Colorimetric Loop-Mediated Isothermal Amplification Assays Accurately Detect blaOXA-23-like and ISAba1 Genes from Acinetobacter baumannii in Pure Cultures and Spiked Human Sera.

Abstract

Carbapenem resistance in Acinetobacter baumannii is a critical global health threat attributed to transferrable carbapenemase genes. Carbapenemase genotyping using polymerase chain reaction (PCR) presents a challenge in resource-limited settings because of its technical requirements. This study designed new loop-mediated isothermal amplification (LAMP) primers using multiple sequence alignment-based workflows, validated the primer performance against multiple target variants in silico, and developed novel LAMP assays (LAntRN-OXA23 and LAntRN-ISAba1) to detect the transferable blaOXA-23-like carbapenemase genes and ISAba1 elements in pure cultures and A. baumannii-spiked serum samples. The designed LAMP primers bind to the conserved regions of their highly polymorphic targets, with their in silico performance comparable with other published primers. The in vitro LAMP assays (using 30 PCR-profiled A. baumannii and 10 standard multidrug-resistant gram-negative isolates) have 100% concordance with the PCR-positive clinical samples, limits of detection as low as 1 pg/µL (200 copies/µL), and specificities of 57.89-100%. Both assays produced positive results when testing DNA samples (extracted using a commercial kit) from blaOXA-23-like and ISAba1-blaOXA-51-like PCR-positive A. baumannii-spiked normal human sera (five set-ups per target). In summary, the LAMP assays accurately detected the target genes and have applications in infection management, control, and point-of-care testing in resource-limited healthcare settings.