Evaluation of a nucleic acid amplification system, GENECUBE, for rapid detection of staphylococcal nuc and mecA in blood culture samples

J Microbiol Methods. 2024 Dec:227:107063. doi: 10.1016/j.mimet.2024.107063. Epub 2024 Oct 22.

Abstract

Objective: This study determined whether the GENECUBE rapid nucleic acid amplification test could directly detect nuc and mecA genes in clinical blood culture samples of Staphylococcus and various other pathogens.

Methods: Between September 2020 and December 2021, 537 blood culture samples from 192 patients with suspected bacteremia were tested using conventional assays (MicroScan WalkAway96 or VITEK 2 systems) and GENECUBE nuc and mecA assays. Isolates from samples with discrepant results between the conventional and GENECUBE assays were further evaluated using MALDI-TOF mass spectrometry, disk diffusion testing using cefoxitin, broth microdilution testing using oxacillin, and sequencing for mecA. Bacterial solutions containing a mixture of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) were prepared to evaluate the limit of detection (LOD) of mecA.

Results: Using conventional assays as the reference, the sensitivity, specificity, and positive and negative predictive values (95 % confidence interval) of GENECUBE were 100 % (96.8-100 %), 100 % (99.1-100 %), 100 % (96.8-100 %), and 100 % (99.1-100 %), respectively, for nuc detection and 100 % (96.1-100 %), 98.9 % (97.4-99.6 %), 94.9 % (88.5-98.3 %), and 100 % (99.2-100 %), respectively, for mecA detection. Sequencing analysis of five samples identified as methicillin-sensitive staphylococci using conventional assays and methicillin-resistant staphylococci using GENECUBE revealed the presence of methicillin-resistant isolates in all samples. The estimated LOD of mecA was 104 colony-forming units (CFU)/mL of MRSE with GENECUBE, compared with 105 CFU/mL with conventional assays.

Conclusion: The GENECUBE assay accurately detected mecA in positive blood culture samples and had higher sensitivity than conventional assays.

Keywords: Blood culture; Methicillin-resistant Staphylococcus aureus; Nucleic acid amplification test; mecA; nuc.

Publication types

  • Evaluation Study

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Bacteremia* / diagnosis
  • Bacteremia* / microbiology
  • Bacterial Proteins* / genetics
  • Blood Culture* / methods
  • Humans
  • Limit of Detection
  • Methicillin-Resistant Staphylococcus aureus / drug effects
  • Methicillin-Resistant Staphylococcus aureus / genetics
  • Methicillin-Resistant Staphylococcus aureus / isolation & purification
  • Microbial Sensitivity Tests
  • Micrococcal Nuclease* / genetics
  • Nucleic Acid Amplification Techniques* / methods
  • Penicillin-Binding Proteins* / genetics
  • Sensitivity and Specificity*
  • Staphylococcal Infections* / diagnosis
  • Staphylococcal Infections* / microbiology

Substances

  • Penicillin-Binding Proteins
  • Bacterial Proteins
  • nuc protein, staphylococcus
  • Micrococcal Nuclease
  • mecA protein, Staphylococcus aureus
  • MecA protein, Staphylococcus epidermidis
  • Anti-Bacterial Agents