Coupling machine learning and high throughput multiplex digital PCR enables accurate detection of carbapenem-resistant genes in clinical isolates

Background: The emergence and spread of carbapenemase-producing organisms (CPO) are a significant clinical and public health concern. Rapid and accurate identification of patients colonised with CPO is essential to adopt prompt prevention measures in order to reduce the risk of transmission. Recent proof-of-concept studies have demonstrated the ability to combine machine learning (ML) algorithms with real-time digital PCR (dPCR) instruments to increase classification accuracy of multiplex assays. From this, we sought to determine if this ML based methodology could accurately identify five major carbapenem-resistant genes in clinical CPO-isolates. Methods: We collected 253 clinical isolates (including 221 CPO-positive samples) and developed a novel 5-plex assay for detection of blaVIM, blaOXA-48, blaNDM, blaIMP and blaKPC. Combining the recently reported ML method "Amplification and Melting Curve Analysis" (AMCA) with the abovementioned multiplex assay, we assessed the performance of the methodology in detecting these five carbapenem-resistant genes. The classification accuracy relies on the usage of real-time data from a single fluorescent channel and benefits from the kinetic and thermodynamic information encoded in the thousands of amplification events produced by high throughput dPCR. Results: The 5-plex showed a lower limit of detection of 100 DNA copies per reaction for each primer set and no cross-reactivity with other carbapenemase genes. The AMCA classifier demonstrated excellent predictive performance with 99.6% (CI 97.8-99.9%) accuracy (only one misclassified sample out of the 253, with a total of 163,966 positive amplification events), which represents a 7.9% increase compared to the conventional ML-based melting curve analysis (MCA) method. Conclusion: This work demonstrates the utility of the AMCA method to increase the throughput and performance of state-of-the-art molecular diagnostic platforms, reducing costs without any changes to instrument hardware. Our findings suggest that, pending additional validation directly from clinical samples, advanced data-driven multiplex dPCR could potentially be integrated in routine clinical diagnostic workflows.

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Coupling Machine Learning and High Throughput Multiplex Digital PCR Enables Accurate Detection of Carbapenem-Resistant Genes in Clinical Isolates

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.775299 ◽

2021 ◽

Vol 8 ◽

Author(s):

Luca Miglietta ◽

Ahmad Moniri ◽

Ivana Pennisi ◽

Kenny Malpartida-Cardenas ◽

Hala Abbas ◽

...

Keyword(s):

Machine Learning ◽

Real Time ◽

High Throughput ◽

Classification Accuracy ◽

Melting Curve ◽

Digital Pcr ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Carbapenem Resistant ◽

Resistant Genes

Rapid and accurate identification of patients colonised with carbapenemase-producing organisms (CPOs) is essential to adopt prompt prevention measures to reduce the risk of transmission. Recent studies have demonstrated the ability to combine machine learning (ML) algorithms with real-time digital PCR (dPCR) instruments to increase classification accuracy of multiplex PCR assays when using synthetic DNA templates. We sought to determine if this novel methodology could be applied to improve identification of the five major carbapenem-resistant genes in clinical CPO-isolates, which would represent a leap forward in the use of PCR-based data-driven diagnostics for clinical applications. We collected 253 clinical isolates (including 221 CPO-positive samples) and developed a novel 5-plex PCR assay for detection of blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM. Combining the recently reported ML method “Amplification and Melting Curve Analysis” (AMCA) with the abovementioned multiplex assay, we assessed the performance of the AMCA methodology in detecting these genes. The improved classification accuracy of AMCA relies on the usage of real-time data from a single-fluorescent channel and benefits from the kinetic/thermodynamic information encoded in the thousands of amplification events produced by high throughput real-time dPCR. The 5-plex showed a lower limit of detection of 10 DNA copies per reaction for each primer set and no cross-reactivity with other carbapenemase genes. The AMCA classifier demonstrated excellent predictive performance with 99.6% (CI 97.8–99.9%) accuracy (only one misclassified sample out of the 253, with a total of 160,041 positive amplification events), which represents a 7.9% increase (p-value <0.05) compared to conventional melting curve analysis. This work demonstrates the use of the AMCA method to increase the throughput and performance of state-of-the-art molecular diagnostic platforms, without hardware modifications and additional costs, thus potentially providing substantial clinical utility on screening patients for CPO carriage.

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