RESUMEN
Chagas disease is still a major public health issue in many Latin American countries. One of the current major challenges is to find an association between Trypanosoma cruzi discrete typing units (DTUs) and clinical manifestations of the disease. In this study, we used a multilocus conventional PCR and quantitative real time PCR (qPCR) approaches to perform the molecular typing and parasite load quantification directly from blood specimens of 65 chronic Chagas disease patients. All patients were recruited at the same health center, but their place of birth were widely distributed in different geographic regions of Brazil. Of the 65 patients, 35 (53.8%) presented positive amplification by real time qPCR, being 20 (30.7%) with the clinical indeterminate form and 15 (23.1%) with the cardiac form of the disease. The parasite load median for all positive patients was 2.54 [1.43-11.14] parasite equivalents/mL (par. Eq./mL), with the load ranging from 0.12 to 153.66 par. Eq./mL. Noteworthy, the parasite load was significantly higher in patients over 70 years old (median 20.05 [18.29-86.86] par. Eq./mL). Using guanidine-EDTA blood samples spiked with reference T. cruzi strains, belonging to the six DTUs, it was possible to genotype the parasite up to 0.5 par. Eq./mL, with high specificity. Of the patients with positive qPCR, it was possible to identify the T. cruzi DTU in 28 patients (80%). For the remaining patients (20%), at least a partial result was obtained. Analysis of specimens showed prevalences of TcVI, TcII and mixed infection TcVI+TcII equal to 40%, 17.1% and 14.3%, respectively. In addition, two patients were infected by TcV, and one patient was coinfected by TcIII+TcVI, These last three patients were in stage A of chronic chagasic cardiomyopathy (CCC), and they were born at the Bahia State (northeast region of Brazil). When T. cruzi genotypes were compared with the parasite load, more elevated parasite loads were observed in patients infected by TcII in general (parasite load median of 7.56 par. Eq./mL) in comparison to patients infected by TcVI (median of 2.35 par. Eq./mL). However, while the frequency of CCC was 50% in patients infected by TcVI and TcV, only 16.7% of patients infected by TcII evolved to CCC. Taking together, our results contribute to update the epidemiological knowledge of T. cruzi DTUs in Brazil, and highlight the age of patient and infection by TcII as important features that lead to the observation of higher parasitemia levels.
Asunto(s)
Enfermedad de Chagas/sangre , Enfermedad de Chagas/epidemiología , Variación Genética , Carga de Parásitos/estadística & datos numéricos , Trypanosoma cruzi/genética , Anciano , Brasil/epidemiología , Enfermedad de Chagas/parasitología , Coinfección/epidemiología , Coinfección/parasitología , Estudios Transversales , ADN Protozoario/genética , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Tipificación Molecular/métodos , Carga de Parásitos/métodos , Parasitemia/epidemiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Inconclusive results of serological diagnosis in Chagas disease have an important impact on blood banks worldwide, reflecting in the high number of discarded bags or in an increased transmission by blood transfusion. Molecular techniques such as qPCR have been used for diagnosis and to monitor Trypanosoma cruzi load in peripheral blood samples. A promising perspective refers to the possibility of parasite DNA detection in serum, taking advantage in using the same samples collected for serological screening. METHODS: In order to evaluate the effectiveness of a qPCR strategy for detecting and quantifying T. cruzi DNA in serum, we selected 40 chronic Chagas disease patients presenting different clinical manifestations: Cardiac (23), Digestive (4), Mixed form [cardiodigestive] (7), and asymptomatic (6). Twenty seronegative individuals from non-endemic areas were included as controls. Samples were extracted using QIAamp DNA mini kit (QIAGEN) and qPCR was performed in a multiplex format with TaqMan probes for the nuclear satellite DNA of T. cruzi and for the human RNase P gene. In addition, DNA migration to serum during blood coagulation was assessed using a commercial exogenous control (Exo IPC, Applied Biosystems) in a separate qPCR reaction. RESULTS: The comparative duplex qPCR analysis revealed that, even with an increase in Ct values, it was possible to detect all DNA targets in serum. In addition, the same linearity range for T. cruzi quantification (from 10(5) to 0.5 par. eq./mL) between serum, blood or culture samples (T. cruzi epimastigotes - Cl Brener strain) was found. When patient samples were evaluated, no significant differences in parasite load between the distinct clinical manifestations were found for both blood and serum samples. Moreover, median values of parasite burden were 1.125 and 1.230 par. eq./mL for serum and blood, respectively. Using serology as gold standard, we found 95% sensitivity for T. cruzi detection in serum and 97.5% for blood, and 100% specificity for both samples. CONCLUSIONS: Taken together, our data indicate the potential of using serum samples for molecular diagnosis and parasite load quantification by qPCR, suggesting its use in reference laboratories for the diagnosis of Chagas disease patients.
Asunto(s)
Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adolescente , Adulto , Enfermedad Crónica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Quantitative real-time PCR (qPCR) is an accurate method to quantify Trypanosoma cruzi DNA and can be used to follow-up parasitemia in Chagas disease (CD) patients undergoing chemotherapy. The Benznidazole Evaluation for Interrupting Trypanosomiasis (BENEFIT) study is an international, multicenter, randomized, double-blinded and placebo-controlled clinical trial to evaluate the efficacy of benznidazole (BZ) treatment in patients with chronic Chagas cardiomyopathy (CCC). One important question to be addressed concerns the effectiveness of BZ in reducing overall parasite load in CCC patients, even in the absence of parasitological cure. This report describes the evaluation of multiple procedures for DNA extraction and qPCR-based protocols aiming to establish a standardized methodology for the absolute quantification of T. cruzi DNA in Guanidine-EDTA blood (GEB) samples. A panel of five primer sets directed to the T. cruzi nuclear satellite DNA repeats (Sat-DNA) and to the minicircle DNA conserved regions (kDNA) was compared in either SYBR Green or TaqMan systems. Standard curve parameters such as, amplification efficiency, coefficient of determination and intercept were evaluated, as well as different procedures to generate standard samples containing pre-established T. cruzi DNA concentration. Initially, each primer set was assayed in a SYBR Green qPCR to estimate parasite load in GEB samples from chronic Chagas disease patients. The results achieved from Bayesian transmutability analysis elected the primer sets Cruzi1/Cruzi2 (p=0.0031) and Diaz7/Diaz8 (p=0.0023) coupled to the QIAamp DNA Kit extraction protocol (silica gel column), as the most suitable for monitoring parasitemia in these patients. Comparison between the parasite burden of 150 GEB samples of BENEFIT patients from Argentina, Brazil and Colombia, prior to drug/placebo administration, was performed using Cruzi1/Cruzi2 primers in a SYBR Green approach. The median parasitemia found in patients from Argentina and Colombia (1.93 and 2.31 parasite equivalents/mL, respectively) was around 20 times higher than the one estimated for the Brazilian patients (0.1 parasite equivalents/mL). This difference could be in part due to the complexity of T. cruzi genetic diversity, which is a factor possibly implicated in different clinical presentations of the disease and/or influencing parasitemia levels in infected individuals from different regions of Latin America. The results of SYBR Green qPCR assays herein presented prove this methodology to be more cost efficient than the alternative use of internal fluorogenic probes. In addition, its sensitivity and reproducibility are shown to be adequate to detect low parasitemia burden in patients with chronic Chagas disease.
Asunto(s)
Cardiomiopatía Chagásica/parasitología , Técnicas de Diagnóstico Molecular/métodos , Carga de Parásitos/métodos , Parasitemia/parasitología , Parasitología/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Trypanosoma cruzi/aislamiento & purificación , Antiprotozoarios/administración & dosificación , Argentina , Brasil , Cardiomiopatía Chagásica/tratamiento farmacológico , Colombia , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Método Doble Ciego , Monitoreo de Drogas/métodos , Monitoreo de Drogas/normas , Humanos , Técnicas de Diagnóstico Molecular/normas , Nitroimidazoles/administración & dosificación , Carga de Parásitos/normas , Parasitemia/tratamiento farmacológico , Parasitología/normas , Placebos/administración & dosificación , Ensayos Clínicos Controlados Aleatorios como Asunto , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Manejo de Especímenes/métodosRESUMEN
The Brazilian Ministry of Health has made tests for HIV1 and HIV2, HTLV I and HTLV II, HCV, HBV, T. cruzi, T. pallidum and Plasmodium in endemic areas, mandatory for all blood collection bags used in the country. However, blood-borne infectious diseases are not investigated in blood recipients before transfusion. For this study, a serological evaluation of recipients before transfusion was carried out. Prior to transfusion, serum samples from 159 blood recipients were analyzed using the same tests used in the serological screening of blood donors. The blood recipients were divided into three groups: Group 1 (G1), patients who had never received blood, Group 2 (G2), patients who had received multiple transfusions and Group 3 (G3) one-off recipients. SPSS v.8 was used for statistical analysis. Values of p<0.05 were taken to be significant. The results showed that 62 blood recipients tested positively for one or more blood-borne infectious diseases. In addition, several recipients were unaware of their serological status before the transfusion. The identification of blood-borne infectious diseases in recipients before transfusion could avoid the State being held responsible by recipients who were unaware that they were carriers of such diseases and only found out about their contamination after transfusion.
O Ministério da Saúde brasileiro determina a realização de testes sorológicos para HIV 1 e 2, HTLV I e II, HCV, HBV, T. cruzi, T. pallidum e Plasmodium nas áreas endêmicas, em todas as bolsas de coleta de sangue utilizadas no País. Entretanto, as doenças infecciosas transmissíveis através do sangue não são investigadas nos receptores de sangue (RS) antes da transfusão. Neste estudo, realizamos uma avaliação sorológica dos RS anterior à transfusão. Amostras de soro de 159 RS foram analisadas aplicando-se os mesmos testes utilizados na triagem sorológica dos doadores de sangue. Os RS foram divididos em três grupos: Grupo 1 (G1), pacientes que nunca receberam sangue, Grupo 2 (G2), pacientes politransfundidos e Grupo 3 (G3) receptores eventuais. Para a análise estatística utilizou-se o programa SPSS v.8. Valores de p<0,05 foram considerados significantes. Os resultados mostraram que 62 RS apresentaram positividade para uma ou mais doenças infecciosas transmissíveis pelo sangue. Além disso, vários RS desconheciam seu estado sorológico anterior à transfusão. A identificação de doenças infecciosas transmissíveis pelo sangue em RS anterior à transfusão poderia evitar a responsabilidade do Estado pelos RS que desconheciam ser portadores de tais doenças e apenas tiveram conhecimento de sua contaminação após a transfusão.