Molecular Methods

This section focuses on the molecular methods used for MRSA/MSSA, VRE, ESBL/CRE screening. Molecular techniques offer high sensitivity, specificity, and can provide rapid results

MRSA/MSSA Screening

• Target: mecA gene (confers methicillin resistance)
• Methods
  • Polymerase Chain Reaction (PCR)
    • Real-Time PCR (qPCR): This is the most common and preferred method. qPCR is a rapid and sensitive technique that amplifies and detects the mecA gene directly from the specimen. Results are usually available within a few hours
    • Conventional PCR: Amplifies the mecA gene, followed by detection using gel electrophoresis. This method is less rapid than qPCR
  • Isothermal Amplification
    • Loop-mediated isothermal amplification (LAMP): This is a rapid and simple method that amplifies the mecA gene at a constant temperature. It can be performed with minimal equipment and provides results in a short time
  • Molecular Hybridization
    • Nucleic acid hybridization assays: Probes specific for the mecA gene can be used to detect the presence of the gene in a sample

VRE Screening

• Target: vanA, vanB, or other van genes (confer vancomycin resistance)
• Methods
  • PCR
    • Real-Time PCR (qPCR): Detects the vanA or vanB genes directly from the specimen
    • Conventional PCR: Amplifies the vanA or vanB genes, followed by gel electrophoresis
  • Isothermal Amplification
    • LAMP: Can be used to detect the vanA or vanB genes
  • Molecular Hybridization
    • Nucleic acid hybridization assays: Probes specific for the vanA or vanB genes can be used to detect the presence of the gene in a sample

ESBL/CRE Screening

• Targets: Genes encoding ESBLs (e.g., blaCTX-M, blaTEM, blaSHV), carbapenemases (e.g., blaKPC, blaOXA-48, blaNDM, blaIMP, blaVIM)
• Methods
  • PCR
    • Real-Time PCR (qPCR): Detects the genes encoding ESBLs and carbapenemases. Multiplex PCR assays can be used to simultaneously detect multiple resistance genes
    • Conventional PCR: Amplifies the genes encoding ESBLs or carbapenemases, followed by gel electrophoresis
  • Multiplex PCR: A single PCR reaction can amplify multiple target sequences simultaneously, enabling the detection of several resistance genes in one test
  • Microarrays: Nucleic acid microarrays can be used to screen for a wide range of resistance genes in a single test
  • Next-Generation Sequencing (NGS): NGS can be used to identify all resistance genes present in a sample, providing a comprehensive picture of the resistance profile

Key Considerations for Molecular Methods

• Advantages
  • Rapid Results: Molecular methods often provide results within hours, which is significantly faster than culture-based methods
  • High Sensitivity: Can detect low levels of organisms or resistance genes
  • High Specificity: Minimize false-positive results by targeting specific genes
  • Direct Detection: Can be performed directly on clinical specimens without the need for culture
• Disadvantages
  • Cost: Molecular methods can be more expensive than culture-based methods, though costs are decreasing
  • Equipment and Expertise: Require specialized equipment and trained personnel
  • Targeted Approach: Only detect the specific resistance genes targeted by the assay. They may not detect novel resistance mechanisms
  • Potential for False Positives: Can occur if there is contamination or if the assay is not properly validated
• Quality Control
  • Positive and Negative Controls: Include positive and negative controls in each run to ensure the assay is working correctly
  • Internal Controls: Use internal controls to monitor for the presence of inhibitors in the sample
  • Validation: Validate the assay before use to determine its sensitivity, specificity, and reproducibility
• Interpretation
  • Molecular tests detect the presence of resistance genes, not necessarily whether the organism is expressing the resistance
  • Phenotypic susceptibility testing is still needed to confirm resistance and guide antibiotic therapy

Key Terms

• PCR (Polymerase Chain Reaction): A molecular technique used to amplify a specific DNA sequence
• qPCR (Quantitative PCR or Real-Time PCR): A PCR method that measures the amount of amplified DNA in real-time, allowing for quantification of the target sequence
• LAMP (Loop-mediated isothermal amplification): An isothermal nucleic acid amplification technique that amplifies DNA with high specificity and efficiency under isothermal conditions
• Multiplex PCR: A PCR reaction that amplifies multiple target sequences simultaneously
• Microarray: A collection of microscopic DNA spots arranged on a solid surface, used to detect the presence of specific DNA sequences
• NGS (Next-Generation Sequencing): High-throughput DNA sequencing technologies that allow for the rapid sequencing of large amounts of DNA
• Gene: A unit of heredity that is transferred from a parent to offspring and is held to determine some characteristic of the offspring
• Primer: A short, single-stranded DNA sequence that is used to initiate DNA synthesis in PCR
• Probe: A short, single-stranded DNA or RNA sequence that is used to detect the presence of a specific DNA or RNA sequence
• Sensitivity: The ability of a test to correctly identify positive results (i.e., the proportion of infected individuals who are correctly identified as infected)
• Specificity: The ability of a test to correctly identify negative results (i.e., the proportion of non-infected individuals who are correctly identified as not infected)