Rapid molecular differentiation of key New Delhi metallo-β-lactamase variants in a setting with emerging high-risk...

The increasing dissemination of bla-New Delhi metallo-β-lactamase (NDM) among high-risk clones and the lack of rapid methods to distinguish between its variants limit real-time epidemiological surveillance and timely infection control responses. This study aimed to develop and evaluate a real-time PCR assay for the rapid differentiation of the most prevalent blaNDM variants (NDM-1, NDM-5, and NDM-14) circulating in our setting.

A multiplex real-time PCR assay targeting 2 polymorphic positions (389 and 460) of blaNDM was designed using reference sequences (FN396876.1, JN104597.1, and KM210086.1). Amplification was performed on a CFX96 system under standardized conditions. The assay was evaluated using 60 well-characterized NDM-producing isolates (20 NDM-1, 20 NDM-5, and 20 NDM-14) and 10 non-NDM as negative controls. All isolates had been previously characterized by whole-genome sequencing, with resistance mechanisms confirmed using CARD and ResFinder databases.

Distinct subtype-specific amplification patterns were observed based on ΔCt values (VIC Ct minus FAM Ct). NDM-1 isolates showed minimal variation between channels at both positions (mean ΔCt 0.15 ± 0.20 at 389; 0.05 ± 0.10 at 460). NDM-14 displayed a marked shift at position 389 (mean ΔCt 4.2 ± 0.5) with negligible differences at 460. In contrast, NDM-5 consistently showed VIC signal dropout at position 460 while maintaining minimal ΔCt at 389 (0.05 ± 0.15). All non-NDM isolates tested negative.

This assay enables rapid and accurate discrimination of key blaNDM variants within a defined epidemiological context. It provides a practical alternative to sequencing-based methods for real-time surveillance and outbreak management. Its implementation may facilitate early detection of high-risk clones, particularly in regions affected by emerging variants such as NDM-14, and support targeted infection control strategies.