Enfermedad de Chagas en una zona no endémicaevaluación de nuevas técnicas diagnósticas
- Gil-Gallardo Parras, Luis Javier
- Manuel Segovia Hernández Director
- María Asunción Iborra Bendicho Director
Defence university: Universidad de Murcia
Fecha de defensa: 27 February 2023
- Pedro Albajar Viñas Chair
- Marina Simón Secretary
- Francisco Javier Nieto Martínez Committee member
Type: Thesis
Abstract
Background: The lack of a gold standard for the serological diagnosis of Chagas disease (CD), as well as the development of new diagnostic tools (PCR, CMIA, ECLIA) challenges the standardization of the diagnosis of CD both in acute and chronic pase. The objective of this work is to evaluate the performance of the new techniques in the diagnosis of chronic CD and congenital CD (CCD). Methods: A pool of sera from patients with CD was analyzed using CMIA for 10 consecutive days to monitor the quality of the results obtained by this technique over time. Different serological techniques (CMIA, IFI, ICT) were evaluated for the detection of anti-Trypanosoma cruzi antibodies using external quality assesment from the PNCQ and the WHO. A comparative study between CMIA and IFI was carried out in order to establish an equivalence between both techniques using sera from patients with CD. Several commercial molecular biology assays (RT-PCR) for the detection of T. cruzi DNA were evaluated, using T. cruzi purified DNA and samples from CD patients. In addition, the performance of CMIA for monitoring clearance of maternal antibodies was evaluated in a cohort of 82 newborns of mothers with CD in a 12-month follow-up study. Finally, the usefulness of eosinophilia in the diagnosis of Strongyloides stercoralis co-infection in CD patients, as well as the adequacy of ELISA in the diagnosis and follow-up of strongyloidiasis were assessed. Results: The internal quality control results indicate that the CMIA technique lacks random or systematic errors (CV 3.48%). The external quality assessment attested the capability of the three techniques studied for the detection of anti-T. cruzi antibodies in the positive samples; false positives results were not obtained. An equivalence between the antibody titers measured by IFI and certain ranges of values (S/CO) of CMIA was observed. All the commercial assays evaluated for the detection of T. cruzi DNA were able to detect up to 1.9x10-2 copies/µL of parasite and had good agreement with in-house PCR in samples from CD patients. Regarding the antibody clearance study in children born to mothers with CD, 81% of children were seronegative by CMIA and 100% by IFI at 9 months of age. At 12 months of age, all children without CCD were negative by both techniques. Finally, the sensitivity and specificity of eosinophilia in the diagnosis of strongyloidiasis was 95.7% and 61.1%, respectively, and a serological cure of 70-80% was obtained by ELISA one year after treatment with ivermectin. Conclusions: The CMIA technique is free of random or systematic errors. The use of external quality controls confirms that the CMIA, IFI and ICT techniques are adequate for the serological diagnosis of EC. Commercial PCR are useful for the detection of T. cruzi DNA both in vitro and in vivo. The CMIA technique is not linear, but an antibody titer could be establish using IFI for a certain range of CMIA values. The CMIA technique is useful in monitoring antibody clearance in healthy infants born to mothers with CD. The detection of anti-S. stercoralis by ELISA is valuable for the diagnosis of strongyloidiasis and for post-treatment follow-up.