Molbio

• MOLECULAR BIOLOGY IN MICROBIOLOGY
• Phenotypic: Routine

  • Colony morphology
  • Enzyme/pigment production
  • Carbohydrate fermentation patterns
• Molecular


  • Genome, transcriptome, proteome
  • Bacteria, fungi, parasites (DNA genomes)
  • Viruses (DNA/RNA genomes)
  • Prions (proteome)
• Specimen collection designed to avoid contamination from environment that yield FALSE POSITIVES


  • Sterile containers
  • Swabs
  • Specialty systems
  • Proprietary systems
  • Anaerobic transport systems
  • Vital transport systems
• Lysis procedures


  • Dependent to microorganisms
  • Mycobacteria and fungi have difficult to lyse thick cell walls
  • Gram positive bacteria have thicker cell wall than gram negative bacteria
  • Mycoplasma have no cell wall
• Concentration of organisms


  • Centrifugation
• Presence of inhibitors


  • Removal and inactivation should be included
  • Removal of RNAses for RNA analysis
• Specimen


  • Blood
  • Sputum
  • Urine
  • CSF
• Blood sample


  • Removal of hemoglobin (Hgb inhibits DNA pol-False Neg)
  • WBC separation by Ficoll-Hypaque
  • Microorganisms: Serum and Plasma
• Sputum sample


  • Obtained by coughing and is examined in the laboratory
  • For respiratory tract infections (RTI)
  • Removal of acidic polysaccharides (Inibits DNA pol)
• Urine


  • Centrifugation
  • Inhibitors DNA Pol: Nitrate, crystals, hemoglobin, Beta-human chorionic gonadotropin
• Quality assurance and quality control


  • Ensures accuracy
  • Integrity of specimens are critical
  • Ensure the absence of inhibitors in the sample
• Controls

  • Positive controls
  • Reagent blank/contamination controls
  • Negative template control
  • Amplification control
• Positive controls


  • Ensures that assay system is working properly
  • Lower (sensitivity control) and Upper limit
• Reagent blank/contamination controls


  • Should always yield negative results
  • All reagents except the target
• Negative template control


  • For studies with non-target organisms
  • Identify the presence of undesired targets while avoiding any reaction with the desired target
• Amplification control


  • Rule out amplification failure
  • To confirm true negative result
  • Examples: groEL, rpoB, recA, gyrB, B-actin, interferon-y, human mitochondrial DNA
• Internal controls


  • Homologous extrinsic
  • Heterologous extrinsic
  • Heterologous intrinsic
• Homologous extrinsic


  Target derived with non-target derived sequence
• Heterologous extrinsic


  Non-target derived
• Heterologous intrinsic


  Non-target that are present in sample
• False positives


  • Carry over of samples
  • Antimicrobial therapy
• Antimicrobial therapy


  To avoid: test 3-6 weeks after therapy
• False negatives


  • Nucleic acid degradation
  • Inhibition of amplification procedures
  • Inhibitors in amplification: Hemoglobin, Lactoferrin, Heparin, Sodium polyanethol sulfonate, Polyamines
• Selection of sequence targets


  • Critical on specificity
  • Specificity is dependent on primers and probes
  • Microorganisms share similar sequences will yield cross reactivity
  • Initial tests use type specific primers/probes
  • Confirmatory tests
• Molecular detection of microorganisms


  • Agarose gel electrophoresis
  • Amplification methods
  • Sequencing
  • Immunoassays
  • Western blots
  • Mass spectrometry
• Bacteria


  • Respiratory
  • Urogenital tract
• Bordetella pertussis


  • Upper respiratory tract
  • Whooping cough
• Legionella pneumophilia


  • Lower respiratory tract
  • Legionnaire's disease
• Mycobacterium tuberculosis


  • Detection (Traditional): Smear and culture, Kinyoun and Ziehl Neelsen stains, Flurochromess
  • qPCR: Targets Rrna internal transcribe spaces (ITS)
• Mycoplasma pneumoniae


  • Multilocus variable-number tandem-repeat (VNTR) analysis
  • Multilocus sequence typing
  • Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)
• Chlamydophila pneumoniae


  • 10% of community-acquired pneumonias
  • Implicated in atherosclerosis & coronary artery disease
  • Detection: qPCR targeting cloned Pst I fragment, 16S rRNA, P1 adhesion gene
• Streptococcus pneumoniae


  • Major cause of community-acquired pneumonias
  • Common cause of bacteremia, sepsis, otitis media, meningitis
  • Detection: PCR tests targeting DNA polymerase gene, plyA, lytA, pbp2a, pbp2b, pspA
• Urogenital tract organisms


  • Neisseria gonorrhoeae: gonorrhea
  • Chlamydia trachomatis: chlamydia
• Urogenital tract detection


  • Target amplification, strand displacement amplification, & transcription-mediated amplification: urethral/cervical swabs, urine, transport vials for PAP smears
  • Culture can be performed along with molecular methods
• Treponema pallidum subsp. Pallidum


  • Agent of syphilis
  • Cannot be grown in vitro
  • Stages: PRIMARY (hard chancre), SECONDARY (disseminated rash), LATENT (asymptomatic), TERTIARY (CNS and CV manifestations)
• Laboratory diagnosis of syphilis


  • Serologic: Presence of antibodies against cariolipin (Rapid plasma regain (RPR), Veneral disease laboratory (VDRL)), Hemagglutination assays, EIAs (fluorescent treponemal antibody absorption-FTA-ABS)
  • PCR assays: Amplification of polA geen
• Mycoplasma


  • Mycoplasma hominis
  • Mycoplasma genitalium
  • Ureaplasma urealyticum
• Mycoplasma


  • Agents of non-gonococcal urethritis
  • PCR assays & laboratory-developed PCR with hybridization
• Traditional virus detection methods


  • Antibodies against the virus (indirect)
  • Presence or absence of viral antigens
  • Growth of a virus in a culture system