RESUMEN
Trypanosoma cruzi, the causative agent of Chagas disease, has a complex life cycle that involves triatomine insects as vectors and mammals as hosts. The differentiation of epimastigote forms into metacyclic trypomastigotes within the insect vector is crucial for the parasite's life cycle progression. Factors influencing this process, including temperature, pH, and nutritional stress, along with specific metabolite availability, play a pivotal role. Amino acids like proline, histidine, and glutamine support cell differentiation, while branched-chain amino acids (BCAAs) inhibit it. Interestingly, combining the pro-metacyclogenic amino acid proline with one of the anti-metacyclogenic BCAAs results in viable metacyclics with significantly reduced infectivity. To explore the characteristics of metacyclic parasites differentiated in the presence of BCAAs, proteomics analyses were conducted. Metacyclics obtained in triatomine artificial urine (TAU) supplemented with proline alone and in combination with leucine, isoleucine, or valine were compared. The analyses revealed differential regulation of 40 proteins in TAU-Pro-Leu, 131 in TAU-Pro-Ile, and 179 in TAU-Pro-Val, as compared to metacyclics from TAU-Pro. Among these, 22%, 11%, and 13% of the proteins were associated with metabolic processes, respectively. Notably, enzymes related to glycolysis and the tricarboxylic acid (TCA) cycle were reduced in metacyclics with Pro-BCAAs, while enzymes involved in amino acid and purine metabolic pathways were increased. Furthermore, metacyclics with Pro-Ile and Pro-Val exhibited elevated enzymes linked to lipid and redox metabolism. The results revealed five proteins that were increased and four that were decreased in common in the presence of Pro+BCAAs, indicating their possible participation in key processes related to metacyclogenesis. These findings suggest that the presence of BCAAs can reshape the metabolism of metacyclics, contributing to the observed reduction in infectivity in these parasites.
Asunto(s)
Aminoácidos de Cadena Ramificada , Prolina , Proteómica , Proteínas Protozoarias , Trypanosoma cruzi , Prolina/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/crecimiento & desarrollo , Aminoácidos de Cadena Ramificada/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Enfermedad de Chagas/parasitología , Proteoma , Animales , Estadios del Ciclo de VidaRESUMEN
Several reptile species have been described as hosts of Trypanosoma cruzi, the causative agent of Chagas disease, and therefore, they have become vertebrates of epidemiological interest. In recent decades, there has been a growing interest in animal welfare, especially in populations with small numbers where lethal sampling could have catastrophic consequences, and non-lethal methodologies have been developed for detecting zoonotic parasites. In this study, we compared three non-lethal sampling methodologies for detecting T. cruzi DNA in 21 captured specimens of the native lizard Liolaemus monticola, collected from the semiarid Mediterranean ecosystem of Chile. Specimens were subjected to xenodiagnosis (XD), tail clipping, and living syringe sampling procedures to evaluate whether lizards could serve as sentinel species for T. cruzi in endemic regions. To detect the protozoan, real-time PCR (qPCR) was performed on the DNA extracted from the samples (intestinal contents, tail tissues, and blood from living syringes). Trypanosoma cruzi DNA was detected in 12 of 21 lizards, considering all three methodologies. By XD, 12 specimens showed infection (57.1 %), and both living syringe and tail sampling methodologies detected only one infected lizard (4.8 %). Therefore, T. cruzi can be detected in lizards by qPCR using the three methodologies but XD is by far the most effective non-lethal detection methodology. The use of tail and living syringe methodologies showed a large underestimation; however, they might be options for monitoring the presence of T. cruzi in lizard populations when large sample sizes are available.
Asunto(s)
Enfermedad de Chagas , ADN Protozoario , Reservorios de Enfermedades , Lagartos , Trypanosoma cruzi , Animales , Lagartos/parasitología , Trypanosoma cruzi/aislamiento & purificación , Trypanosoma cruzi/genética , Chile/epidemiología , ADN Protozoario/análisis , ADN Protozoario/aislamiento & purificación , Enfermedad de Chagas/veterinaria , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/epidemiología , Reservorios de Enfermedades/parasitología , Reservorios de Enfermedades/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Zoonosis/parasitologíaRESUMEN
The present study aimed to verify the impact of etiological treatment on the genotype-specific serological diagnosis of chronic Chagas disease patients (CH), using the Chagas-Flow ATE IgG1 methodology. For this purpose, a total of 92 serum samples from CH, categorized as Not Treated (NT, n = 32) and Benznidazole-Treated (Bz-T, n = 60), were tested at Study Baseline and 5Years Follow-up. At Study Baseline, all patients have the diagnosis of Chagas disease confirmed by Chagas-Flow ATE IgG1, using the set of attributes ("antigen/serum dilution/cut-off"; "EVI/250/30%"). The genotype-specific serodiagnosis at Study Baseline demonstrated that 96% of patients (44/46) presented a serological profile compatible with TcII genotype infection. At 5Years Follow-up monitoring, NT and Bz-T presented no changes in anti-EVI IgG1 reactivity. However, significant differences were detected in the genotype-specific IgG1 reactivity for Bz-T. The most outstanding shift comprised the anti-amastigote TcVI/(AVI), anti-amastigote TcII/(AII) and anti-epimastigote TcVI/(EVI) reactivities. Regardless no changes in the genotype-specific serology of NT (TcI = 6%; TcII = 94%), distinct T. cruzi genotype-specific sero-classification was detected for Bz-T samples at 5Years Follow-up (TcII = 100%) as compared to Baseline (TcII = 97%; TcVI = 3%). The anti-trypomastigote TcI/(TI) was the attribute accountable for the change in genotype-specific sero-classification. In conclusion, our findings of dissimilar T. cruzi genotype-specific serology upon Bz-treatment re-emphasize the relevance of accomplishing the genotype-specific serodiagnosis during clinical pos-therapeutic management of chronic Chagas disease patients.
Asunto(s)
Anticuerpos Antiprotozoarios , Enfermedad de Chagas , Genotipo , Inmunoglobulina G , Nitroimidazoles , Tripanocidas , Trypanosoma cruzi , Humanos , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Nitroimidazoles/uso terapéutico , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Inmunoglobulina G/sangre , Anticuerpos Antiprotozoarios/sangre , Masculino , Femenino , Persona de Mediana Edad , Adulto , Tripanocidas/uso terapéutico , Pruebas Serológicas , Enfermedad Crónica , Anciano , Adulto JovenRESUMEN
BACKGROUND: Chagas disease (CD), caused by Trypanosoma cruzi, poses a major global public health challenge. Although vector-borne transmission is the primary mode of infection, oral transmission is increasingly concerning. METHODS: This study utilized long-amplicon-based sequencing (long-ABS), focusing on the 18S rRNA gene, to explore T. cruzi's genetic diversity and transmission dynamics during an acute CD outbreak in Colombia, an area without domestic infestation. RESULTS: Analyzing samples from five patients and five T. cruzi-positive marsupial samples, we identified coinfections between T. cruzi and Trypanosoma rangeli, mixed T. cruzi DTUs, suggesting possible links between human and marsupial T. cruzi infections. Coexistence of TcI, TcIV and T. rangeli suggests marsupial secretions as the possible source of T. cruzi transmission. Our investigation revealed diversity loss in DTUs TcIV and T. rangeli in humans after infection and in marsupial samples after culture. CONCLUSION: These findings provide significant insights into T. cruzi dynamics, crucial for implementing control and prevention strategies.
Asunto(s)
Enfermedad de Chagas , Brotes de Enfermedades , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Marsupiales , ARN Ribosómico 18S , Trypanosoma cruzi , Enfermedad de Chagas/transmisión , Enfermedad de Chagas/epidemiología , Enfermedad de Chagas/parasitología , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Humanos , Animales , Marsupiales/parasitología , ARN Ribosómico 18S/genética , Colombia/epidemiología , Masculino , Coinfección/epidemiología , Coinfección/parasitología , Coinfección/transmisión , Trypanosoma rangeli/genética , Femenino , Adulto , ADN Protozoario/genéticaRESUMEN
We assessed the diversity of triatomines, the rates of natural infection, and the discrete typing units (DTUs) of Trypanosoma cruzi isolated from them in two municipalities in the state of Sergipe, Brazil. Active searches for triatomines were conducted in the peridomicily and wild enviroments of 10 villages within the two municipalities. Triatomines were taxonomically identified and their feces were extracted using the abdominal compression method. Parasite detection was performed using optical microscopy. For Trypanosoma cruzi genotyping via PCR-FFLB, 151 samples of the subspecies Triatoma brasiliensis macromelasoma and Triatoma brasiliensis were isolated from both municipalities. In total, 505 triatomines were collected, with Triatoma brasiliensis macromelasoma being the most frequent species (58.81 %). Triatoma b. brasiliensis was the only species in both peridomestic and wild environments. Regarding the other species, T. pseudomaculata was found only in the peridomestic environment; and T. b. macromelasoma and Psammolestes tertius were found in the wild environment. Three Discrete Typing Units were identified: TcI (87.51 %) detected in T. b. brasiliensis and T. b. macromelasoma, TcI+TcIII (10.41 %) in T. b. macromelasoma, and TcI+Trypanosoma rangeli (2.08 %) in T. b. macromelasoma. It is concluded that T. b. macromelasoma is the species collected most frequently in the studied region and the one that presents the highest rates of natural infection, highlighting its epidemiological importance for the vectorial transmission of Chagas disease in Sergipe.
Asunto(s)
Enfermedad de Chagas , Genotipo , Insectos Vectores , Triatoma , Trypanosoma cruzi , Animales , Brasil , Trypanosoma cruzi/genética , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/aislamiento & purificación , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/transmisión , Enfermedad de Chagas/epidemiología , Triatoma/parasitología , Triatoma/clasificación , Insectos Vectores/parasitología , Insectos Vectores/clasificación , Heces/parasitología , HumanosRESUMEN
BACKGROUND: Vectorial transmission through hematophagous triatomine insects remains the primary mode of Chagas Disease contagion. These insects have become increasingly common in urban environments. Therefore, this study aimed to report an encounter of triatomines with trypanosomatid infection in a vertical residential condominium in Rio Branco, the capital of the state of Acre, in the western Brazilian Amazon. METHODS: Triatomines were collected from residents and sent to the municipality's Entomological Surveillance sector. Trypanosomatid positivity was evaluated using optical microscopy, followed by species and genotype identification using molecular biology techniques. RESULTS: Twenty-five adult triatomine specimens were collected from two of three condominium buildings invading apartments from the 2nd to 13th floors. Six specimens were identified as Rhodnius sp. and 19 as R. montenegrensis. Among these, molecular tests were conducted on seven specimens, with five testing positive for Trypanosoma cruzi, all belonging to genotype TcI. CONCLUSIONS: These findings underscore the need for further studies to better understand the invasive capacity of these insects in these environments and the mechanisms involved in this process.
Asunto(s)
Enfermedad de Chagas , Insectos Vectores , Rhodnius , Trypanosoma cruzi , Animales , Brasil , Insectos Vectores/clasificación , Insectos Vectores/parasitología , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Enfermedad de Chagas/transmisión , Rhodnius/clasificación , Rhodnius/genética , Rhodnius/parasitología , Genotipo , Vivienda , HumanosRESUMEN
Glycosylation is one of the most structurally and functionally diverse co- and post-translational modifications in a cell. Addition and removal of glycans, especially to proteins and lipids, characterize this process which has important implications in several biological processes. In mammals, the repeated enzymatic addition of a sialic acid unit to underlying sialic acids (Sia) by polysialyltransferases, including ST8Sia2, leads to the formation of a sugar polymer called polysialic acid (polySia). The functional relevance of polySia has been extensively demonstrated in the nervous system. However, the role of polysialylation in infection is still poorly explored. Previous reports have shown that Trypanosoma cruzi (T. cruzi), a flagellated parasite that causes Chagas disease (CD), changes host sialylation of glycoproteins. To understand the role of host polySia during T. cruzi infection, we used a combination of in silico and experimental tools. We observed that T. cruzi reduces both the expression of the ST8Sia2 and the polysialylation of target substrates. We also found that chemical and genetic inhibition of host ST8Sia2 increased the parasite load in mammalian cells. We found that modulating host polysialylation may induce oxidative stress, creating a microenvironment that favors T. cruzi survival and infection. These findings suggest a novel approach to interfere with parasite infections through modulation of host polysialylation.
Asunto(s)
Enfermedad de Chagas , Ácidos Siálicos , Sialiltransferasas , Trypanosoma cruzi , Trypanosoma cruzi/genética , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/fisiología , Sialiltransferasas/metabolismo , Sialiltransferasas/genética , Enfermedad de Chagas/parasitología , Ácidos Siálicos/metabolismo , Humanos , Animales , GlicosilaciónRESUMEN
Panstrongylus geniculatus is the most widely distributed species of Panstrongylus in Brazil and merits attention from vector control programs due to its potential for domiciliation. Specimens infected with Trypanosoma cruzi have already been reported in both peridomiciliary and domiciliary environments. Building on these findings, we report, for the first time, the presence of P. geniculatus in the state of Alagoas and provide an updated dichotomous key (based on cytogenetic data) for species in Alagoas. Panstrongylus geniculatus has been identified in Boca da Mata, Joaquim Gomes, and Novo Lino. In light of the recent notification of Rhodnius domesticus and this record of P. geniculatus, we present an updated identification key enabling the differentiation of all species in Alagoas. Emphasis is placed on the importance of correctly identifying triatomine species because it is crucial for the development of effective control measures, thereby aiding in the mitigation of Chagas disease.
Asunto(s)
Enfermedad de Chagas , Insectos Vectores , Panstrongylus , Animales , Brasil/epidemiología , Enfermedad de Chagas/transmisión , Enfermedad de Chagas/epidemiología , Insectos Vectores/genética , Insectos Vectores/clasificación , Insectos Vectores/parasitología , Panstrongylus/genética , Panstrongylus/parasitología , Trypanosoma cruzi/genética , Masculino , Femenino , HumanosRESUMEN
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.
Asunto(s)
Enfermedad de Chagas , Placenta , Trofoblastos , Trypanosoma cruzi , Humanos , Trofoblastos/parasitología , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/fisiología , Femenino , Embarazo , Placenta/parasitología , Enfermedad de Chagas/parasitología , Línea Celular , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Técnicas de Cultivo Tridimensional de Células/métodosRESUMEN
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.
Asunto(s)
Enfermedad de Chagas , Coinfección , Helmintiasis , Parasitosis Intestinales , Trypanosoma cruzi , Humanos , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Coinfección/parasitología , Coinfección/epidemiología , Coinfección/inmunología , Enfermedad de Chagas/epidemiología , Enfermedad de Chagas/complicaciones , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/sangre , Enfermedad de Chagas/inmunología , Animales , Adulto , Estudios Transversales , Masculino , Femenino , Parasitosis Intestinales/epidemiología , Parasitosis Intestinales/parasitología , Parasitosis Intestinales/complicaciones , Parasitosis Intestinales/inmunología , Persona de Mediana Edad , Helmintiasis/complicaciones , Helmintiasis/parasitología , Helmintiasis/epidemiología , Helmintiasis/inmunología , Adulto Joven , Adolescente , Argentina/epidemiología , Estudios Seroepidemiológicos , Strongyloides stercoralis/inmunología , Strongyloides stercoralis/aislamiento & purificación , Parasitemia/parasitología , Parasitemia/epidemiología , Células Th2/inmunología , Niño , Estrongiloidiasis/epidemiología , Estrongiloidiasis/parasitología , Estrongiloidiasis/complicaciones , Estrongiloidiasis/inmunología , Estrongiloidiasis/sangre , Anciano , Citocinas/sangre , Anticuerpos Antiprotozoarios/sangreRESUMEN
BACKGROUND: Vertical transmission of Trypanosoma cruzi represents approximately 20% of new Chagas disease cases. Early detection and treatment for women of childbearing age and newborns is a public health priority, but the lack of a simple and reliable diagnostic test remains a major barrier. We aimed to evaluate the performance of a point-of-care loop-mediated isothermal amplification (LAMP) assay for the detection of T cruzi. METHODS: In this proof-of-concept study, we coupled a low-cost 3D printer repurposed for sample preparation and amplification (PrintrLab) to the Eiken T cruzi-LAMP prototype to detect vertically transmitted T cruzi, which we compared with standardised PCR and with the gold-standard algorithm (microscopy at birth and 2 months and serological study several months later). We screened pregnant women from two hospitals in the Bolivian Gran Chaco province, and those who were seropositive for T cruzi were offered the opportunity for their newborns to be enrolled in the study. Newborns were tested by microscopy, LAMP, and PCR at birth and 2 months, and by serology at 8 months. FINDINGS: Between April 23 and Nov 17, 2018, 986 mothers were screened, among whom 276 were seropositive for T cruzi (28·0% prevalence, 95% CI 25·6-31·2). In total, 224 infants born to 221 seropositive mothers completed 8 months of follow-up. Congenital transmission was detected in nine of the 224 newborns (4·0% prevalence, 1·9-7·5) by direct microscopy observation, and 14 more cases were diagnosed serologically (6·3%, 3·6-10·3), accounting for an overall vertical transmission rate of 10·3% (6·6-15·0; 23 of 224). All microscopy-positive newborns were positive by PrintrLab-LAMP and by PCR, while these techniques respectively detected four and five extra positive cases among the remaining 215 microscopy-negative newborns. INTERPRETATION: The PrintrLab-LAMP yielded a higher sensitivity than microscopy-based analysis. Considering the simpler use and expected lower cost of LAMP compared with PCR, our findings encourage its evaluation in a larger study over a wider geographical area. FUNDING: Inter-American Development Bank.
Asunto(s)
Enfermedad de Chagas , Transmisión Vertical de Enfermedad Infecciosa , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Trypanosoma cruzi , Humanos , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/transmisión , Enfermedad de Chagas/epidemiología , Enfermedad de Chagas/parasitología , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Recién Nacido , Bolivia/epidemiología , Femenino , Embarazo , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Prueba de Estudio Conceptual , Sensibilidad y Especificidad , AdultoRESUMEN
INTRODUCTION: Chagas disease caused by Trypanosoma cruzi (T cruzi) found in the Americas is often missed during the early stage of infection due to lack of sensitive diagnostic tests. The classic immunological and parasitological tests often fail in the acute phase due to the nonspecific and low antibody level nature of the infection and in the chronic phase due to low levels of trypanosomes in the blood. For successful control strategies, there must be a sensitive and specific diagnostic test. OBJECTIVE/METHODS: We have demonstrated the possibility (proof of concept) of detecting T cruzi-specific repeat DNA via polymerase chain reaction (PCR) by (1) spiking 15 urine samples collected from volunteers free of prior infection with 3 different concentrations of T cruzi (3 strains), Trypanosoma brucei, and Trypanosoma rhodesiense (African strain) genomic DNA and (2) from filtered collected clinical samples from Argentina. Three sets of primers were used. RESULTS: Our approach detected repeat DNA specific for T cruzi strains from 1 clinical sample by 2 sets of primer and from spiked urine by all 3 sets of primer but not the African species. A serial dilution (spiking) also was performed on T cruzi strains to detect sensitivities of the assay. One set of primers constantly detected satellite DNA for all T cruzi strains from 70 pg/µl to 175 fg/µl. CONCLUSIONS: We were able to demonstrate the feasibility of detecting T cruzi-specific DNA from filtered urine samples by sensitive and specific PCR assay. Besides the evident increased sensitivity and specificity of primers, our approach can be used to explore Chagas prevalence in endemic areas - especially in congenital Chagas newborn screening - and in the acute phase.
Asunto(s)
Enfermedad de Chagas , ADN Protozoario , Reacción en Cadena de la Polimerasa , Trypanosoma cruzi , Humanos , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/orina , ADN Protozoario/orina , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Sensibilidad y Especificidad , Argentina/epidemiología , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Enfermedad de Chagas , Enfermedades de los Perros , Ensayo de Inmunoadsorción Enzimática , Sensibilidad y Especificidad , Pruebas Serológicas , Trypanosoma cruzi , Animales , Perros , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/veterinaria , Enfermedad de Chagas/parasitología , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/genética , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Anticuerpos Antiprotozoarios/sangre , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genéticaRESUMEN
Background: Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi and vectored by triatomines, affects millions of people worldwide. In endemic countries including Mexico, infections in domestic animals, such as dogs, may affect the risk of human disease when they serve as a source of infection to vectors that subsequently infect humans. Materials and Methods: We conducted a cross-sectional study of 296 dogs from two cities near the northern and southern borders of Mexico: Reynosa, Tamaulipas, and Tuxtla Gutierrez, Chiapas. Infection was measured based on testing of blood using T. cruzi quantitative PCR (qPCR) and up to three antibody detection assays. The StatPak immunochromatographic assay was used to screen samples and the indirect fluorescent antibody (IFA) and multiplex microsphere immunoassay (MIA) tests were used as secondary tests on all samples that screened positive and a subset of negatives. Serologic positivity was defined based on reactivity on at least two independent tests. Results: Of the 280 samples tested for parasite DNA, two (0.7%) were positive, one of which (0.4%) was confirmed as T. cruzi discrete typing unit TcIV. Overall, 72 (24.3%) samples were reactive for T. cruzi antibodies via StatPak of which 8 were also positive using MIA and 2 were also positive using IFA (including one of the PCR-positive dogs). Overall, nine dogs (3.4%) met study criteria of positivity based on either/both serology or PCR tests. Positive dogs were found in both regions of Mexico; five (2.7%) from Reynosa and four (3.6%) from Tuxtla Gutierrez. We found no association between infection status and state of origin, sex, age group, breed group, neighborhood, and whether other pets lived in the home. Conclusion: Our results re-emphasize dogs' utility as sentinels for T. cruzi in Mexico and underscore the need for improved veterinary diagnostic tests and parasite surveillance at the household level in endemic countries.
Asunto(s)
Enfermedad de Chagas , Enfermedades de los Perros , Trypanosoma cruzi , Animales , Perros , Enfermedad de Chagas/veterinaria , Enfermedad de Chagas/epidemiología , Enfermedades de los Perros/epidemiología , Enfermedades de los Perros/parasitología , Trypanosoma cruzi/aislamiento & purificación , Trypanosoma cruzi/genética , México/epidemiología , Estudios Transversales , Masculino , Femenino , Anticuerpos Antiprotozoarios/sangreRESUMEN
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.
Asunto(s)
Enfermedad de Chagas , Trasplante de Corazón , Linfopenia , Parasitemia , Reacción en Cadena de la Polimerasa , Trypanosoma cruzi , Humanos , Trasplante de Corazón/efectos adversos , Masculino , Persona de Mediana Edad , Femenino , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Estudios Retrospectivos , Recuento de Linfocitos , Enfermedad de Chagas/complicaciones , Reacción en Cadena de la Polimerasa/métodos , Adulto , Factores de Riesgo , Factores de Tiempo , Valor Predictivo de las Pruebas , Cardiomiopatía Chagásica/cirugía , Cardiomiopatía Chagásica/sangre , Curva ROCRESUMEN
P21 is a protein secreted by all forms of Trypanosoma cruzi (T. cruzi) with recognized biological activities determined in studies using the recombinant form of the protein. In our recent study, we found that the ablation of P21 gene decreased Y strain axenic epimastigotes multiplication and increased intracellular replication of amastigotes in HeLa cells infected with metacyclic trypomastigotes. In the present study, we investigated the effect of P21 in vitro using C2C12 cell lines infected with tissue culture-derived trypomastigotes (TCT) of wild-type and P21 knockout (TcP21-/-) Y strain, and in vivo using an experimental model of T. cruzi infection in BALB/c mice. Our in-vitro results showed a significant decrease in the host cell invasion rate by TcP21-/- parasites as measured by Giemsa staining and cell count in bright light microscope. Quantitative polymerase chain reaction (qPCR) analysis showed that TcP21-/- parasites multiplied intracellularly to a higher extent than the scrambled parasites at 72h post-infection. In addition, we observed a higher egress of TcP21-/- trypomastigotes from C2C12 cells at 144h and 168h post-infection. Mice infected with Y strain TcP21-/- trypomastigotes displayed higher systemic parasitemia, heart tissue parasite burden, and several histopathological alterations in heart tissues compared to control animals infected with scrambled parasites. Therewith, we propose that P21 is important in the host-pathogen interaction during invasion, cell multiplication, and egress, and may be part of the mechanism that controls parasitism and promotes chronic infection without patent systemic parasitemia.
Asunto(s)
Enfermedad de Chagas , Proteínas Protozoarias , Trypanosoma cruzi , Animales , Humanos , Ratones , Línea Celular , Enfermedad de Chagas/parasitología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Interacciones Huésped-Parásitos , Ratones Endogámicos BALB C , Parasitemia , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Trypanosoma cruzi/fisiología , Trypanosoma cruzi/metabolismo , VirulenciaRESUMEN
Phospholipases A2 (PLA2) comprise a superfamily of enzymes that specifically catalyze hydrolysis of the ester bond at the sn-2 position of glycerophospholipids, generating lysophospholipids and fatty acids. In Rhodnius prolixus, one of the main vectors of the Chagas's disease etiologic agent Trypanosoma cruzi, it was previously shown that lysophosphatidylcholine, a bioactive lipid, found in the insect's saliva, contributes to the inhibition of platelet aggregation, and increases the production of nitric oxide, an important vasodilator. Due to its role in potentially generating LPC, here we studied the PLA2 present in the salivary glands of R. prolixus. PLA2 activity is approximately 100 times greater in the epithelium than in the contents of salivary glands. Our study reveals the role of the RpPLA2XIIA gene in the insect feeding performance and in the fatty acids composition of phospholipids extracted from the salivary glands. Knockdown of RpPLA2XIIA significantly altered the relative amounts of palmitic, palmitoleic, oleic and linoleic acids. A short-term decrease in the expression of RpPLA2III and RpPLA2XIIA in the salivary glands of R. prolixus was evident on the third day after infection by T. cruzi. Taken together, our results contribute to the understanding of the role of PLA2 in the salivary glands of hematophagous insects and show that the parasite is capable of modulating even tissues that are not colonized by it.
Asunto(s)
Fosfolipasas A2 , Rhodnius , Glándulas Salivales , Trypanosoma cruzi , Animales , Rhodnius/parasitología , Rhodnius/enzimología , Rhodnius/genética , Glándulas Salivales/parasitología , Glándulas Salivales/enzimología , Glándulas Salivales/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/enzimología , Fosfolipasas A2/metabolismo , Fosfolipasas A2/genética , Ácidos Grasos/metabolismo , Enfermedad de Chagas/parasitología , Insectos Vectores/parasitología , Insectos Vectores/enzimologíaRESUMEN
Background: Marsupials and rodents are the most important wild and synanthropic hosts of Trypanosoma cruzi due to the high frequency of infection, maintenance of diverse genetic populations of the parasite, and their close proximity to interact with both transmission cycles, sylvatic and peridomestic. Our aim was to identify the discrete typing units (DTU) of T. cruzi from different wild and synanthropic hosts in two regions of Mexico and to carry out a review of historical data focusing on current knowledge on the diversity and T. cruzi DTUs of host species. Materials and Methods: One hundred fifteen samples were obtained from two areas in Tabasco and Nayarit state. The presence of T. cruzi was evaluated by PCR. Results: The 12.6% (12/95) of samples from Tabasco and 65% (13/20) from Nayarit were found to be positive for parasite DNA. All the sequences analyzed were grouped in T. cruzi DTU I; low nucleotide diversity was observed in Tabasco (π = 0.00566, and Ï´ = 0.00632), while high genetic diversity was observed in Nayarit sequences, up to 8.63 (π) to 11.10 (Ï´) times greater than Tabasco sequences. Genetic flow and migration between Tabasco, and Nayarit were scarce (FST = 0.37329 and Nm = 0.42), and genetic exchange was observed only between nearby areas. The bibliographic review of hosts in Mexico, together with our data, shows a heterogeneous T. cruzi prevalence in Chiroptera and domestic animals. For Atelidae and Canids, prevalence is generally below 25%. However, a high prevalence, greater than 25% and up to 100%, was recorded in Didelphimorphia, and Rodentia. Few studies in regions of Mexico have been described as infected with the parasite; in these, the genetic group with the highest prevalence is the DTU I. Conclusion: Marsupials and rodents are important reservoirs of T. cruzi; DTU I was frequently reported; however, recent genetic and reservoir studies have demonstrated the presence of greater diversity of genetic groups.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Trypanosoma cruzi/genética , Trypanosoma cruzi/aislamiento & purificación , Trypanosoma cruzi/clasificación , Animales , México/epidemiología , Enfermedad de Chagas/epidemiología , Enfermedad de Chagas/veterinaria , Enfermedad de Chagas/transmisión , Animales Salvajes/parasitología , Mamíferos/parasitología , Variación Genética , Roedores/parasitologíaRESUMEN
BACKGROUND: Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES: In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS: Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS: The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS: Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.
Asunto(s)
Proteínas Fluorescentes Verdes , Tripanocidas , Trypanosoma cruzi , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Proteínas Fluorescentes Verdes/genética , Tripanocidas/farmacología , Nitroimidazoles/farmacología , Pruebas de Sensibilidad Parasitaria , Animales , Concentración 50 Inhibidora , Evaluación Preclínica de Medicamentos , Supervivencia Celular/efectos de los fármacosRESUMEN
BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, whose genetic structure is divided into six discrete typing units (DTUs) known as TcI-TcVI. In the Yucatan Peninsula, Mexico, information regarding the DTUs circulating in wild mammals is scarce, while this is important knowledge for our understanding of T. cruzi transmission dynamics. METHODS: In the current study, we sampled wild mammals in a sylvatic site of the Yucatan Peninsula and assessed their infection with T. cruzi by PCR. Then, for infected mammals, we amplified and sequenced nuclear and mitochondrial T. cruzi genetic markers for DTU identification. RESULTS: In total, we captured 99 mammals belonging to the orders Chiroptera, Rodentia and Didelphimorphia. The prevalence of infection with T. cruzi was 9% (9/99; 95% CI [5, 16]), and we identified TcI in a Jamaican fruit bat, Artibeus jamaicensis. Moreover, we fortuitously identified Trypanosoma dionisii in another Jamaican fruit bat and detected an unidentified Trypanosoma species in a third specimen. While the latter discoveries were not expected because we used primers designed for T. cruzi, this study is the first to report the identification of T. dionisii in a bat from Yucatan, Mexico, adding to a recent first report of T. dionisii in bats from Veracruz, and first report of this Trypanosoma species in Mexico. CONCLUSION: Further research is needed to enhance our knowledge of T. cruzi DTUs and Trypanosoma diversity circulating in wildlife in Southeastern Mexico.