Diagnostic Methods Infectious Diseases

  1. Serology
    Science characterizing & measuring antibodies, antigens & other immunological substances in body fluids (primarily serum)
  2. 2 Uses of Serology in Infectious Disease
    • Diagnose a current or acute infection
    • Determine disease susceptibility or immunity
  3. Why choose serology?
    • Time: culture may take days to weeks
    • Specimen: difficult or painful to obtain
    • Availability: culture impossible
    • Sensitivity/Specificity: in some instances, lower with culture
    • Serological assays are particularly useful in the diagnosis of viruses, because viruses are small (Gram stain not applicable), & must use cell culture techniques, rather than traditional culture media
  4. Viruses: Serologic Diagnosis
    • Epstein Barr Virus
    • Hepatitis A, B, C, D
    • Measles
    • Rubella
    • Mumps
    • Arbovirus
    • Parvovirus B19
    • Respiratory viruses
    • HIV
    • Hantavirus
  5. Antigens
    • Specific proteins found on the surfaces of bacteria, viruses, fungi, & parasites
    • Provoke an immune response
    • “THEM”
  6. Antibodies
    • Also called immunoglobulins
    • Nomenclature: Ig
    • Gamma globulin part of serum proteins
    • Secreted by clones of primed B cells
    • “us”
  7. Inactivation Methods
    Antibodies inactivate antigens by 4 main methods: Complement Fixation, Neutralization, Agglutination, Precipitation
  8. Complement Fixation
    Proteins attach to antigen surface & cause holes to form, which results in cell lysis
  9. Neutralization
    Binding to specific sites to prevent attachment
  10. Agglutination
  11. Precipitation
    Forcing insolubility & settling out of solution
  12. 5 Antibody Classes
    • IgA: Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents colonization by pathogens. Also found in saliva, tears, and breast milk.
    • IgD: Functions mainly as an antigen receptor on B cells that have not been exposed to antigens. It has been shown to activate basophils and mast cells to produce antimicrobial factors.
    • IgE: Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms.
    • IgG: In its four forms, provides the majority of antibody-based immunity against invading pathogens. The only antibody capable of crossing the placenta to give passive immunity to fetus.
    • IgM: Expressed on the surface of B cells and in a secreted form with very high avidity. Eliminates pathogens in the early stages of B cell mediated (humoral) immunity before there is sufficient IgG.
    • 3 are formed in response to infection: IgM, IgG, IgA
  13. IgM After Infection
    • Appears in serum in 1-2 weeks
    • Persists for 2-3 months
    • Consistent with current or recent infection
    • Largest size
    • Does not cross the placenta
  14. IgG After Infection
    • Appears 2-3 weeks after infection
    • May persist for life
    • May represent somewhat recent infection or immunity
    • Most common circulating antibody (by far)
    • Only antibody that crosses the placenta
  15. IgA After Infection
    • Antibodies formed in response to infections of cells with secretory activity (cells lining the respiratory, GI and GU tracts)
    • Secretory IgA is released at the infection site, then goes to bloodstream
    • Not frequently measured
  16. Antibody Titers
    • Amount of antibody at a particular dilution of patient serum
    • Results expressed as titer: 1:2, 1:4, 1:8, 1:16, 1:32, etc.
    • Higher dilution is consistent with higher level of antibody in patient serum
  17. Acute & Convalescent Antibody Titers
    • Used to assess IgG levels at 2 phases
    • Acute phase: first serum sample collected after exposure or symptom onset, may be asymptomatic
    • Convalescent phase: serum sample collected 2-4 weeks later
    • Simultaneous assay of both sera is most meaningful
  18. Interpreting IgG Results in Diagnosing Acute Infection
    • Compare acute & convalescent results
    • 4-fold rise in titer of paired sera collected 2-4 weeks apart verifies recent infection & is considered diagnostically significant
  19. Interpreting Antibody Results
    • For some infections, a single serum result above a certain value may be significant & diagnostic of infection
    • Some require additional testing to confirm a diagnosis
  20. Serologic Diagnosis of Infections
    • Advantages: Specimen (usually serum) is easy to obtain, Serologic tests are widely available, Ease of specimen transport, Serologic Diagnosis of Infections
    • Disadvantages: IgG tests require acute & convalescent sera in some disease states, IgM tests can have false-positive & false-negative results, 2-3 week delay in diagnosis in infections with short incubation period
  21. Epidemiologic vs Clinical Relevance
    • Public Health Issues / Outbreak: Influenza as example; individual patient management unchanged, but serology guides vaccination for next flu season
    • Treatment as Standard of Care: confirming diagnosis may not change treatment plan; treat based on history & clinical suspicion
  22. Immunity
    • Protection against infectious disease by the immune response
    • Generated by Immunization or Previous infection
  23. Susceptibility
    • Lack of adequate immunity
    • Possible reasons: No prior infection, No prior immunization, Waning immunity, Incompetent or compromised immune system
  24. Interpretation
    • Interpretation of serology results may depend on type of test/assay used
    • Separation of IgM from IgG
    • Concern with IgM antibody assays
    • False-negative IgM result if patient has both IgM & IgG to same antigen
    • False-positive IgM result if Rheumatoid Factor present, or cross-reaction Abs to other viral infections or autoimmune dz
    • To prevent, perform extra step in lab to separate IgM & IgG prior to IgM assay
  25. Antibody Detection Methods
    • Immunoprecipitation
    • Complement Fixation
    • Neutralization
    • Particle agglutination (PA) / Agglutination inhibition (PAI)
    • Immunofluorescence
    • Enzyme immunoassay
    • Radioimmunoassay
  26. Immunoprecipitation
    • Formation of insoluble complexes too large to remain in suspension
    • Soluble antigens are mixed with a specific antibody
    • Not commonly used in ID
    • Is used to detect M-spike for multiple myeloma.
  27. Complement Fixation
    • Not frequently used.
    • Inactivation (fixation) of complement after binding of complement factors to antigen-antibody immune complexes
    • If bound, complement is not available to induce lysis of sheep RBCs
    • Slow assay
    • Examples: used to dx fungal infections, VZV
  28. Neutralization
    • Infectivity of a microorganism or activity of a toxin is neutralized by specific antibody
    • Rarely used for diagnostic purposes
    • Used for screening populations of patients from immunity or past exposure
    • Example: used to test efficacy of new vaccines against viruses
  29. Particle Agglutination / Particle Agglutination Inhibition
    • PA: antigen-coated particles are mixed with patient serum dilutions then examined for macroscopic PA
    • PAI: competition between antibody-coated particles and serum antibodies for a limited number of Ag-binding sites
    • Simple & fast assay
    • Examples: fungal infections, ABO blood typing
  30. Immunofluorescence
    • Apply a dilution of patient serum to a slide containing infectious agent or cells expressing antigens of the infectious agent, then add anti-human IgG or IgM Ab conjugate, then examine microscopically or with UV light fluorescence
    • Very sensitive, not very specific
    • Examples: bacterial infections (Borrelia, Rickettsia, Ehrlichia), HSV, CMV, EBV, Parvovirus
  31. Enzyme Immunoassay
    • EIA, ELISA
    • Indirect assay, uses an anti-human IgG or IgM antibody-enzyme conjugate
    • Endpoint is color development read by spectrophotometer or eye
    • Very sensitive antibody detection assay
    • Commonly used to detect viruses.
  32. Radioimmunoassay (RIA)
    • Very sensitive antibody detection assay
    • Assay principle same as EIA, EXCEPT; Endpoint reading requires gamma or beta scintillation counter; Special handling & disposal of radioactive waste is required
    • Specimens: serum; (occasionally other specimens, such as spinal fluid, urine or oral mucosal transudate)
  33. Alert Values in Serology
    • Lab will alert you immediately of positive results.
    • Influenza & RSV EIA (in season)
    • Positive HIV antibody & Western Blot
    • Positive results called from CDC or NC State Laboratory of Public Health
    • Includes syphilis tests such as RPR, VDRL and FTA-AVS, HIV
  34. Empiric Treatment
    • Initiation of treatment prior to establishment of a firm diagnosis.
    • Always obtain culture (if applicable) prior to initiation of antibiotics
    • Evidence Based Medicine…know your pathogen & antibiotic susceptibility
  35. Normal Flora
    Indigenous bacteria that reside symbiotically (protective) on human skin or mucous membranes.
  36. Sterile Sites
    Sites within human body that are considered sterile or “free” of microbes (blood, brain, muscle tissue).
  37. Exogenous pathogens
    • Infectious agent that resides outside host
    • multiple sources in environment…water, soil, foods & other animals (also humans)
  38. Endogenous pathogens
    • Infectious agent resides within host’s indigenous flora (becomes pathogenic)
    • eg. HIV+ patient who is immunocompromised
  39. Eye: Aqueous/Vitreous Humor
    • Culture for bacterial suspect…don’t forget viral, parasitic, fungal & AFB possibilities
    • Swabs appropriate for suspicion of bacterial conjunctivitis
    • Fluid in sterile container for other infections
  40. Ear: Bacterial, Viral
    • Otitis Externa (swimmer’s ear): Specimen required, aerobic swab
    • Otitis Media: Usually treated empirically unless chronic infection (usually assoc. with anaerobes)
    • If chronic, then tympanocentesis or mastoid bone (if obtained peri-operatively)
    • Use anaerobic culture medium
  41. Eyes, Ears & Sinuses
    • Sinuses: Normally sterile
    • Preceding Viral Infection most frequent
    • Normal Respiratory pathogens predominate (following viral URI)
    • Usually a clinical diagnosis unless severe
    • Severe sinusitis cultures obtained by maxillary sinus aspiration (avoid normal flora contamination) anaerobic media
  42. Upper Respiratory Tract
    • Throat: Pharyngitis & Tonsillitis
    • Most cases occur in combination with viral infections (influenzae, Epstein-Barr, or CMV)
    • Primary bacterial agent is Group A Streptococcus.
    • Treat to avoid sequelae of Rheumatic Fever or Glomerulonephritis
    • Throat swab (aerobic) is obtained
  43. Bronchitis
    Inflammation of tracheo-bronchial tree
  44. Acute Bronchitis
    Associated with viral URI almost always, exception is peds
  45. Whooping Cough (Bordetella pertussis)
    Requires special collection with wire NP (nasopharyngeal) swab & specific media in lab (or PCR test)
  46. Bronchitis (Adult)
    • Rarely Rx antibiotics
    • Acute: Viral Upper Respiratory Tract Infection, so culture is not appropriate.
    • Chronic: Affects 10 to 25% adults (smokers, exposures, depressed immune system). Mostly viral.
    • Resist the temptation to treat with antibiotics unless absolutely indicated.
  47. Pneumonia
    • Inflammation of lower respiratory tract involving the lung airways.
    • Viral, Bacterial (Typical & Atypical), Fungal, AFB & Parasitic (immuno-compromised patients with HIV).
    • Specimens: Sputum, Endotracheal Aspirates, Bronchoscopy wash/brush
    • Gram Stain denotes specimen quality
  48. Gastroenteritis
    Inflammation of GI tract caused by viral, parasitic, or bacterial infection or bacterial toxin.
  49. Stool
    • May require serial (2 or 3 specs) due to intermittent organism shedding
    • Specimens may require additives or preservatives for certain specialized testing
  50. Genital Tract
    • Infections classified as Exogenous (STIs) or Endogenous (imbalance of normal genital flora)
    • Female Lower Tract: vulva, vagina & cervix.
    • Female Upper Tract: uterus, fallopian tubes, ovaries & abdominal cavity.
    • Male Lower Tract: urethra; Upper Tract epididymis, prostate or testicles.
    • Transport swabs often require special media
    • Urinary Tract Infections (UTIs) include Lower Tract (bladder & urethra) or Upper Tract (ureters & kidneys).
    • Female anatomy favors infection
    • Primarily bacteria (rarely AFB) parasitic due to genital tract contamination.
    • Specs: Clean catch, catheters (indwelling or straight) supra-pubic aspirate
  51. Urine Cultures
    • Immediately refrigerate sterile containers.
    • Culture inoculation is calibrated to allow quantification to define clinical significance. Complicated schematic.
    • >100,000 CFU/ml of 1 or 2 organisms
    • >10,000 CFU/ml of 1 pathogen (males)
    • >3 orgs = mixed flora/contamination
  52. Wound Cultures
    • Depends on anatomic site & Superficial vs. Deep anatomy
    • Remember Normal Flora for site that is cultured. Swab? (aer & ana) or media.
  53. Bone
    • Always sterile
    • Tissue: sterility depends on location
    • Tissue ALWAYS better than a swab!
  54. Bacteremia
    • Bacteria in Bloodstream
    • Transient: Incidental or Spontaneous (e.g. Dental Proc, Urine Cath, I&D of Abscess, Indwelling devices (IV, Cardiac, GI)
    • Continuous: organisms consistently released (septic shock, endocarditis)
    • Intermittent: Bacteria released about 45 minutes prior to febrile episode (e.g. undrained abscess)
  55. Septic Shock
    Bacteremia causes circulatory changes that reduce tissue perfusion & result in multi-organ failure
  56. Endocarditis
    Infection of endocardium (membrane lining of heart chambers & valves) *bacteria + “defective” valve
  57. Vegetation
    Web of bacteria, platelets, fibrin & inflammatory cells entrapped on heart valve seed bacteria into bloodstream @ slow, continuous rate.
  58. BLOOD
    • Pathogens from all 4 major groups: Bacteria (aerobic, anaerobic & fastidious organisms), fungi, parasites and viruses.
    • Important to obtain 2 to 3 “sets” of Blood Cultures with fever spike 38.5 °C
    • Aseptic technique to avoid contamination!
  59. Body Fluids
    • All Body Fluids should be sterile
    • Most Body Fluids collected by sterile needle aspiration of fluid
    • Follow guidelines of transport media & volume of fluid necessary for specific tests based on laboratory protocol.
    • Know what you are looking for & cover all your bases!
  60. CSF
    • 4 Routes of Infection
    • Hematogenous spread (most common)
    • Extension of infected site (otitis media)
    • Anatomic defect in CNS structure (trauma or surgery)
    • Direct intraneural (least common-rabies)
  61. Encephalitis
    Inflammation of the brain parenchyma (typically viral)
  62. Meningoencephalitis
    • Concomitant meningitis & encephalitis (lower WBC counts than meningitis…lymphocytes)
    • Often, Viral Encephalitis & Aseptic Meningitis clinically indistinguishable
  63. Meningitis
    • Infection within the subarachnoid space.
    • 2 Categories
    • Purulent: CSF contains large # of WBC’s (neutrophils)…cause is typically bacterial
    • Aseptic: CSF contains large # of lympho-cytes or other mononuclear cells…usually viral & self limiting
    • CSF collected by aseptic lumbar puncture…3 to 4 sterile tubes delivered immediately to lab for multiple tests.
    • Appropriate handling is imperative!
    • Bacterial, Fungal, AFB & sometimes parasitic studies requested in Micro.
    • Gram Stain Stat, India ink vs. serology, AFB stain, wet prep for amoebas rarely
  64. Antimicrobial Susceptibility
    • MIC-Minimal Inhibitory Concentration
    • Lowest concentration of drug that inhibits visual growth of bacteria
    • Most common automated method for antibiotic susceptibility testing
    • Useful in guiding antibiotic prescribing
    • Resistance is “community” specific
    • MICs are standardized by national agency by bacteria & achievable serum levels
    • Interpretation of Susceptibility Results
    • S = Susceptible – appropriate for Rx
    • I = Intermediate - gray zone (e.g. certain antibiotics are “concentrated” in urine)
    • R = Resistant – serum levels to “kill” aren’t achievable or due to resistance mechanisms
    • Useful for bacterial cultures to distinguish gram positive & gram nega-tive infections rapidly. Remember to distinguish what is pathogenic from “normal flora” in non-sterile specimens.
    • Gram stains may guide antibiotic therapy (i.e. some drugs are directed @ G+ vs. G-)
    • Gram Stain - Bacteria NOT visualized
    • Organisms that exist almost exclusively within host cells (obligate intracellular), e.g. Chlamydia
    • Organisms that lack a cell wall, e.g. Myco-plasma & ureaplasma
    • Organisms that are of insufficient dimension to be resolved by light microscopy, e.g. Spirochetes
  66. Sputum Gram Stain
    To ascertain quality of specimen (ie culture or reject)
  67. Gram Stains
    • Primarily to detect bacteria (G- & G+), based on “classic” morphology, may “tentatively” ID bug
    • Fungi/Yeasts & elements sometimes visualized
    • WBCs & Epithelial cells, mucus & cellular debris also visualized.
  68. Acid Fast Stain
    • Specific for subset of bacteria whose cell walls contain long-chain fatty (mycolic) acids, which render cells resistant to decolorization with acid alcohol e.g. Mycobacterium
    • Gram stain may show faintly stained “beaded” G+ rods for Mycobacterium
  69. Acid Fast Stain
    Certain other bacteria “partially” stain acid fast, e.g. Legionella, Nocardia, Rhodococcus & parasitic cysts of Cryptosporidium & Isospora
  70. Acid Fast Stain
    Direct examination of sputum only positive about 60% of time in patients who grow M. tuberculosis
  71. India Ink Prep
    • Historical staining technique for Cryptococcus neoformans (fungal infection, performed on CSF)
    • Latex agglutination more sensitive & specific (India Ink <50% accurate)
    • Some C. neoformans species lack capsule & thus have a false negative India Ink
    • Microbiology Stains
  72. KOH (Potassium Hydroxide) Prep
    • Utilized for rapid detection of fungi by dissolving host proteins & enhancing visualization of fungal elements
    • The “Traditional” Method…calcofluor white fluorescent stain is replacing KOH in some labs
  73. Wet Preps
    • Primarily used for direct visualization of Trichomonas vaginalis (common parasitic STI malodorous, greenish vaginal discharge)
    • Wet prep is still primary ID method found in most labs, serological testing is rarely used.
Card Set
Diagnostic Methods Infectious Diseases
Diagnostic Methods Infectious Diseases