Molecular Methods

Molecular methods have transformed the diagnosis of lower respiratory tract infections, offering significant advantages over traditional culture techniques. These methods are particularly valuable for rapid identification, detecting non-culturable pathogens, and assessing antibiotic resistance

General Principles

• Targeted Approach: Molecular methods detect pathogens by identifying their unique genetic material (DNA or RNA)
• Key Advantages
  • High Sensitivity and Specificity: PCR, etc. can detect even small amounts of pathogens
  • Rapid Turnaround Time: Results are often available within hours
  • Detection of Non-Culturable Organisms: Pathogens that are difficult or impossible to culture
  • Multiplexing: Detects multiple pathogens
  • Detection of Resistance Genes: Can identify specific resistance genes
• Applications
  • Identifying bacteria, viruses, and fungi
  • Detecting antimicrobial resistance
  • Quantifying the microbial load
• Limitations
  • Cost: Can be expensive
  • Technical Expertise and Specialized Equipment: Requires specialized equipment and training
  • Can Detect Non-Viable Organisms: May detect DNA from non-viable organisms
  • Not all resistance mechanisms are identified

Molecular Methods

Polymerase Chain Reaction (PCR)

• Principle: Amplifies a specific DNA target sequence
• Steps
  • DNA Extraction
  • Specific Primers
  • PCR Amplification
  • Detection
• Applications
  • Detecting bacteria, viruses, and fungi
  • Detecting resistance genes
• Variations
  • Multiplex PCR: Many targets in a single test
  • Real-Time PCR: Quantitative results
• Advantages
  • Rapid turnaround time
  • Can detect non-culturable organisms
• Disadvantages
  • Cost
  • Technical expertise is required

Reverse Transcription PCR (RT-PCR)

• Detects RNA Viruses: This is performed by detecting RNA
• Mechanism
  • RNA to DNA: Reverse transcription
  • PCR Amplification: Amplified DNA is detected
• Applications
  • Detection of viral infections
• Advantages
  • High sensitivity and specificity
  • Can detect non-culturable organisms
  • Multiplexing capabilities
• Disadvantages
  • Cost
  • Specialized equipment and training
  • Cannot always provide antibiotic susceptibility information

Next-Generation Sequencing (NGS)

• Comprehensive Method: Used to identify pathogens when conventional methods are non-diagnostic
• Mechanism
  • Extraction
  • Library Preparation
  • Sequencing and Analysis
• Applications
  • Unbiased identification
  • Novel or unusual pathogens
• Advantages
  • Comprehensive analysis
  • New or rare pathogens
• Disadvantages
  • High cost
  • Complex data analysis

Targeted Pathogens in Lower Respiratory Infections

• Bacteria
  • Streptococcus pneumoniae
  • Haemophilus influenzae
  • Moraxella catarrhalis
  • Legionella pneumophila
  • Mycoplasma pneumoniae
  • Chlamydophila pneumoniae
  • Staphylococcus aureus
  • Pseudomonas aeruginosa
  • Acinetobacter spp.
  • Klebsiella pneumoniae
  • Burkholderia cepacia complex
• Viruses
  • Influenza A and B viruses
  • Respiratory syncytial virus (RSV)
  • Adenovirus
  • Rhinovirus
  • Human metapneumovirus
  • SARS-CoV-2
• Fungi
  • Pneumocystis jirovecii (formerly Pneumocystis carinii)
• Other agents

Applications by Respiratory Specimen

• Sputum: Many molecular methods can be used
• Endotracheal Aspirate (ETA): Many molecular methods can be used
• Bronchoalveolar Lavage (BAL): Many molecular methods can be used
• Bronchial Wash (BW): Many molecular methods can be used

Molecular Tests Performed

• PCR
  • Pneumonia Panels
  • Viral Panels
  • Identification of resistant genes
• RT-PCR
  • Viral Respiratory Panels
• Other
  • Rapid tests

Role in Diagnosis and Management

• Rapid Diagnosis: Rapid and accurate diagnosis
• Improved Outcomes: Early, targeted therapy
• Identification of Non-Culturable Organisms
• Multiplex Testing
• Monitoring Treatment Response: Quantitative PCR

Key Terms

• Molecular Method: A method that detects the presence of specific DNA or RNA sequences
• Polymerase Chain Reaction (PCR): A molecular method to amplify specific DNA sequences
• Reverse Transcription PCR (RT-PCR): A PCR method that detects RNA
• Primer: A short DNA sequence that binds to a specific region of DNA
• Multiplex PCR: A PCR method that detects multiple targets simultaneously
• Real-Time PCR (qPCR): A PCR method that quantifies DNA during amplification
• Next-Generation Sequencing (NGS): A high-throughput sequencing method
• Nucleic Acid Extraction: The process of isolating DNA or RNA from a sample
• Sensitivity: The ability of a test to correctly identify a pathogen
• Specificity: The ability of a test to correctly identify the absence of a pathogen
• False Positive: A test result that indicates the presence of a pathogen when it is not actually present
• False Negative: A test result that indicates the absence of a pathogen when it is actually present
• Viral Load: The amount of virus present in a sample
• Antimicrobial Therapy: The use of antibiotics or antifungals to treat infections
• Amplification: The process of making multiple copies of a DNA or RNA sequence
• Target: The specific DNA or RNA sequence that is amplified by PCR
• Multidrug-Resistant Organisms (MDROs): Microorganisms resistant to multiple antibiotics
• Sputum: Secretions from the lower respiratory tract
• Endotracheal Aspirate (ETA): Secretions collected through an endotracheal tube
• Bronchoalveolar Lavage (BAL): Fluid obtained from the lungs via bronchoscope
• Bronchial Wash (BW): Fluid collected from the bronchi via bronchoscope
• Pneumonia: Lung infection
• Pneumocystis jirovecii: Opportunistic fungus
• Rapid Test: Any test that has a rapid result
• Influenza A and B: Common viruses that cause the flu
• RSV: Respiratory syncytial virus
• SARS-CoV-2: Virus that causes Covid-19
• Human metapneumovirus: Virus that causes respiratory illness
• Chlamydophila pneumoniae: Bacteria that causes respiratory illness
• Mycoplasma pneumoniae: Bacteria that causes respiratory illness