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ELISA for Chagas disease
What Is Chagas Disease
Chagas disease is a chronic, systemic, parasitic infection caused by the protozoan Trypanosoma cruzi. According to estimates by the Pan American Health Organization and the World Health Organization (WHO), 7.7-10 million people are chronically infected with T. cruzi and 10,000-14,000 deaths per year are caused by Chagas disease . Approximately 60-70% of the chronic patients have no clinical symptoms (indeterminate form), whereas 30-40% either have or will develop cardiomyopathy, digestive megasyndromes or both. Chagas disease ranks among the world’s most neglected diseases and is considered to be the parasitic infection with the greatest socioeconomic impact in Latin America, being responsible for an estimated US$ 1.2 billion in lost productivity annually .In the last four decades, controlling vector and blood transfusion-transmission of T. cruzi has been successful in several endemic countries. However, other transmission mechanisms, such as congenital, transplantation and oral, may sustain infection. Furthermore, population migration has caused Chagas disease to be a worldwide public health issue.
New treatments for Chagas disease are urgently needed. The only two drugs available, benznidazole (BZ) and nifurtimox (NF), require prolonged treatment, are often poorly tolerated and have variable efficacy in the chronic phase of the disease, which is the predominant clinical presentation encountered .The reasons for treatment failures are unknown, but may be due to different treatment evaluation methods, incomplete treatment administration, variable drug susceptibility among T. cruzi strains and characteristics of the host’s immune system .Nevertheless, several non-randomised clinical studies demonstrate the efficacy of etiological treatment to reduce antibody titres and the progression of chronic Chagas heart disease .Two double-blind, placebo-controlled clinical trials with a large cohort of patients, TRAENA and BENEFIT (Figure), are assessing whether BZ therapy improves prognosis for patients with chronic Chagas disease.
ELISA For Chagas Disease
For all blood collections, the parents or person responsible for each student received instructions and signed the Instructed and Free Terms of Consent.
Three parameters were used for sample calculation: I) the proportion of individuals infected with T. cruzi in the age group studied in the national investigation of Chagas disease between 1975 and 19809; II) an acceptable error of 5%; and III) the number of schoolchildren in the rural zone of the municipality. In the absence of data related to the first parameter, a proportion of 41-50% of the total schoolchildren was used. The sample size was increased to 50% for greater reliability and was higher than that recommended for the period by the Health Ministry9. The number of children by age group and by school was calculated to estimate who should be examined. Children were stratified by school and age. Each child received a number for identification. A simple random selection was performed using the statistics program Epi Info 6.0 to calculate the final sample size. Thus, the sample size determined for this study was 603 individuals. The children were evaluated according to the recommendations of the Health Ministry7 for the laboratory diagnosis of T. cruzi infection or suspected Chagas disease.
During the first phase, blood samples collected on filter paper were evaluated by an ELISA test using blood eluate as in previous studies10. Next, venous blood from all participants considered positive or equivocal during the first phase was tested along with 10% of the negative samples to confirm the ELISA results. The following conventional serological tests were employed: enzyme-linked immunosorbent assay (ELISA), indirect hemaglutination assay (IHA) and indirect immunofluorescence (IIF) assay. The ELISA test was performed with serum samples diluted to 1:80, alkaline antigen of T. cruzi Y strain epimastigote forms and human anti-immunoglobulin G (IgG) labeled with peroxidase diluted to 1:10,000. The reaction was quantified by spectrometry (Bio-Rad model 680) at a wavelength of 490nm. Positive and negative controls were processed in parallel. The cut-off was established as the mean of the absorbance of ten negative control sera + three times the standard deviation calculated for each plate. Serum samples with absorbance values above the cut-off were considered positive.
Kits used for IHA and IIF tests were purchased from Bio-Mérieux and Bio-Manguinhos/FIOCRUZ/RJ, respectively, and the techniques were performed according to the manufacturer's instructions. The results were considered positive when tests of at least two types were positive, uncertain when the results of the tests were discordant and negative when two tests were not reactive according to WHO9 and Brazilian Ministry of Health11 recommendations.
Chagas Disease Treatment
In conjunction with high priority research on treatment for Chagas disease, the development of diagnostic tests for early determination of therapeutic responses and cure is urgently required (WHO 2012). A Strategic Translation Award from The Wellcome Trust is funding a three-year study to identify new biological markers for the evaluation of therapeutic response in Chagas disease (Figure). The WHO, the Special Programme for Research and Training in Tropical Diseases and the Pan-American Health Organization have sponsored an international study to evaluate polymerase chain reaction (PCR) methods detecting T. cruzi DNA in blood samples of patients with Chagas disease . Further work is still needed to validate these methods through prospective studies in different settings, such as post-treatment follow-up. Nevertheless, alternatives to PCR are clearly needed.
Especially with the benefit of new drugs, international consensus is required to scale up diagnosis and treatment, to define criteria for treatment and to provide access to drugs. Currently, treatment coverage for Chagas disease patients is unacceptably low because many guidelines exclude chronic patients who are undoubtedly the most vulnerable majority.
For efficient R&D, highly effective collaborations are required to optimise use of limited funding. Continued efforts to focus and improve drug discovery will greatly benefit both the research community and the population burdened with Chagas disease.
ELISA For Chagas Disease Related Studies
1.Bianca Zingales et al.(2014).Drug discovery for Chagas disease should consider Trypanosoma cruzi strain diversity.Mem Inst Oswaldo Cruz 0074-0276.
2.Duarte LF et al.(2014).Comparison of seven diagnostic tests to detect Trypanosoma cruzi infection in patients in chronic phase of Chagas disease.Colomb Med (Cali). 45(2):61-6.
3.Adriana dos Santos et al.(2014).Evaluation of the Chagas Disease Control Program in Açucena Municipality, Rio Doce Valley, State of Minas Gerais, Brazil.Rev. Soc. Bras. Med. Trop. vol.47 no.2 .