Carbapenemase Detection in Clinical Isolates: Development of Agglutination Tests as a Diagnostic Alternative

• Grant number: 23/12960-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2024
• Status: Discontinued
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Ana Cristina Gales
• Grantee: André Valêncio Siqueira
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated scholarship(s): 24/13835-8 - Development and Validation of a CRISPR-Cas12-Based Diagnostic Platform for Rapid Detection of Carbapenemase Encoding Genes, BE.EP.DR

Abstract

The treatment of carbapenemase-producing Enterobacteriaceae (CPE) involves the combination of ²-lactam antibiotics already used in clinical practice and new carbapenemase inhibitors. Due to the selective action spectrum among carbapenemases, it becomes necessary to differentiate between them for the proper prescription of antimicrobials. However, the lack of rapid tests has hindered the implementation of appropriate antibiotic therapy, increasing hospital costs, resistance rates, and patient mortality. The latex agglutination test is based on the principle of antigen-antibody binding (Ag-Ab) coupled with latex nano particles, which, upon contact with the target antigen, triggers an agglutination reaction. Agglutination tests are quick (around 3 minutes), easy to perform, and do not require specialized equipment or technicians for execution. Various agglutination tests are available for recognizing different clinically relevant antigens in infectious diseases; however, there are no commercially available agglutination tests for the detection of carbapenemases. Therefore, our goal is to develop, standardize, and validate a rapid latex agglutination test platform based on the epidemiology of antimicrobial resistance in the national territory. To achieve these objectives, we will select four clinically relevant carbapenemase enzymes present in the Brazilian epidemiology, namely KPC-2, KPC-160, NDM, and OXA-23. The genes encoding these enzymes will be confirmed and subsequently cloned into an expression vector. The enzymes will then be expressed and purified. Polyclonal antibodies will be produced through the immunization of New Zealand rabbits. The antibodies will be purified using affinity columns and conjugated to latex particles. Different sample preparation methodologies will be tested for optimization and rapid test execution. Validation will involve different strains of carbapenemase-producing and non-producing Enterobacteriaceae, as well as blind testing of clinical samples. With the results obtained, we will present a new rapid test for the detection and differentiation of carbapenemases based on the worldwide epidemiology of carbapenemases produced by Enterobacteriaceae. We hope that this test will be cost-effective, easy to perform, and available for clinical laboratories.