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1.
PLoS Negl Trop Dis ; 17(10): e0011643, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37801449

RESUMO

BACKGROUND: Chagas disease (ChD) is the most important endemy in Latin America. Some patients, develop chronic Chagasic cardiopathy (CCC) years after the acute phase. It is unknown if patients infected by the oral route have higher risk of developing early CCC. METHODS AND FINDINGS: A prospective cohort study was conducted to assess morbidity and mortality during 10 years observation in 106 people simultaneously infected and treated in the largest known orally transmitted ChD outbreak in 2007. A preschooler died during the acute phase, but thereafter was no mortality associated to ChD. All acute phase findings improved in the first-year post-treatment. Each person was evaluated 8.7 times clinically, 6.4 by electrocardiogram (ECG)/Holter, and 1.7 by echocardiogram. Based on prevalence, the number of people who had any abnormalities (excluding repolarization abnormalities and atrial tachycardia which decreased) was higher than 2007, since they were found at least once between 2008-2017. However, when we evaluated incidence, except for clinical bradycardia and dizziness, it was observed that the number of new cases of all clinical and ECG findings decreased at the end of the follow-up. Between 2008-2017 there was not incidence of low voltage complex, 2nd degree AV block, long QT interval, left bundle branch block or left ventricular dysfunction that allowed the diagnosis of CCC. Total improvement prevailed over the persistence of all clinical and ECG/Holter findings, except for sinus bradycardia. Incomplete right bundle branch block, sinus bradycardia and/or T-wave inversion were diagnosed persistently in 9 children. The second treatment did not have significant influence on the incidence of clinical or ECG/Holter findings. CONCLUSIONS: At the end of the 10-year follow-up, there were not clinical or ECG/Holter criteria for classifying patients with CCC. The incidence of arrhythmias and repolarization abnormalities decreased. However, special attention should be paid on findings that not revert as sinus bradycardia, or those diagnosed persistently in all ECG as sinus bradycardia, incomplete right bundle branch block or T-wave inversion. Early diagnosis and treatment may have contributed to the rapid improvement of these patients. In ChD follow-up studies prevalence overestimates the real dimension of abnormalities, the incidence looks as a better indicator.


Assuntos
Bradicardia , Doença de Chagas , Criança , Humanos , Bradicardia/epidemiologia , Bloqueio de Ramo/epidemiologia , Seguimentos , Estudos Prospectivos , Arritmias Cardíacas , Doença de Chagas/complicações , Doença de Chagas/epidemiologia , Eletrocardiografia , Surtos de Doenças
2.
PLoS Negl Trop Dis ; 17(4): e0011290, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37058542

RESUMO

BACKGROUND: Chagas disease or American trypanosomiasis, a neglected tropical disease, is a persistent Public Health problem in Latin America and other, non-endemic, countries. Point-of-care (POC) sensitive methods are still needed to improve and extend early diagnosis in acute infections such as congenital Chagas disease. The objective of this study was to analytically evaluate in the lab the performance of a qualitative POC molecular test (Loop-mediated isothermal amplification (LAMP), Eiken, Japan) for rapid diagnosis of congenital Chagas disease employing FTA cards or Whatman 903 filter paper as solid supports for small-scale volumes of human blood. METHODOLOGY/PRINCIPAL FINDINGS: We used human blood samples artificially infected with cultured T. cruzi strains to assess the analytical performance of the test in comparison with liquid blood anticoagulated with heparin. The DNA extraction process was evaluated using the ultrarapid purification system PURE manufactured by Eiken Chemical Company (Tokio, Japan) over artificially infected liquid blood or different amounts of dried blood spot (DBS) 3- and 6-mm pieces of FTA and Whatman 903 paper. LAMP was performed on a AccuBlock (LabNet, USA) heater or in the Loopamp LF-160 incubator (Eiken, Japan), and visualization of results was either done at naked eye, using the LF-160 device or P51 Molecular Fluorescence Viewer (minipcr bio, USA). Best conditions tested showed a limit of detection (LoD) with 95% accuracy (19/20 replicates) of 5 and 20 parasites/mL, respectively for heparinized fluid blood or DBS samples. FTA cards showed better specificity than Whatman 903 filter paper. CONCLUSIONS/SIGNIFICANCE: Procedures to operate LAMP reactions from small volumes of fluid blood or DBS in FTA were standardized for LAMP detection of T. cruzi DNA. Our results encourage prospective studies in neonates born to seropositive women or oral Chagas disease outbreaks to operationally evaluate the method in the field.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Recém-Nascido , Humanos , Feminino , Trypanosoma cruzi/genética , Estudos Prospectivos , Sensibilidade e Especificidade , Técnicas de Amplificação de Ácido Nucleico/métodos , Doença de Chagas/diagnóstico , Doença de Chagas/congênito
3.
ACS Infect Dis ; 9(3): 582-592, 2023 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-36780430

RESUMO

The oral transmission of Chagas disease (oCD) in Venezuela announced its appearance in 2007. Different from other populations affected by oCD and despite close supervision during treatment with nitroheterocyclic drugs, the result was treatment failure. We studied genetic features of natural bloodstream parasite populations and populations after treatment of nine patients of this outbreak. In total, we studied six hemoculture isolates, eight Pre-Tx blood samples, and 17 samples collected at two or three Post-Tx time-points between 2007 and 2015. Parasitic loads were determined by quantitative polymerase chain reaction (qPCR), and discrete typing units (DTU), minicircle signatures, and Tcntr-1 gene sequences were searched from blood samples and hemocultures. Half-maximal inhibitory concentration (IC50) values were measured from the hemocultures. All patients were infected by TcI. Significant decrease in parasitic loads was observed between Pre-Tx and Post-Tx samples, suggesting the evolution from acute to chronic phase of Chagas disease. 60% of intra-DTU-I variability was observed between Pre-Tx and Post-Tx minicircle signatures in the general population, and 43 single-nucleotide polymorphisms (SNPs) were detected in a total of 12 Tcntr-1 gene sequences, indicative of a polyclonal source of infection. SNPs in three post-Tx samples produced stop codons giving rise to putative truncated proteins or displaced open reading frames, which would render resistance genes. IC50 values varied from 5.301 ± 1.973 to 104.731 ± 4.556 µM, demonstrating a wide range of susceptibility. The poor drug response in the Pre-Tx parasite populations may be associated with the presence of naturally resistant parasite clones. Therefore, any information that can be obtained on drug susceptibility from in vitro assays, in vivo assays, or molecular characterization of natural populations of Trypanosoma cruzi becomes essential when therapeutic guidelines are designed in a given geographical area.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Humanos , Trypanosoma cruzi/genética , Venezuela/epidemiologia , Genótipo , Doença de Chagas/tratamento farmacológico , Doença de Chagas/epidemiologia , Doença de Chagas/parasitologia , Surtos de Doenças , Imunidade Inata
4.
Microorganisms ; 10(5)2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35630354

RESUMO

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected-namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen's kappa index and Bland-Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = -0.58-0.62] and -0.04 [CI = -0.45-0.37] and a Cohen's kappa coefficient of 0.78 [CI = 0.60-0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.

5.
Bol. malariol. salud ambient ; 62(5): 908-918, 2022. ilus, graf
Artigo em Espanhol | LILACS, LIVECS | ID: biblio-1418933

RESUMO

La enfermedad de Chagas (ECh) es una parasitosis del grupo de enfermedades desatendidas de la OMS. Endémica del continente americano, la transmisión se realiza en ciclos selvático, peridomiciliario y domiciliario. Epidemiológicamente, los caninos y felinos constituyen una fuente importante de infección y son centinelas de la transmisión. El perro es un hospedador común e importante del parásito ya que la presencia y número de caninos infectados en la vivienda del hombre constituyen factores de riesgo de transmisión doméstica de Trypanosoma cruzi. El presente estudio reporta la seroprevalencia de la infección por T. cruzi en la bioregión centro norte de Venezuela (Distrito Capital, Chichiriviche de la Costa del Estado La Guaira y parte del Estado Miranda), en 301 perros y 49 gatos empleando el ensayo inmunoenzimatico (ELISA). La prevalencia global en perros fue del 30,2 % en las tres zonas estudiadas mientras que en gatos fue de 40,8 %. Con relación al sexo de los animales, se encontró una prevalencia general de perros hembras del 27,6 % y para los perros machos del 33,1%. Los gatos machos presentaron una prevalencia mayor que las hembras en todas las localidades. Tanto en perros como en gatos la distribución de seropositividad fue mayor en animales intradomicilio. Se evidenció diferencia en los valores de ELISA-IgG para las poblaciones de perros muestreados en la localidad de Petare comparado con perros presentes en la localidad de Aricagua (perros de caza), (p=0,006). En líneas generales, esta última localidad presentó una media de densidad óptica para la prueba de ELISA-IgG de 0,959 [0,369 - 1,975]. La presencia de perros y gatos infectados es un factor de riesgo actual de infección por T. cruzi para el hombre tanto en el medio rural como en el urbano(AU)


Chagas disease (ChD) is a parasitic infection in the WHO Neglected Diseases group. Endemic to the american continent, transmission takes place in sylvatic, peridomiciliary and domestic cycles. Epidemiologically, canines and felines constitute an important source of infection and are sentinels of transmission. The dog is a common and important host of the parasite, since the presence and number of infected canines in the man's house are risk factors for domestic transmission of Trypanosoma cruzi. This study reports the seroprevalence of T. cruzi infection in the north-central bioregion of Venezuela (Capital District, Chichiriviche de la Costa, La Guaira State, and part of Miranda State), in 301 dogs and 49 cats using the immunoenzymatic assay (ELISA). The overall prevalence in dogs was 30.2 % in the three studied areas, while in cats it was 40.8 %. Regarding the sex of the animals, a general prevalence of 27.6 % for female dogs and 33.1% for male dogs was found. Male cats presented a higher prevalence than females in all localities. In both, dogs and cats, the distribution of seropositivity was greater in indoor animals. There was of a difference in ELISA-IgG values for the populations of dogs sampled in the town of Petare compared to dogs present in the town of Aricagua (hunting dogs), (p=0.006). In general, this last locality presented a mean optical density for the ELISA-IgG test of 0.959 [0.369 - 1.975]. The presence of infected dogs and cats is a current risk factor for T. cruzi infection for man in both of them in the rural and in the urban environment(AU)


Assuntos
Animais , Trypanosoma cruzi , Gatos , Estudos Soroepidemiológicos , Cães , Doenças Parasitárias , Epidemiologia , Doenças Transmissíveis , Doença de Chagas , Área Urbana
6.
STAR Protoc ; 2(3): 100703, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34505085

RESUMO

The pathogen Trypanosoma cruzi differentiates from epimastigotes (E) into infective metacyclic trypomastigotes (MTs) to invade the mammalian cell. This process, called metacyclogenesis, is mimicked in vitro by nutrient starvation or incubation with minimal media. Here, we describe an alternative protocol for metacyclogenesis by incubating E forms in a biphasic medium supplemented with human blood. Although time consuming, this procedure yields fully differentiated MTs without the presence of intermediate forms, even for cultures that have been maintained as E for years.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Estágios do Ciclo de Vida/fisiologia , Trypanosoma cruzi/genética , Proteínas de Protozoários , Trypanosoma cruzi/citologia , Trypanosoma cruzi/metabolismo
7.
Front Cell Infect Microbiol ; 11: 723549, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34458163

RESUMO

The clinical evolution of patients with chronic Chagas disease (CCD) is mainly associated with an excessive inflammation and a defective immunomodulatory profile caused by the interaction between T. cruzi and the host. Regulatory B (Breg) cells exert immune suppression mostly through IL-10 production (B10 cells), but also through IL-10-independent mechanisms. Previously, we demonstrated that CCD patients with cardiomyopathy show changes in the ex vivo Breg cell phenotypic distribution although maintain IL-10 production capacity. Here, we sought to identify potential alterations on Breg cells upon in vitro stimulation. Isolated B cells from CCD patients with or without cardiomyopathy and non-infected (NI) donors were stimulated with T. cruzi lysate or CpG + CD40L, and characterized by flow cytometry based on the expression of CD24, CD27, CD38, and the regulatory molecules IL-10 and PD-L1. IL-10 and IL-17 secretion in the supernatant of B cells was evaluated by ELISA. Data showed that T. cruzi stimulation diminished the expression of CD24 and CD38 on CD27- B cells while reducing the percentage of CD24high inside CD27+ B cells. Furthermore, T. cruzi induced a regulatory B cell phenotype by increasing B10 cells and IL-10 secretion in all the groups. The innate-like B10 cells expansion observed in patients with cardiomyopathy would be associated with CD27- B10 cell subsets, while no predominant phenotype was found in the other groups. Patients with cardiomyopathy also displayed higher IL-17 secretion levels in T. cruzi-activated B cells. CpG + CD40L stimulation revealed that B cells from CCD patients and NI donors had the same ability to differentiate into B10 cells and secrete IL-10 in vitro. Additionally, CCD patients showed an increased frequency of CD24-CD27- B cells and a reduction in the percentage of CD24highCD27+ Breg cells, which appeared to be inversely correlated with the presence of T. cruzi DNA in blood. Finally, CCD patients exhibited a higher frequency of PD-L1+ B cells in T. cruzi-stimulated samples, suggesting that IL-10-independent mechanisms could also be tangled in the control of inflammation. Altogether, our results provide evidence about the potential role of Breg cells in the immune response developed against T. cruzi and its contribution to chronic Chagas cardiomyopathy.


Assuntos
Linfócitos B Reguladores , Cardiomiopatia Chagásica , Doença de Chagas , Trypanosoma cruzi , Humanos , Inflamação
8.
Acta Trop ; 222: 106034, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34224715

RESUMO

Trypanosoma cruzi uses various mechanisms of infection to access humans. Since 1967, food contaminated with metacyclic trypomastigotes has triggered several outbreaks of acute infection of Chagas disease by oral transmission. Follow-up studies to assess the effectiveness of anti-parasitic treatment of oral outbreaks are rather scarce. Here, we report a 10-year laboratory follow-up using parasitological, serological, and molecular tests of 106 individuals infected in 2007 of the largest known outbreak of orally transmitted Chagas disease, which occurred in Caracas city, Venezuela. Before treatment (2007), specific IgA, IgM and IgG, were found in 71% (75/106), 90% (95/106) and 100% (106/106), respectively, in addition to 21% (9/43) parasitemia, Complement Mediated Lysis (CML) in 98% (104/106) and 79% (34/43) parasitic DNA for PCR. Blood culture detected parasitemia up to 18 months post-treatment in 6% (6/106) of the patients. In 2017, the original number of cases in the follow-up decreased by 46% and due to the country's economic situation, not all the trials could be carried out in the entire population. During follow-up, IgA and IgM disappeared promptly, with IgM persisting in 19% (20/104) of the patients three years after treatment. The anti-T. cruzi IgG remained positive 10 years later in 41% (20/49) of the individuals evaluated. CML remained positive seven years later in 79% (65/82) of the cases. PCR positive cases decreased after treatment but progressively recovered, being positive in 69% (32/46) of the individuals evaluated in 2017. The group of children (under 18 years of age) showed the highest PCR positivity with 76% (26/34) of the cases, but their parasitic load tended to diminish, while in adults the parasitic load regained their initial values. The simultaneous evaluation of serological tests and PCR of the patients allowed us to separate patients among responders and non-responders to the anti-parasitic treatment, and this information prompted us to apply a second anti-parasitic treatment in the group of non-responders. In this population not subjected to the like lihood of re-infection, adult patients were more likely to be non-responders when compared to children. These results suggest that rigorous laboratory follow-up with T. cruzi infectious biomarkers is essential to detect cases of parasite persistence.


Assuntos
Doença de Chagas , Adulto , Anticorpos Antiprotozoários/análise , Biomarcadores , Doença de Chagas/diagnóstico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/epidemiologia , Criança , Surtos de Doenças , Seguimentos , Humanos , Estudos Soroepidemiológicos , Falha de Tratamento , Venezuela/epidemiologia
9.
Vet Parasitol Reg Stud Reports ; 24: 100557, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-34024373

RESUMO

This work aimed to conduct a first PCR-based approach for differential diagnosis of kinetoplastidean infections in dogs. Diagnosis of Kinetoplastid infections in domestic animals is difficult, since parasitemia is intermittent and signs are nonspecific; it is mainly based on parasitological smears or concentration techniques, which lack sensitivity and depend on operator` expertise. Dogs are relevant reservoirs in transmission of Kinetoplastids; they function as sentinels to detect active transmission cycles before they involve humans. Trypanosoma cruzi, Trypanosoma evansi, and various species of Leishmania genus are multi-host parasites, capable of parasitizing dogs among a vast number of reservoirs. An algorithm based on sequential Real-Time PCR-High Resolution Melting (HRM) (qPCR-HRM) assays directed at 24S alpha ribosomal DNA, ITS1 and Hsp70 designed to distinguish among T. cruzi, T. rangeli, T. evansi and Leishmania spp. was tested in fourteen dogs with suspicion of kinetoplastid diseases. A qPCR control of DNA integrity in the tested sample, targeted to the mammalian interphotoreceptor retinoid-binding protein (IRBP) gene fragment was incorporated to the algorithm. T. evansi was detected in four dogs and L. infantum in one. Two of five qPCR positive cases were smear negative. Smear and T. evansi qPCR positive cases corresponded to animals that died despite being treated, indicating the association of parasitemia with disease severity. This laboratory tool increases the possibility of confirming outbreaks of kinetoplastid diseases with zoonotic potential and identify the etiological agents involved.


Assuntos
Leishmania , Trypanosoma cruzi , Lobos , Animais , Cães , Leishmania/genética , Mesopotâmia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Trypanosoma cruzi/genética
10.
Front Cell Infect Microbiol ; 11: 665063, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33996636

RESUMO

We aimed to characterize the genetic constitution of natural T. cruzi populations involved in an Oral Chagas Disease (OCD) outbreak at a rural school of the community of Chichiriviche de la Costa, Venezuela, which affected patients did not respond to the etiological treatment. Peripheral blood samples and/or hemocultures were obtained from twenty-nine OCD patients at time of diagnosis or along nine years of Post-treatment (Tx) follow-up. The IgG serology, T. cruzi discrete typing units (DTU), satellite DNA-qPCR parasitic loads, and minicircle signatures were determined at Pre-Tx and after Tx. The serological titles and parasitic loads changed after treatment, with a significant decrease of IgG titers (Spearman's r value= -0.961) and median parasite loads from 2.869 [IQR = 2.113 to 3.720] to 0.105 [IQR = -1.147 to 1.761] log10 par eq. /mL at Pre-Tx and Post-Tx, respectively, suggesting infection evolution from acute to chronic phase, without seroconversion or parasitological eradication, which was indicative of treatment failure. All patients were infected with T. cruzi DTU I populations. At Pre-Tx their median Jaccard genetic distances were 0.775 [IQR = 0.708 to 0.882], decreasing in genetic variability towards the end of follow-up (Mann-Whitney U test p= 0.0031). Interestingly, no Post-Tx minicircle signature was identical to its Pre-Tx counterpart population in a same patient, revealing selection of parasite subpopulations between the primary infection and Post-Tx. The parasitic populations isolated from hemocultures showed a lower number of bands in the minicircle signatures with respect to the signatures obtained directly from the patients' blood samples, demonstrating a process of parasitic selection and reduction of the population variability that initially infected the patients. Decrease of parasitic loads after treatment as well as Pre- and Post-Tx intra-TcI diversity might be a consequence of both, natural evolution of the acute infection to the chronic phase and persistence of refractory populations due to Tx selection.


Assuntos
Doença de Chagas , Trypanosoma cruzi , DNA de Protozoário , Seguimentos , Humanos , Carga Parasitária , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/genética
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