Laboratory Techniques in Diagnostic Microbiology

Diagnosis of Infections

  • The process involves moving from specimen collection to obtaining results and finally to effective treatment.
  • Diagnosis relies on history, clinical examination, and laboratory investigations.
  • Laboratory investigations include microbiological and virological tests, as well as hematological and biochemical analyses.
  • Additional investigations like X-rays and scans may be necessary.

Importance of History

  • A detailed patient history is crucial because the lab focuses on likely pathogens based on this information.
  • Key elements include symptoms, duration (acute vs. chronic), travel history (risk of tropical diseases, vaccinations), contact with animals (zoonotic infections), contact with infected individuals (tuberculosis, meningitis), and exposure to specific foods or drugs (including antibiotic treatment).

Good Quality Samples

  • Collecting specimens at the right time is vital i.e., before commencing antibiotics, acute and convalescent sample for serology.
  • The right type of sample should be collected.
  • Specimens must be collected carefully to minimize contamination.
  • Samples should reach the lab promptly.
  • Clinical data provided with the sample must be relevant.

Well Collected Specimens

  • Poor quality specimens can lead to misleading results; for example, saliva instead of sputum, contaminated mid-stream urine, or contaminated blood cultures.
  • The quality of specimens directly influences the usefulness of lab procedures, emphasizing the principle of "rubbish in = rubbish out".

Optimal Timing for Specimen Collection

  • Culture specimens for bacteria/viruses should ideally be taken before starting antimicrobial treatment.
  • For serology, both acute and convalescent samples are needed to show a rise in antibody titer.
  • Some pathogens don't survive long at room temperature, while normal flora may proliferate, leading to inaccurate results if processing is delayed.

Role of the Laboratory

  • Ensuring samples are processed quickly, accurately, and cost-effectively.
  • Daily communication between lab staff and clinicians.
  • Providing advice on appropriate sample types and antimicrobial therapy.
  • Infection control measures.
  • Creating antimicrobial and infection control guidelines.

Classification and Identification of Bacteria

  • Culture is usually the gold standard.
  • Initial identification involves microscopy and growth characteristics.
  • Further identification uses biochemical tests, antigen detection, and toxin demonstration.
  • Indirect detection methods include immunofluorescence, nucleic acid amplification (PCR), and serological diagnosis (four-fold rise in specific IgM).

Bacterial Growth Requirements

  • Bacteria require carbon (organic or inorganic), nitrogen (organic or inorganic), an energy source (oxidation of organic/inorganic compounds, or sunlight), and a hydrogen source.
  • Most bacteria grow at 37^{\circ}C in air; some need added CO_2, and some are anaerobic.

Microscopy of Bacterial Colonies

  • Gram stain: differentiates bacteria based on cell wall structure (Gram-positive = purple, Gram-negative = pink).
  • Morphology: identifies rods/cocci, pairs/clusters/chains.
  • Special stains: Ziehl-Neelsen for Mycobacteria, cotton blue for fungi, darkfield microscopy for spirochetes.

Bacterial Cell Walls

  • Gram-positive: Thick peptidoglycan layer, teichoic acids, and lipoteichoic acids (LTA).
  • Gram-negative: Thin peptidoglycan layer, outer membrane containing lipopolysaccharide (LPS) and porins, and periplasmic space.

Gram Stain Procedure

  • Application of crystal violet (primary stain).
  • Iodine (mordant) is added.
  • Alcohol wash (decolorization step).
  • Safranin (counterstain) is applied.

Basic Classification for Clinical Microbiologists

  • Gram Positive Cocci
  • Gram Positive Bacilli
  • Gram Negative Cocci
  • Gram Negative Bacilli
  • Anaerobes
  • Spirochetes
  • Mycobacteria

Gram-Positive Cocci – Clustering (Staphylococci)

  • S. aureus: causes severe soft tissue infections, bacteremia, endocarditis, necrotizing pneumonia, and nosocomial outbreaks.
  • S. epidermidis: normal skin commensal, opportunistic pathogen causing infections in immunocompromised patients and prosthetic device infections.

Virulence Factors of S. aureus

  • Enzymes: coagulase, catalase, hyaluronidase, fibrinolysin, lipase, nuclease, penicillinase.
  • Other: slime production, capsule, cell wall components.
  • Toxins: cytotoxins, exfoliative toxin, toxic shock syndrome toxin, enterotoxins.

Laboratory Detection of S. aureus

  • Catalase-positive.
  • Coagulase and DNAase positive.

Gram-Positive Cocci – Chains (Streptococci)

  • α-hemolytic: S. oralis, S. salivaris, S. mitis (normal oral commensals causing subacute endocarditis), S. pneumoniae (severe pneumonia, bacteremia, and meningitis).
  • β-hemolytic: Groups A, B, C, D, and G streptococci - S. pyogenes (Group A, severe SSTI), S. agalactiae (Group B, neonatal sepsis), S. bovis / Enterococci (Group D, UTI, biliary sepsis).

Streptococci and Hemolysis

  • β-hemolysis: complete red cell lysis (e.g., S. pyogenes, S. agalactiae).
  • α-hemolysis: partial lysis (e.g., S. pneumoniae, S. mutans).
  • non-hemolytic streptococcus: No lysis.

Lancefield Antigens

  • β-hemolytic streptococci are divided into groups A to G based on Lancefield antigen detection on their surface.

    • S. pyogenes: Lancefield Group A
    • S. agalactiae: Lancefield Group B

Gram-Positive Bacilli

  • Clostridium (C. tetani, C. perfringens, C. botulinum, C. difficile).
  • Bacillus (B. anthracus, B. cereus).
  • Corynebacterium (C. diphtheriae).
  • Listeria (L. monocytogenes).

Clostridial Diseases

  • Anaerobic, spore-forming organisms.
  • Toxin-mediated diseases.
    • C. perfringens: gas gangrene, clostridial myonecrosis, food poisoning.
    • C. tetani: tetanus (paralysis).
    • C. botulinum: wound/systemic botulism.
    • C. difficile: antibiotic-associated diarrhea.

Gram-Negative Cocci (Moraxellaceae)

  • Respiratory pathogens: Haemophilus influenzae, Moraxella catarrhalis, Neisseria meningitidis.
  • Genitourinary pathogens: Neisseria gonorrhoeae.
  • Opportunistic pathogens (hospitals): Acinetobacter baumannii.
  • Most have fastidious growth requirements, needing specialized media with lysed blood and factors X (Haemin) and V (NAD).
  • Rapid tests exist for meningococcal disease.

Gram-Negative Rods (Enterobacterales)

  • Examples: E. coli, Klebsiella, Enterobacter, Serratia, Citrobacter, Morganella
  • Can cause asymptomatic colonization, wound infections, UTIs, intra-abdominal infections, nosocomial pneumonia, bacteremia/septicemia, and neurosurgical meningitis.

Enterobacterales - Obligate vs. Opportunistic Pathogens

  • Obligate pathogens: Salmonella spp. (S. typhi, S. paratyphi), Shigella spp. (S. dysenteriae, S. flexneri, S. sonnei, S. boydii).
  • Opportunistic pathogens: Escherichia coli, Klebsiella pneumoniae, Enterobacter spp. (E. aerogenes, E. cloacae), Yersinia spp. (Y. enterocolitica, Y. pseudotuberculosis).

Gram Negative Bacteria - Metabolism

  • ATP generation from organic compounds.
  • Respiration: breakdown of diverse substrates, generating large amounts of ATP using O_2 (aerobic) or nitrate (anaerobic) as electron acceptors.
  • Fermentation: fewer substrates, more intermediates, smaller ATP yield without O_2.

Gram-Negative Bacilli – Enterobacterales vs. Non-Fermenters

  • Enterobacterales ('coliforms'): aerobically ferment sugars.
  • Non-fermenters: Pseudomonas aeruginosa, Stenotrophomonas, Burkholderia; environmental/opportunistic pathogens with oxidative metabolism.

Cytochrome Oxidase

  • Oxidative bacteria possess cytochrome oxidase, activating cytochrome c in the electron transport chain and reducing O2 to H2O.
  • Oxidase positive: Pseudomonas, Neisseria, Campylobacter.
  • Oxidase negative: Enterobacterales, most Gram positives, anaerobes.

Fermentation of Carbohydrates

  • Bacteria differ in ability to ferment monosaccharides, polysaccharides, and alcohols.
  • Monosaccharides: arabinose, ribose, fructose, galactose, mannose.
  • Disaccharides: sucrose (glucose + fructose), maltose (glucose + glucose), lactose (glucose + galactose).
  • Useful for species-level identification but may need up to 20 reactions.

Other Metabolic Tests

  • Many tests based on carbohydrate metabolism, utilization of inorganic/organic carbon sources, and amino acid metabolism.
  • Detection of key intermediates in biochemical pathways.

Methyl Red and Voges-Proskauer Tests

  • Methyl Red (MR) test: detects mixed acid fermentation (pH < 4.4 is positive).
  • Voges-Proskauer (VP) test: detects butylene glycol pathway and acetoin production.
  • Examples of organisms:
    • Klebsiella sp., Enterobacter sp., and Serratia sp. are VP positive.
    • E. coli, Shigella sp., and Salmonella sp. are MR positive.

Multitest Identification Systems

  • e.g., API 20E, API 20NE: combine dozens of organic/inorganic substrates.
  • Color changes indicate utilization or breakdown.
  • Widely used for primary identification.

Automated Systems

  • 30-45 wells with substrates or antimicrobials.
  • Fully automated; can process up to 120 cards, with identification in 4-8 hours.
  • Initially developed for identifying organisms in space.

Mycobacteria Characteristics

  • 'Waxy' cell wall (lipid-rich) leads to:
    • Resistance to drying and hydrophobic.
    • Resistance to antibiotics and disinfectants.
    • Resistance to acids and alkalis.
  • 'Acid and alcohol fast': impermeable to standard stains; survives in macrophages.

Mycobacterial Cell Wall

  • Complex structure containing mycolic acids, arabinogalactan, peptidoglycan, and lipids.
  • Does not take up Gram stain; detected by Ziehl-Neelsen (ZN) stain, which stains bacteria bright red.

Fungi

  • Eukaryotic organisms with plant-like characteristics, but unable to perform photosynthesis.
  • Cell wall composed mainly of chitin.
  • More than 100,000 species, less than 200 are pathogenic.
  • Can be unicellular (yeasts) or multicellular (molds).

Yeast Form

  • Candida albicans grows as single cells, dividing by budding.
  • May cause vaginal or skin infections in immunocompromised individuals or those on antibacterial therapy.

Mold Form

  • Grows as filaments (hyphae) forming a mesh (mycelium).
  • Reproduces by forming spores, which can cause infections.
  • Aspergillosis infects immunocompromised people; hyphae may impede lung function, causing damage and severe pneumonia.

Serological / Immunological Diagnosis

  • Look for at least a four-fold rise in antibody titer in acute infection or presence of IgM.
  • Especially useful in diagnosing viral infections or when growing presumptive bacteria is difficult.

Diagnostic Micro History

  • Robert Koch needed to isolate bacterial pathogens.
  • Fannie Eilshemius Hesse suggested agar use (1881).
  • Richard Petri developed the Petri dish (1887).
  • Gram stain introduced in 1882
  • MacConkey plate developed in 1905

Mass Spectrometry in Bacterial Diagnostics

  • Early ID studies focused on metabolites, enzymes, and toxins.
  • Commercial systems emerged between 1996-2006.
  • Current applications involve direct ID of bacteria and virulence factor analysis (2006-present).

MALDI-TOF MS

  • MALDI: Matrix Assisted Laser Desorption Ionization.

  • TOF: Time-of-Flight.

  • Allows bacterial identification in minutes!

  • Relies on organisms being present in database.

MALDI-TOF MS Protocol

  • Inoculum preparation.
  • Centrifugation Resuspension.

Phenotypic tests – Point of Care

  • Acidometric Tests Carba NP / Blue.
  • Immuno-Chromatographic Lateral flow.
  • Chromogenic ESBL-ve ESBL +ve.
  • Rule in or Rule out at the point of Decision.

Molecular Techniques

  • Molecular techniques have been widely used in virology departments for diagnosis of infections for many years (viruses are often difficult to culture).
  • Many microbiology departments are now using molecular techniques to diagnose certain infections (e.g using this on “infected” tissue which is culture negative to try and find the infecting organism).

Molecular Tests – PCR

  • Increasingly multiplexed.
  • Gel Based – Individual target.
  • Multiplex PCR available for simultaneous detection of multiple targets.

Real-Time PCR

  • Evalution of a new ral-time PCR assay (Check-Direct CPE) for rapid detection of KPC, OXA-48, VIM and NDM carbapenamases using spiked rectal swabs

Cepheid GeneXpert

  • Sputum liquefaction and inactivation with 2:1 sample reagent

PNA FISH

  • 90 min. identification and differentiation of E. coli, K. pneumoniae and P. aeruginosa from GNR-positive blood cultures.
  • E. coli (35%), K. pneumoniae (20%), P. aeruginosa (15%).

Microarrays

  • Array based platformns
  • Indentibac 72 resistance genes.

Check MDR-ESBL / Carba Arrays

  • Check Points Database Viewer.

PCR-ESI Mass Spectrometry

  • Converting Masses to Base Composition
  • MS Analysis and Signal Processing
  • Search database and generate report

PLEX-ID Assay Menu

  • Biodefense and Routine Monitoring
  • Food Safety

Whole Genome Sequencing

  • Rapidly decreasing in cost < £50 / genome

Center for Genomic Epidemiology

  • Identification of acquired antibiotic resistance genes.
  • Prediction of a bacteria's pathogenicity towards human hosts.
  • Prediction of bacterial species using the S16 ribosomal DNA sequence

Sequencing of Bacterial Genomes

  • Sequencing of Bacterial Genomes is Now Cheap and Easy!
  • What’s the organism?
  • How can we treat it?
  • Have we seen it before?

NHS UK Standards for Microbiology Investigations

  • Investigation of urine

The End of Culture?

  • URINES