MICROBIO 1.5

General Principles of Laboratory Diagnostics

• Rapid diagnostic tests (RDTs) offer immediate results at the point of care, increasing efficiency and speeding up diagnosis.
• RDTs reduce the risk of a patient getting sicker before a correct diagnosis, by speeding up identification of organisms and susceptibility to various antimicrobials.
• Some RDTs are valuable in low-resource settings, replacing complicated laboratory tests.
• RDTs help fight antimicrobial resistance by enabling faster administration of appropriate therapies.
• RDT sensitivity is generally 50-70% compared to gold standards, while specificity for influenza is ~90-95%.
• Delay in administering antibiotics for septic shock patients reduces their survival rate by 7.6 percentage points for each hour delay.

Class Objectives

• Students should describe the four diagnostic principles, assess the performance of different diagnostic tests, and understand when to use tests based on diagnostic protocols.
• Students should understand the meaning of results and how to diagnose infections by culture.
• Students should be able to describe molecular and genetic approaches to studying bacteria.

Assessing the Performance of Diagnostic Tests

• True Positive: Pathogen positive, test positive
• True Negative: Pathogen negative, test negative
• False Positive: Pathogen negative, test positive (Type I Error)
• False Negative: Pathogen positive, test negative (Type II Error)
• Sensitivity quantifies a test's ability to correctly identify those with a disease by calculating true positives divided by true positives + false negatives.
• Specificity measures the test's ability to correctly identify those without the disease, based on calculating true negatives divided by true negatives + false positives.
• Positive Predictive Value (PPV) measures the probability of disease given a positive test, obtained by true positives divided by true positives + false positives.
• Negative Predictive Value (NPV) measures the probability of no disease given a negative test, calculated by true negatives divided by true negatives + false negatives.

Diagnostic Sensitivity and Specificity

• The reliability of a test needs to be understood before clinical application or use.
• No test is perfect, all tests produce false positives and false negatives.
• How important a test is, depends on the sensitivity and specificity of that particular test.
• Sensitivity: Probability that the test will be positive in a patient who has the disease.
• Specificity: Probability that the test will be negative in a patient who does not have the disease.

True vs False

• A true positive result correctly predicts a positive outcome.
• A true negative result correctly predicts a negative outcome.
• A false positive result incorrectly predicts a positive outcome.
• A false negative result incorrectly predicts a negative outcome.

Sensitivity and Specificity Example

• In an example relating to student health, data was used to calculate sensitivity and specificity of a diagnostic test for a feared condition.
• The example, with data provided (1593, 729, 473, 2907), shows that sensitivity is 77% and specificity is 80%.

The Four Diagnostic Principles

• Microscopic examination of patient samples
• Cultivation and identification of microorganisms from patient samples.
• Measuring a pathogen-specific immune response in the patient.
• Detection of pathogen-specific macromolecules in patient samples

Diagnosing Infections by Microscopy- Stains

• Gram stains and acid-fast stains detect the presence of bacteria in normally sterile body fluids (CSF, blood, urine).
• Less useful on non-sterile body sites (like skin).
• Stains help identify bacteria, and this helps with choosing appropriate antibiotics.
• Giemsa stains are helpful for diagnosing systemic protozoal infections.
• Lugol's iodine stains assist in identifying intestinal helminthes (intestinal worms).
• Silver stains aid the diagnosis of systemic fungal infections.

Diagnosing Infections by Microscopy - Antibody-Based Identification

• Specific antibodies enhance microscopic identification accuracy
• Monoclonal antibodies create most specific tests.
• Polyclonal antibodies can be less specific.
• Direct immunofluorescence: fluorophore is conjugated to the antibody.
• Indirect immunofluorescence: unlabeled primary antibody is added to bind secondary antibody.

Diagnosing Infections by Culture

• Culturing microbes on agar-based media (aerobic/anaerobic) and broth media for identification
• Blood cultures directly inoculate blood into nutrient broth.
• Subcultures and transfers to agar for identification
• Phenotypic properties, including motility; nutrient utilization and enzymes produced.
• Antibody-based techniques.
• Selective media identify specific cultures.

Common Cultures: EMB and MacConkey Agar

• EMB (Eosin Methylene Blue) Agar and MacConkey Agar help culture identification based on lactose fermentation in bacteria.
• Differences in color indicate if the bacteria are lactose fermenting or non-lactose fermenting.

Antimicrobial Sensitivity Testing

• Antimicrobial sensitivity tests determine bacterial susceptibility to antimicrobial agents.

Measuring the Antibody Response to Infection - Western Blot

• Serology-examining blood serum to check for immune response.
• Western blots: one of the most specific serologic methods for detecting specific antibodies to pathogens.
• Protein bands are transferred to a membrane, then patient serum is added, and antibodies are visualized.

Measuring the Antibody Response to Infection - ELISA

• Enzyme-linked immunosorbent assay (ELISA)
• Solid-phase assay fixes pathogens to a solid surface for patient antibody detection.
• Enzyme-labeled anti-antibodies bind to patient antibodies present to detect and measure color reactions for quantifying antigen presence.

Test Your Knowledge #1

• Patient with suspected E. coli infection from a salad recall warrants a stool culture using Bile Esculin Agar.

Diagnosing Infection by Detecting Pathogen Macromolecules (& Genetic Testing)

• A) Antigen detection tests
• B) Nucleic Acid-Based Diagnosis of Infection
• C) Microarrays
• D) Next-Gen Sequencing

Antigen Detection Tests

• Specific antibodies capture antigens in a patient's sample.
• Simple agglutination assays detect pathogens by observing clumping.
• Positive: antigen binds to antibodies, clumping occurs.
• Negative: no antigen binding, no clumping.
• Prozone: too much antigen, false negative reaction possible
• Latex agglutination tests utilize antibody-coated latex beads to detect capsular material by observing clumping.

Antigen Detection Tests-ELISA & EIA

• Enzyme-linked immunosorbent assays (ELISAs) and enzyme immunoassays (EIAs) quantify antigens in solutions and visually detect antigens.
• Immobilized antigens (or antibodies) create a solid support for patient sample incubation and detection of a color change.

Test Your Knowledge #2

• Requires identification of antibiotic resistance based on the provided antimicrobial sensitivity testing diagram.

Nucleic Acid-Based Diagnosis of Infection

• DNA, when heated, separates into single strands.
• DNA strands reconnect (hybridize) because they are complementary sequences.
• Probes (short single-stranded DNA sequences) can hybridize to a target sequence.
• The DNA probe test was an early nucleic acid-based test.
• In situ hybridization works on tissue samples.

Nucleic Acid Amplification

• Amplification of nucleic acids from trace amounts.
• Steps include heat denaturation, annealing, synthesis of complementary strands, and amplified sequence formation (amplicons).

Polymerase Chain Reaction (PCR)

• Real-time PCR (qPCR) quantifies DNA.
• Targeted for specific DNA segments using primers.
• Fluorescent-labeled probes quantify DNA target sequences.
• Combines amplification steps for rapid detection.
• Useful for identifying viruses and bacteria.

- PCR has a nuanced practical use in context.
- It is useful for patients needing treatment fast, such as those with bacterial infections and requiring quick treatment.
- Used to identify new viral or infections for those who have not responded to prior treatment.

PCR Debate

• Use of PCRs depends on context and clinical urgency.
• Broad-spectrum treatment is often needed when patients need rapid treatment for bacterial infections.
• PCR is helpful for patient follow up; determining if infections are viral or bacterial.

COVID-19 Testing Overview

• Testing overview for COVID-19 includes RT-PCR, Serology, and Antigen testing.
• RT-PCR amplifies viral RNA, is highly sensitive and specific.
• Serology measures antibodies to viral proteins; takes days for positive results.
• Antigen tests detect viral proteins (high specificity but less sensitive).

Microarrays

• Bacteria have conserved sequences useful for amplification to detect species by hybridizing to microarrays.

Next-Generation Sequencing (NGS)

• Bacterial genome sequence determination from samples.
• Information on resistance, virulence, & typing is obtained from the runs; beneficial for outbreak investigations.

Test Your Knowledge #3

• Requires interpreting images of latex agglutination tests as positive or negative.

ALSO A SCIENTIST

• Jane Hinton, DVM, was one of two African-American women to earn a DVM degree from the University of Pennsylvania.
• She was a granddaughter of slaves.
• She opened a laboratory and developed training courses for women in the field.
• Co-developed Mueller-Hinton agar.
• Mueller-Hinton agar became a gold standard for antibiotic testing.