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

ABSTRACT: 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 bla_IMP, bla_KPC, bla_NDM, bla_OXA-48, and bla_VIM. 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.

SUBMITTER: Miglietta L

PROVIDER: S-EPMC8650054 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and objectivesAcinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-detecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.Materials and methodsLAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla SHV-1 , bla PER-1 , bla TEM-1, AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic identification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilutions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.ResultsAmong one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL producers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla SHV-1 positive, (40%) bla PER-1, (85%) aac (6')-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla SHV-1 positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.ConclusionWherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detection rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. baumannii in these clinical isolates showed frequent commonality of genotypes.

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

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