RESUMO
BACKGROUND: Measurement of the success of antitrypanosomal treatment for Chagas disease is difficult, particularly in the chronic phase of the disease, because anti-Trypanosoma cruzi antibodies persist in serum for prolonged periods. We studied the effects of nifurtimox administered by two different treatment regimens on the T. cruzi calcium-binding flagellar protein F29 in children diagnosed with Chagas disease measured using an enzyme-linked immunosorbent assay (ELISA) technique (ELISA F29). METHODS AND PRINCIPAL FINDINGS: In a phase 3, randomized, double-blind, parallel-group, historically controlled study (ClinicalTrials.gov NCT02625974), blood samples obtained from children diagnosed with Chagas disease and treated with nifurtimox for either 60 days or 30 days were analyzed using an ELISA with an F29 recombinant protein as the antigen, as well as conventional serological tests (recombinant ELISA and indirect hemagglutination assay). In an exploratory approach, serological response to nifurtimox treatment was evaluated for 4 years post-treatment. In both treatment groups, the number of patients with negative ELISA F29 values increased over the period of observation. The incidence rate of negative seroconversion using ELISA F29 was 22.94% (95% CI: 19.65%, 26.63%) in the 60-day treatment group and 21.64% (95% CI: 17.21%, 26.86%) in the 30-day treatment group. In the subpopulation of patients who tested seropositive for F29 before nifurtimox treatment, 88 patients (67.7%) in the 60-day regimen and 39 patients (59.1%) in the 30-day regimen were F29 seronegative at 4 years post-treatment. All patients who had a positive ELISA F29 test at baseline and seroconverted to negative measured by conventional serology reached seronegativity in ELISA F29 earlier than in conventional serology. CONCLUSIONS: The results demonstrate a serological response to treatment with nifurtimox measured by the ELISA F29 test in children diagnosed with Chagas disease. The F29-based ELISA can be considered a potential early marker of response to antitrypanosomal therapy for Chagas disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT02625974.
Assuntos
Doença de Chagas , Tripanossomicidas , Humanos , Criança , Nifurtimox/uso terapêutico , Tripanossomicidas/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Doença de Chagas/diagnóstico , Doença de Chagas/tratamento farmacológico , Anticorpos Antiprotozoários , BiomarcadoresRESUMO
BACKGROUND AND OBJECTIVE: The real prevalence of congenital Chagas disease is undefined because of difficulties in the detection of Trypanosoma cruzi by microscopic examination. The aim of this study was to determine the diagnostic accuracy of two molecular diagnostic tools, qPCR and LAMP, in the diagnosis of congenital Chagas disease in a clinical setting. METHODS: To this end, we conducted a prospective cohort study in a tertiary care center, of infants under 9 months of age, born in Buenos Aires to women with Chagas disease. Blood samples were collected for microscopic examination and molecular diagnosis at baseline. If negative, infants were followed up until 9 months of age to determine a final diagnosis by serology. In-house qPCR and LAMP previously validated were challenged as index tests. RESULTS: A total of 154 participants were potentially eligible, 120 of whom were enrolled. Finally, 102 (66.2%) of them fulfilled the follow-up. The diagnosis of congenital Chagas disease was confirmed in 13 infants and excluded in 89. Both the sensitivity and specificity of the qPCR were 100.0% (95% confidence interval 75.3-100.0 and 95% confidence interval 95.9-100.0, respectively), whereas the sensitivity and specificity of LAMP were 69.2% (95% confidence interval 38.6-90.9) and 100% (95% confidence interval 95.9-100.0), respectively. CONCLUSIONS: The qPCR agreed with the current diagnostic algorithm, and was a reliable and sensitive tool to detect congenital Chagas disease earlier, providing an appropriate and timely identification of infected infants requiring treatment. LAMP was able to detect congenital Chagas disease in infected infants by naked-eye visualization in accordance with a microscopic examination. The advantages of molecular diagnostic tools should be taken into account by the health system to improve congenital Chagas disease diagnosis.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Doença de Chagas/congênito , Doença de Chagas/diagnóstico , Feminino , Humanos , Lactente , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Trypanosoma cruzi/genéticaRESUMO
trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/diagnóstico , Doença de Chagas/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Trypanosoma cruzi/isolamento & purificação , Animais , Doença de Chagas/sangue , Doença de Chagas/parasitologia , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Doenças do Cão/sangue , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Glicoproteínas/análise , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Neuraminidase/análise , Neuraminidase/genética , Neuraminidase/imunologia , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Fatores de Virulência/sangue , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
The efficacy of specific chemotherapy in congenital Chagas disease before the first year of life ranges between 90 and 100%. Between this age and 15 years of age, the efficacy decreases to around 60%. Therefore, early infection detection is a priority in vertical transmission. The aim of this work was to assess whether polymerase chain reaction (PCR) plays a predictive role in the diagnosis of congenital Chagas disease as compared to conventional parasitological and serological methods. To this end, we studied a total of 468 children born to Trypanosoma cruzi seroreactive mothers came from Argentina, Bolivia and Paraguay, who lived in the city of Buenos Aires and suburban areas (Argentina), a non-endemic area of this country. These children were assessed by PCR from 2004 to 2009 with the specific primers Tcz1 and Tcz2, and 121 and 122. PCR allowed detecting 49 T. cruzi-positive children. Eight of these 49 children were excluded from the analysis: six because they did not complete follow-up and two because the first control was performed after 12 months of age. Parasitological methods allowed detecting 25 positive children, 7 of whom had been earlier diagnosed by PCR (1.53±2.00 vs. 6.71±1.46 months; p=0.0002). Serological methods allowed detecting 16 positive children, 12 of whom had been earlier diagnosed by PCR (1.46±1.48 vs. 11.77±4.40 months; p<0.0001). None of the children negative by PCR was positive by serological or parasitological methods. This study shows that PCR allows early diagnosis in congenital Chagas disease. At present, an early positive PCR is not indicative for treatment. However, a positive PCR would alert the health system to search only those infected infants diagnosed by early PCR and thus generate greater efficiency in the diagnosis and treatment of congenital T. cruzi infection.
Assuntos
Doença de Chagas/congênito , Doença de Chagas/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Parasitologia/métodos , Reação em Cadeia da Polimerase/métodos , Trypanosoma cruzi/isolamento & purificação , Adulto , Argentina , Diagnóstico Precoce , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Gravidez , Testes Sorológicos/métodos , Trypanosoma cruzi/genética , Adulto JovemRESUMO
Tc13Tul antigen is expressed in the mammalian stages of Trypanosoma cruzi, the etiological agent of Chagas' disease. Here, we designed and validated an enzyme-linked immunosorbent assay using the recombinant Tc13Tul (Tc13Tul-ELISA) and found that it had 82.5% sensitivity and 97.05% of specificity. To evaluate whether the decrease in antibodies against Tc13Tul may be used as an early marker of the effect of chemotherapy with benznidazole, sera from 30 T. cruzi-infected children were evaluated by Tc13Tul-ELISA before and after benznidazole treatment. While in Group A (6 months-4 years old, n = 16) the decrease of more than 30% of Tc13Tul-ELISA values showed a sensitivity similar to that of conventional serology (CS); in Group B, (5-12 years old, n = 14) the decrease of Tc13Tul-ELISA values was a better parameter than negativization of CS to monitor the impact of treatment. Therefore, the dosage of anti-Tc13Tul antibodies may be useful as a methodology complementary to CS to evaluate chagasic patients undergoing chemotherapy with benznidazole.
Assuntos
Antígenos de Protozoários/sangue , Doença de Chagas/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática/métodos , Nitroimidazóis/uso terapêutico , Trypanosoma cruzi/isolamento & purificação , Anticorpos Antiprotozoários/sangue , Doença de Chagas/imunologia , Criança , Pré-Escolar , Humanos , Lactente , Sensibilidade e Especificidade , Testes Sorológicos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Congenital transmission (CT) has acquired relevance in Chagas disease (CHD). A cohort of pregnant CHD women (4,355) and their babies were studied in the period 1994-2004. Children were excluded when they had received blood transfusions, or were born or had been in endemic areas; CT rate was 6.1%. Babies were diagnosed between months 1 and 5 in 68.9% of the cases and between months 6 and 12 in 31.1%. In the latter group, parasitemia was detected in 94% and serology in 74.7%. Between months 6 and 9, parasitemia diagnosed 36.2% (P = 0.000) more cases than serology. If serology had been the diagnosis method, those children would have been considered CT free. Taking the overall outcomes, 38.1% of babies were CT free, and 55.8% did not complete the follow-up. Establishing CT as a public health priority and improving first-line health service, congenital CHD coverage could be more efficient in endemic countries.
Assuntos
Doença de Chagas/congênito , Complicações Parasitárias na Gravidez/epidemiologia , Trypanosoma cruzi , População Urbana , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Argentina/epidemiologia , Doença de Chagas/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Vigilância da População , Gravidez , Estudos Soroepidemiológicos , Fatores de Tempo , Trypanosoma cruzi/imunologiaRESUMO
The Trypanosoma cruzi karyotype shows an extensive chromosomal size polymorphism. Absence of condensed mitotic chromosomes and chromatin fragility are characteristic features of T. cruzi which would allow DNA breaks and chromosomal rearrangements during cell proliferation. We have investigated by pulsed field gel electrophoresis (PFGE) eventual changes in chromosomal size during exponential and stationary phases of T. cruzi epimastigotes in culture, in G0 trypomastigotes and throughout the cell cycle in synchronized epimastigotes. T. cruzi molecular karyotype was stable throughout the cell cycle and during differentiation. Thus, the chromosomal size polymorphism previously reported in T. cruzi contrasts with the stability of the molecular karyotype observed here and suggests that chromosomal rearrangements leading to changes in chromosomal size are scarce events during the clonal propagation of this parasite.