Development of an immunochromatographic test for detection of carbapenemases in Enterobacteria

• Grant number: 21/01158-3
• Support Opportunities: Regular Research Grants
• Start date: July 01, 2021
• End date: June 30, 2024
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Ana Cristina Gales
• Grantee: Ana Cristina Gales
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated researchers: Marcelo Ferreira Marcondes Machado ; Roxane Maria Fontes Piazza

Abstract

The approval of new antimicrobials for the treatment of infections caused by gram-negative bacteria producing carbapenemases of classes A, C, and D resulted in the need for laboratory tests that are capable of differentiating classes of beta-lactamases. The immunochromatographic test is based on the antigen-antibody binding for the differentiation of several substances, and its use has shown good results in previous studies. However, the immunochromatographic tests developed for the detection of carbapenemases are based on the epidemiology of high-income countries, therefore, they are not completely adapted to the Brazilian reality. Therefore, our goal is the development, standardization, and validation of a rapid immunochromatographic test based on the epidemiology of antimicrobial resistance observed in the national territory. To achieve these objectives, we will select two carbapenemase enzymes of clinical interest present in Brazilian epidemiology, namely: KPC-2 and SPM-1. The genes of these enzymes will be confirmed and, later, cloned into an expression vector. After, the enzymes will be expressed and purified. Polyclonal antibodies will be produced from the immunization of New Zealand rabbits and monoclonal antibodies from Balb/c mice. The mice will be sacrificed for the removal of popliteal lymph nodes, which will be fused to obtain hybridomas. These will be cloned and classified according to the antibodies produced. The antibodies will be purified on affinity columns, and conjugate on colloidal gold particles. For the manufacture of the immunochromatographic platform, we will use glass, cellulose, fiber, and nitrocellulose HiFlow papers. These papers will be treated and overlaid on a plastic base. The standardization of the test will be based on the evaluation of different parameters, such as the antibody concentration. For validation, different strains of carbapenemase-producing and non-producing enterobacteria will be used. We expect to develop a new rapid laboratory test for the detection and differentiation of carbapenemases, based on the worldwide epidemiology of carbapenemases produced by enterobacteria. We hope that this test is inexpensive, easy to perform, and that it can be made available to clinical laboratories. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

VALENCIO, ANDRE; DA SILVA, MIRIAM APARECIDA; SANTOS, FERNANDA FERNANDES; POLATTO, JULIANA MOUTINHO; MACHADO, MARCELO MARCONDES FERREIRA; PIAZZA, ROXANE MARIA FONTES; GALES, ANA CRISTINA. Capture ELISA for KPC Detection in Gram-Negative Bacilli: Development and Standardisation. MICROORGANISMS, v. 11, n. 4, p. 14-pg., 2023-04-01. (21/01158-3)