  Citation: Muñoz-Calderón, A.A.; Besuschio, S.A.; Wong, S.; Fernández, M.; García Cáceres, L.J.; Giorgio, P.; Barcan, L.A.; Markham, C.; Liu, Y.E.; de Noya, B.A.; et al. Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease. Microorganisms 2022, 10, 909. https://doi.org/10.3390/ microorganisms10050909 Academic Editor: José Ma. Alunda Received: 1 March 2022 Accepted: 11 April 2022 Published: 26 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). microorganisms Communication Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease Arturo A. Muñoz-Calderón 1,† , Susana A. Besuschio 1,† , Season Wong 2 , Marisa Fernández 3 , Lady J. GarcíaCáceres 1 , Patricia Giorgio 4 , Laura A. Barcan 5 , Cole Markham 2 , Yanwen E. Liu 2 , Belkisyole Alarcón de Noya 6 , Silvia A. Longhi 1 and Alejandro G. Schijman 1, * 1 Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI-CONICET), Buenos Aires 1428, Argentina; arturomc35@gmail.com (A.A.M.-C.); bsusanaalicia@gmail.com (S.A.B.); ladyjuliette1108@gmail.com (L.J.G.C.); longhi.ingebi@gmail.com (S.A.L.) 2 AI Biosciences, Inc., College Station, TX 77845, USA; season.wong@aibiosciences.com (S.W.); markhamc@gmail.com (C.M.); yanwenliu1997@gmail.com (Y.E.L.) 3 Hospital de Enfermedades Infecciosas “Dr. Francisco J. Muñiz”, Buenos Aires 1282, Argentina; marisa.fernandez@gmail.com 4 Servicio de Infectología, Hospital Británico de Buenos Aires, Buenos Aires 1280, Argentina; patrilougiorgio@gmail.com 5 Sección Infectología, Departamento de Medicina, Hospital Italiano, Buenos Aires 1199, Argentina; labarcan@gmail.com 6 Instituto de Medicina Tropical, Universidad Central de Venezuela, Caracas 1053, Venezuela; belkisuole@gmail.com * Correspondence: schijman@dna.uba.ar † These authors contributed equally to this work. Abstract: 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. Keywords: loop-mediated isothermal amplification; real-time PCR; Trypanosoma cruzi; Chagas-HIV; orally transmitted Chagas disease; primary infection after transplant in seropositive donor-seronegative recipients; Chagas disease reactivation 1. Introduction Chagas disease (CD), a neglected tropical disease (NTD) caused by the protozoan Trypanosoma cruzi, affects about 7 million people worldwide, mainly in endemic areas of Microorganisms 2022, 10, 909. https://doi.org/10.3390/microorganisms10050909 https://www.mdpi.com/journal/microorganisms