Lecture 59: Intro to Infectious Disease

Why Perform Diagnostic Testing?

• Determine the presence or absence of disease
• Gauge severity
• Monitor response to therapy (e.g., localize disease in body)
• Inform prognosis
• Note: Must consider the whole picture and understand pathogenesis of organisms

Selection of the Best Diagnostic Test

• Helps reach a diagnosis swiftly
• Allows therapy initiation sooner
• Decreases medical costs
• Helps prevent diagnostic errors
  • Many diagnostic errors result from ordering the wrong test or submitting the wrong sample

All Tests Are Not Created Equal

• No test is perfect!
• Tests are compared to a reference standard (sensitivity & specificity)
  • Newer terminology: "Reference standard" instead of "gold standard"
  • Necropsy is the gold standard for most infectious diseases
• Predictive values vary with disease prevalence

2 x 2 Tables

• Sensitivity = TP / (TP + FN)
  • The proportion of disease-positive patients that test positive
  • Example: 100 dogs have an infection and 90 test positive. Sensitivity = 90/100 = 90%
  • Highly sensitive test: many false positives → negative test rules out disease
• Specificity = TN / (TN + FP)
  • The proportion of disease-negative patients that test negative
  • Example: 100 dogs are NOT infected, 95 of those test negative. Specificity = 95/100 = 95%
  • Highly specific test: many false negatives → positive test rules in disease
• Ideal Test: Highly sensitive & highly specific
  • Few false negatives & few false positives
  • Almost no diagnostic tests are like this

Predictive Values

• More helpful to inform clinical decisions than just sensitivity & specificity
• Positive Predictive Value = TP / All positives
  • Proportion of test positives that have the disease
• Negative Predictive Value = TN / All negatives
  • Proportion of test negatives that have the disease

Example: New Virus in Cats

• Virus affects 10 of every 1000 cats
• Diagnostic test with 90% sensitivity and 90% specificity
• If a cat tests positive, what are the chances the cat actually has the disease?
  • PPV = 9/108 = 8.3%
• Test is valuable when the result is negative: very confident that the cat does not have the disease
  • NPV = 891/892 = 99.9%

Infectious Disease Tests

Organism Detection

• Antibody Detection
  • Assess for direct evidence of an organism's presence
  • Assesses for whole organisms, antigen, or nucleic acid (DNA or RNA)
  • Advantages:
    • Positive implies presence of organism
    • Can localize the disease process
    • Sensitive in immunocompromised
    • Quantification of organism numbers may be possible
  • Disadvantages:
    • False positives possible
    • Positive test doesn’t always imply disease
    • Low sensitivity for some infections

Cytology & Histopathology

• Performed on impression smears, aspirates, bodily fluids, or tissue samples
• Special stains (e.g., IFA) can be applied
• Can identify organisms directly
• Can be very insensitive

Culture

• Majority performed with cell-free culture
• Allows for organism identification & antimicrobial susceptibility testing
• Techniques:
  • Morphology
  • Biochemical testing
  • MALDI-TOF: Isolate bacteria from culture → shoot with lasers to break up organism → each organism has its own “fingerprint”
• Pitfalls:
  • False negatives with low specimen sizes, inadequate incubation, or after ABX admin
  • Some organisms are unculturable, fastidious, or need special labs
  • Loss of viability with storage & transport
  • False positives from contamination
  • Can be expensive

Immunoassays Detecting Antigen

• Detect organism proteins
• Method:
  • ELISA
  • Lateral flow assay (LFA)
  • Direct fluorescent antibody (DFA)
  • Latex agglutination (LA)
  • Enzyme immunoassay (EIA)
• Examples:
  • FeLV SNAP test
  • Parvovirus fecal antigen SNAP test
  • Giardia fecal antigen DFA
  • Heartworm test
  • Cryptococcus latex agglutination test
• Pitfalls:
  • False negatives with low antigen levels
  • False positives from cross-reactions
  • Variable sensitivity & specificity

Nucleic Acid Assays - PCR

• Detects organism DNA or RNA
• Need organism-specific primers + polymerase enzymes
• Can detect in two ways:
  • Endpoint (qualitative)
  • Real-time (quantitative)
• Indicated when:
  • Difficult or dangerous to culture
  • Need rapid results
  • Prior to robust antibody production
  • In immunocompromised animals
• Pitfalls:
  • False negatives if insufficient sample
  • False negative if there is strain variation
  • Inhibition of enzymes from bodily fluids
  • False negative from degradation of nucleic acid
  • False positives from contamination

How Do You Choose?

• Organism Test: Acute disease, immunocompromised host
• Antibody Test: Chronic, persistent infection, undetectable organisms
• Sensitive Test: For screening purposes, to rule OUT disease
• Specific Test: To confirm a diagnosis, to rule IN a disease