43S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1ß, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer. Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1ß, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43S368 and Cx43S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1ß, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells. Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease.

New insights into the pro-oxidant mechanism of dehydroleucodine on Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is one of the most important neglected diseases in Latin America. The limited use of the current nitro-derivative-based chemotherapy highlights the need for alternative drugs and the identification of their molecular targets. In this study, we investigated the trypanocidal effect of the sesquiterpene lactone dehydroleucodine (DhL) and its derivatives, focusing on the antioxidative defense of the parasites. DhL and two derivatives, at lesser extent, displayed antiproliferative effect on the parasites. This effect was blocked by the reducing agent glutathione (GSH). Treated parasites exhibited increased intracellular ROS concentration and trypanothione synthetase activity, accompanied by mitochondrial swelling. Although molecular dynamics studies predicted that GSH would not interact with DhL, 1H-NMR analysis confirmed that GSH could protect parasites by interacting with the lactone. When parasites overexpressing mitochondrial tryparedoxin peroxidase were incubated with DhL, its effect was attenuated. Overexpression of cytosolic tryparedoxin peroxidase also provided some protection against DhL. These findings suggest that DhL induces oxidative imbalance in T. cruzi, offering new insights into potential drug targets against this parasite.

A guide for the generation of repositories of clinical samples for research on Chagas disease.

Chagas disease, caused by the parasite Trypanosoma cruzi, affects over 6 million people, mainly in Latin America. Two different clinical phases, acute and chronic, are recognised. Currently, 2 anti-parasitic drugs are available to treat the disease (nifurtimox and benznidazole), but diagnostic methods require of a relatively complex infrastructure and trained personnel, limiting its widespread use in endemic areas, and the access of patients to treatment. New diagnostic methods, such as rapid tests (RDTs) to diagnose chronic Chagas disease, or loop-mediated isothermal amplification (LAMP), to detect acute infections, represent valuable alternatives, but the parasite's remarkable genetic diversity might make its implementation difficult. Furthermore, determining the efficacy of Chagas disease treatment is complicated, given the slow reversion of serological anti-T. cruzi antibody reactivity, which may even take decades to occur. New biomarkers to evaluate early therapeutic efficacy, as well as diagnostic tests able to detect the wide variety of circulating genotypes, are therefore, urgently required. To carry out studies that address these needs, high-quality and traceable samples from T. cruzi-infected individuals with different geographical backgrounds, along with associated clinical and epidemiological data, are necessary. This work describes the framework for the creation of such repositories, following standardised and uniform protocols, and considering the ethical, technical, and logistic aspects of the process. The manual can be adapted according to the resources of each laboratory, to guarantee that samples are obtained in a reproducible way, favouring the exchange of data among different work groups, and their generalizable evaluation and analysis. The main objective of this is to accelerate the development of new diagnostic methods and the identification of biomarkers for Chagas disease.

DNA lesions that block transcription induce the death of Trypanosoma cruzi via ATR activation, which is dependent on the presence of R-loops.

Trypanosoma cruzi is the etiological agent of Chagas disease and a peculiar eukaryote with unique biological characteristics. DNA damage can block RNA polymerase, activating transcription-coupled nucleotide excision repair (TC-NER), a DNA repair pathway specialized in lesions that compromise transcription. If transcriptional stress is unresolved, arrested RNA polymerase can activate programmed cell death. Nonetheless, how this parasite modulates these processes is unknown. Here, we demonstrate that T. cruzi cell death after UV irradiation, a genotoxic agent that generates lesions resolved by TC-NER, depends on active transcription and is signaled mainly by an apoptotic-like pathway. Pre-treated parasites with α-amanitin, a selective RNA polymerase II inhibitor, become resistant to such cell death. Similarly, the gamma pre-irradiated cells are more resistant to UV when the transcription processes are absent. The Cockayne Syndrome B protein (CSB) recognizes blocked RNA polymerase and can initiate TC-NER. Curiously, CSB overexpression increases parasites' cell death shortly after UV exposure. On the other hand, at the same time after irradiation, the single-knockout CSB cells show resistance to the same treatment. UV-induced fast death is signalized by the exposition of phosphatidylserine to the outer layer of the membrane, indicating a cell death mainly by an apoptotic-like pathway. Furthermore, such death is suppressed in WT parasites pre-treated with inhibitors of ataxia telangiectasia and Rad3-related (ATR), a key DDR kinase. Signaling for UV radiation death may be related to R-loops since the overexpression of genes associated with the resolution of these structures suppress it. Together, results suggest that transcription blockage triggered by UV radiation activates an ATR-dependent apoptosis-like mechanism in T. cruzi, with the participation of CSB protein in this process.

Humans seropositive for Trypanosoma cruzi co-infected with intestinal helminths have higher infectiousness, parasitaemia and Th2-type response in the Argentine Chaco.

BACKGROUND: The Gran Chaco ecoregion is a well-known hotspot of several neglected tropical diseases (NTDs) including Chagas disease, soil-transmitted helminthiasis and multiparasitic infections. Interspecific interactions between parasite species can modify host susceptibility, pathogenesis and transmissibility through immunomodulation. Our objective was to test the association between human co-infection with intestinal parasites and host parasitaemia, infectiousness to the vector and immunological profiles in Trypanosoma cruzi-seropositive individuals residing in an endemic region of the Argentine Chaco. METHODS: We conducted a cross-sectional serological survey for T. cruzi infection along with an intestinal parasite survey in two adjacent rural villages. Each participant was tested for T. cruzi and Strongyloides stercoralis infection by serodiagnosis, and by coprological tests for intestinal parasite detection. Trypanosoma cruzi bloodstream parasite load was determined by quantitative PCR (qPCR), host infectiousness by artificial xenodiagnosis and serum human cytokine levels by flow cytometry. RESULTS: The seroprevalence for T. cruzi was 16.1% and for S. stercoralis 11.5% (n = 87). We found 25.3% of patients with Enterobius vermicularis. The most frequent protozoan parasites were Blastocystis spp. (39.1%), Giardia lamblia (6.9%) and Cryptosporidium spp. (3.4%). Multiparasitism occurred in 36.8% of the examined patients. Co-infection ranged from 6.9% to 8.1% for T. cruzi-seropositive humans simultaneously infected with at least one protozoan or helminth species, respectively. The relative odds of being positive by qPCR or xenodiagnosis (i.e. infectious) of 28 T. cruzi-seropositive patients was eight times higher in people co-infected with at least one helminth species than in patients with no such co-infection. Trypanosoma cruzi parasite load and host infectiousness were positively associated with helminth co-infection in a multiple regression analysis. Interferon-gamma (IFN-γ) response, measured in relation to interleukin (IL)-4 among humans infected with T. cruzi only, was 1.5-fold higher than for T. cruzi-seropositive patients co-infected with helminths. The median concentration of IL-4 was significantly higher in T. cruzi-seropositive patients with a positive qPCR test than in qPCR-negative patients. CONCLUSIONS: Our results show a high level of multiparasitism and suggest that co-infection with intestinal helminths increased T. cruzi parasitaemia and upregulated the Th2-type response in the study patients.

Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Introduction: Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. Methods: We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring ß-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Results: Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 µm observed at 5 days. Functionality, assessed through ß-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. Discussion: This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

Cladoic Acid, an Anti-Trypanosoma cruzi Polyketide from Cladosporium sp. TP-F2020.

A new polyketide, cladoic acid, was isolated from a fungus of the genus Cladosporium. The structure of the highly oxygenated trans-decalin ring with an all-E triene side chain was elucidated by extensive spectroscopic analysis. The unique chair/twist-boat conformation of the trans-decalin core and the flexibility of the B-ring were demonstrated by computer-aided conformational analysis. Cladoic acid was active against Trypanosoma cruzi and inhibited the proliferation of amastigotes and epimastigotes with IC50 values of 27 and 46 µM, respectively, but it did not show any appreciable activity against P388 murine leukemia cells, bacteria, or fungi, indicating it is a potential candidate for drug development against Chagas disease.

In Vitro and In Silico Evaluation of the Leishmanicidal and Trypanocidal Activities of Lignan Methylpiperitol Isolated from Persea Fulva.

Neglected Tropical Diseases are a significant concern as they encompass various infections caused by pathogens prevalent in tropical regions. The limited and often highly toxic treatment options for these diseases necessitate the exploration of new therapeutic candidates. In the present study, the lignan methylpiperitol was isolated after several chromatographic steps from Persea fulva L. E. Koop (Lauraceae) and its leishmanicidal and trypanocidal activities were evaluated using in vitro and in silico approaches. The chemical structure of methylpiperitol was defined by NMR and MS spectral data analysis. The antiprotozoal activity of methylpiperitol was determined in vitro and indicated potency against trypomastigote forms of Trypanosoma cruzi (EC50 of 4.5±1.1 mM) and amastigote forms of Leishmania infantum (EC50 of 4.1±0.5 mM), with no mammalian cytotoxicity against NCTC cells (CC50>200 mM). Molecular docking studies were conducted using six T. cruzi and four Leishmania. The results indicate that for the molecular target hypoxanthine phosphoribosyl transferase in T. cruzi and piteridine reductase 1 of L. infatum, the methylpiperitol obtained better results than the crystallographic ligand. Therefore, the lignan methylpiperitol, isolated from P. fulva holds potential for the development of new prototypes for the treatment of Neglected Tropical Diseases, especially leishmaniasis.

Drug screening and development cascade for Chagas disease: an update of in vitro and in vivo experimental models.

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.

Nonsteroidal Anti-Inflammatory Drugs and Experimental Chagas Disease: An Unsolved Question.

Chagas disease is a parasitic disease caused by the protozoan Trypanosoma cruzi with an acute, detectable blood parasites phase and a chronic phase, in which the parasitemia is not observable, but cardiac and gastrointestinal consequences are possible. Mice are the principal host used in experimental Chagas disease but reproduce the human infection depending on the animal and parasite strain, besides dose and route of administration. Lipidic mediators are tremendously involved in the pathogenesis of T. cruzi infection, meaning the prostaglandins and thromboxane, which participate in the immunosuppression characteristic of the acute phase. Thus, the eicosanoids inhibition caused by the nonsteroidal anti-inflammatory drugs (NSAIDs) alters the dynamic of the disease in the experimental models, both in vitro and in vivo, which can explain the participation of the different mediators in infection. However, marked differences are founded in the various NSAIDs existing because of the varied routes blocked by the drugs. So, knowing the results in the experimental models of Chagas disease with or without the NSAIDs helps comprehend the pathogenesis of this infection, which still needs a better understanding.

Antimicrobial activity of NK cells to Trypanosoma cruzi infected human primary Keratinocytes.

Infection with the protozoan parasite Trypanosoma cruzi is causative for Chagas disease, which is a highly neglected tropical disease prevalent in Latin America. Humans are primary infected through vectorial transmission by blood-sucking triatomine bugs. The parasite enters the human host through mucous membranes or small skin lesions. Since keratinocytes are the predominant cell type in the epidermis, they play a critical role in detecting disruptions in homeostasis and aiding in pathogen elimination by the immune system in the human skin as alternative antigen-presenting cells. Interestingly, keratinocytes also act as a reservoir for T. cruzi, as the skin has been identified as a major site of persistent infection in mice with chronic Chagas disease. Moreover, there are reports of the emergence of T. cruzi amastigote nests in the skin of immunocompromised individuals who are experiencing reactivation of Chagas disease. This observation implies that the skin may serve as a site for persistent parasite presence during chronic human infection too and underscores the significance of investigating the interactions between T. cruzi and skin cells. Consequently, the primary objective of this study was to establish and characterize the infection kinetics in human primary epidermal keratinocytes (hPEK). Our investigation focused on surface molecules that either facilitated or hindered the activation of natural killer (NK) cells, which play a crucial role in controlling the infection. To simulate the in vivo situation in humans, an autologous co-culture model was developed to examine the interactions between T. cruzi infected keratinocytes and NK cells. We evaluated the degranulation, cytokine production, and cytotoxicity of NK cells in response to the infected keratinocytes. We observed a strong activation of NK cells by infected keratinocytes, despite minimal alterations in the expression of activating or inhibitory ligands on NK cell receptors. However, stimulation with recombinant interferon-gamma (IFN-γ), a cytokine known to be present in significant quantities during chronic T. cruzi infections in the host, resulted in a substantial upregulation of these ligands on primary keratinocytes. Overall, our findings suggest the crucial role of NK cells in controlling acute T. cruzi infection in the upper layer of the skin and shed light on keratinocytes as potential initial targets of infection.

Evaluation of chimeric recombinant antigens for the serodiagnosis of Trypanosoma cruzi in dogs: a promising tool for Chagas disease surveillance.

BACKGROUND: Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). METHODS: We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. RESULTS: The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. CONCLUSIONS: Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease.

Absolute Lymphocyte Count: a Predictor of Positive Serum PCR for Trypanosoma cruzi in Patients with Chagas Disease Undergoing Heart Transplantation. / Contagem Absoluta de Linfócitos: um Preditor de PCR Sérica Positiva para o Trypanosoma cruzi em Pacientes com Chagas Submetidos ao Transplante Cardíaco.

BACKGROUND: It is unknown whether lymphopenia is a risk factor for the reactivation of Chagas disease in heart transplantation (HTx), as recently described in the reactivation of cytomegalovirus in transplant patients. OBJECTIVE: To evaluate whether lymphopenia in the perioperative period of heart transplantation is related to early Trypanosoma cruzi parasitemia. METHODS: This observational, retrospective study analyzed a sample from January 2014 to January 2023). Parasitemia was evaluated in the first 3 months after HTx using serum polymerase chain reaction (PCR) and compared with the total lymphocyte count in the perioperative period of HTx using receiver operating characteristic curves. Baseline characteristics were compared with PCR for Chagas using independent Cox proportional hazards models. A significance level of 5% was adopted. RESULTS: The sample (n = 35) had a mean age of 52.5 ± 8.1 years, and 22 patients (62.8%) had positive PCR for Chagas. The mean lowest lymphocyte values in the first 14 days after HTx were 398 ± 189 and 755 ± 303 cells/mm3 in patients with and without parasitemia, respectively, within 3 months after HTx (area under the curve = 0.857; 95% confidence interval: 0.996 to 0.718, sensitivity and specificity of 83.3% and 86.4%). A cutoff value of less than 550 lymphocytes/mm3 was determined as a risk factor for the presence of parasitemia. Patients with lymphocytes < 550 units/mm3 in the first 14 days after HTx presented positive PCR in 80% of cases. For every increase of 100 lymphocytes/mm3, the risk of PCR positivity was reduced by 26% (hazard rate ratio = 0.74; 95% confidence interval: 0.59 to 0.93, p = 0.009). CONCLUSION: There was an association between lymphopenia in the perioperative period of HTx and early T. cruzi parasitemia detected by PCR.

FUNDAMENTO: É desconhecido se a linfopenia é fator de risco para a reativação da doença de Chagas no transplante cardíaco (TxC), como recentemente descrito na reativação de citomegalovírus em pacientes transplantados. OBJETIVO: Avaliar se a linfopenia no perioperatório do TxC está relacionada à parasitemia precoce pelo Trypanosoma cruzi. MÉTODOS: Amostra analisada (janeiro de 2014 a janeiro de 2023) em estudo observacional e retrospectivo. A parasitemia foi avaliada nos primeiros 3 meses após o TxC por meio da reação em cadeia da polimerase sérica (PCR) e comparada com a contagem total de linfócitos no perioperatório do TxC por curvas ROC. Comparadas características de base com a PCR Chagas por modelos de risco proporcionais de Cox independentes. Nível de significância adotado de 5%. RESULTADOS: Amostra (n = 35) apresentou idade média de 52,5 ± 8,1 anos e PCR Chagas positiva em 22 pacientes (62,8%). As médias dos menores valores de linfócitos nos primeiros 14 dias do TxC foram 398 ± 189 e 755 ± 303 células/mm3 em pacientes com e sem parasitemia nos 3 meses após o TxC, respectivamente (área sob a curva = 0,857; intervalo de confiança de 95%: 0,996 a 0,718, sensibilidade e especificidade de 83,3% e 86,4%). Determinado valor de corte inferior a 550 linfócitos/mm3 como fator de risco para presença de parasitemia. Pacientes com linfócitos < 550 unidades/mm3 nos primeiros 14 dias do pós-TxC apresentaram PCR positiva em 80% dos casos. Para cada aumento de 100 linfócitos/mm3, o risco de positividade da PCR é reduzido em 26% (razão de riscos = 0,74; intervalo de confiança de 95%: 0,59 a 0,93, p = 0,009). CONCLUSÃO: Houve associação entre a linfopenia no perioperatório do TxC com a parasitemia precoce pelo T. cruzi detectada por PCR.

Screening of synthetic 1,2,3-triazolic compounds inspired by SRPIN340 as anti-Trypanosoma cruzi agents.

BACKGROUND: The current treatments for Chagas disease (CD) include benznidazole and nifurtimox, which have limited efficacy and cause numerous side effects. Triazoles are candidates for new CD treatments due to their ability to eliminate T. cruzi parasites by inhibiting ergosterol synthesis, thereby damaging the cell membranes of the parasite. METHODS: Eleven synthetic analogs of the kinase inhibitor SRPIN340 containing a triazole core (compounds 6A-6K) were screened in vitro against the Tulahuen strain transfected with ß-galactosidase, and their IC50, CC50, and selectivity indexes (SI) were calculated. Compounds with an SI > 50 were further evaluated in mice infected with the T. cruzi Y strain by rapid testing. RESULTS: Eight compounds were active in vitro with IC50 values ranging from 0.5-10.5 µg/mL. The most active compounds, 6E and 6H, had SI values of 125.2 and 69.6, respectively. These compounds also showed in vivo activity, leading to a reduction in parasitemia at doses of 10, 50, and 250 mg/kg/day. At doses of 50 and 250 mg/kg/day, parasitemia was significantly reduced compared to infected untreated animals, with no significant differences between the effects of 6E and 6H. CONCLUSIONS: This study identified two new promising compounds for CD chemotherapy and confirmed their activity against T. cruzi.