Clinical Microbiology

Diagnosis of infectious diseases

1. Clinical presentation: examination, history
   1. symptoms & signs
   2. Medical history
   3. Demographic information, occupation\
   4. Travel, animal contact
2. Differential diagnosis
   • Anatomy
   • Etiology
3. Clinical laboratory testing

Isolation/culture of pathogens

1. Specimen collection

   1. Sterile swabs

   2. Needle aspiration

   3. Intubation

   4. Catheters

2. Specimen handling & transport

   1. Labeling

   2. Preservation

   3. Storage & shipping

3. Culturing the pathogen

   aerobic vs. anaerobic media

   • Thioglycollate medium for Clostridium

   selective and differential media: many types

   1. Blood agar: differential for Streptococcus, Staphylococcus

   2. Eosin Methylene Blue agar: s/d for enteric bacteria

   3. MacConkey agar (s/d for enterobacteria)

   4. Thayer Martin agar: s/d for Neisseria, Francisella

   5. Mannitol Salt agar: s/d for Staphylococcus

   6. Lowenstein-Jensen medium for Mycobacterium

   7. Salmonella-Shigella (XLD) agar - selective/differential

   8. CHROMagar- differential for Candida spp., other microbes

   9. Sabouraud dextrose agar: fungi

   enriched media for fastidious microbes

   • Blood agar: Streptococcus, Bordetella

   • Chocolate agar: Neisseria, Haemophilus

   cultivation in host cells

   • Rickettsia, Chlamydia: cell cultures

   • Viruses: cell cultures, chicken eggs

   unculturable or impractical-to-culture pathogens

   • Rickettsia: too dangerous

   • Treponema, Borrelia, Chlamydia, Mycoplasma - difficult to culture

   • Mycobacterium, fungi - grow too slowly

Identification of pathogens

1. Direct Microscopic examination

   1. Gram stain: Shape, arrangement, endospores

      • Streptococcus

      • Clostridium botulinum

      • Bacillus anthracis

      • coliforms

   2. Mycobacteria: Acid-fast (Ziehl-Neelson)

   3. Protozoa: Trichrome, Toluidine blue, iron hematoxylin

      • stained specimen with Giardia

      • Trypanosomes in blood: Wright-Giemsa stain

      • Plasmodium in RBCs

      • Cryptosporidium: Modified acid-fast stain

   4. Fungi: 

      • Rhizopus in a nasal swab

      • Aspergillosis

      • Cryptococcus: India ink (negative stain)

   5. Viruses

      • Cytology: virus-induced cytopathic effect

        • Syncytia: HSV, HIV, VZV

        • Inclusions: Rabies, CMV, HSV

      • Electron microscopy

   6. Special techniques

      • Darkfield microscope: Treponema, Leptospira

       

2. Fluorescence microscopy

   1. Protozoa: Plasmodium in red blood cells, Cryptosporidium, Giardia

   2. Viruses: Cytomegalovirus; Herpes; Hepatitis C

   3. Bacteria: S. pyogenes, Bordetella, francisella, Legionella, Chlamydia

3. Growth and biochemical characteristics (e.g. API-20 tests)

   • Fermentation: enterobacteria

   • Catalase: Streptococcus vs. Staphylococcus

   • Coagulase: S. aureus 

   • Gelatinase: Clostridium, Serratia, Pseudomonas

   • Hydrogen sulfide production: Salmonella

   • Indole/methyl red/VP: Klebsiella

   • Lipase: Clostridium

   • Oxidase: Neisseria

   • Amylase: Bacillus

4. Immunological tests

   1. Immunoprecipitation

      • Radial immunoprecipitation: determine antigen (or antibody) concentration

      • Ouchterlony technique: determine antigen relatedness

      • Immunoelectrophoresis

   2. Direct Fluorescent Antigen test: 

      • syphilis

      • Legionella

   3. Immunohistology: Rickettsia

   4. Rapid detection kits

      • E. coli O157:H7 (vs Surface antigens)

      • HIV (detects HIV antibodies)

      • Influenza

      • COVID

      • Toxins (e.g. C. diff)

   5. Liposomes: rapid antibody tests

      • RSV

      • Influenza

   6. ELISA: soluble antibody or antigen

      • fungi, bacteria, viruses, toxins

   7. Radioimmunoassay (RIA): quantitative

   8. Agglutination tests: antigen or antibody-coated latex beads

      1. Staphaurex

5. Bacteriophage typing: mainly for reference lab

6. Molecular methods: 

   1. DNA hybridization: tuberculosis, E. coli, Chlamydia, Mycoplasma

      • In-situ hybridization (eg CMV)

      • Dot blot

      • Southern blot (DNA), Northern blot (RNA)

   2. Polymerase chain reaction

      • RT-PCR: RNA viruses (1993 Hantavirus outbreak)

      • Real-time PCR: quantitation (HIV viral plasma load)

      • Branched chain DNA assay

      • TAM, SDA, LAMP: different amplification methods

   3. Restriction fragment length polymorphism (RFLP)

      • Strain differentiation (HSV 1 vs 2)

   4. Plasmid fingerprinting

   5. Microarray technology

   6. SDS-PAGE: protein separation

      • Western blots

   7. MALDI-TOF: protein characterization for species ID

Key Points

1. Bacterial infections: Culture, microscopy, growth
2. Viral infections: cytopathology, immunoserology, genetic tests
3. Fungal & parasitic infections: microscopy

References & Links

1. http://www.cdc.gov/ncidod/eid/
2. http://medic.med.uth.tmc.edu/path/00001450.htm