MICROBIO 1.5

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Questions and Answers

What does sensitivity measure in a diagnostic test?

The ability of the test to differentiate between similar diseases

The probability that the test will be positive in patients who have the disease (correct)

The probability that the test is accurate without errors

The probability that the test will be negative in non-diseased individuals

What does specificity assess in the context of diagnostic testing?

The probability that the result will be negative in patients without the disease (correct)

The probability of a positive result among diseased patients

The likelihood of false negatives in a test result

The accuracy of the test in detecting true disease cases

Which statement is true regarding false positives?

They are measured by the test's specificity metric.

They represent patients who truly have the disease according to the test results.

They occur when the test correctly identifies a disease that is not present.

They are instances where the test incorrectly indicates the presence of a disease. (correct)

Which principle is related to detecting pathogen-specific immune responses?

Measurement of a pathogen-specific immune response in the patient (D) Signup and view all the answers

Which statement correctly describes specificity in diagnostic testing?

It is the ability to correctly identify those without the disease (C) Signup and view all the answers

How is positive predictive value (PPV) defined?

The percentage of true positives among all positive test results (D) Signup and view all the answers

What is a false negative in the context of diagnostic testing?

Test indicates disease absence when present (D) Signup and view all the answers

When evaluating the performance of diagnostic tests, which principle indicates how well the test identifies those who do not have the disease?

Specificity (B) Signup and view all the answers

What is the primary method used by Western blot to separate pathogen antigens?

Electrophoresis (B) Signup and view all the answers

In an ELISA test, what indicates that antibodies have successfully bound to the antigen?

The enzyme's production of color (C) Signup and view all the answers

Which agar medium would be most suitable for culturing E. coli to support a suspected infection?

MacConkey agar (D) Signup and view all the answers

What is a characteristic feature of antigen detection tests?

They use specific antibodies to capture antigens. (A) Signup and view all the answers

Which of the following immunoglobulins can a secondary antibody in an ELISA be made specific for?

IgG or IgM among others (A) Signup and view all the answers

What is a common application of nucleic acid-based diagnosis?

Amplifying pathogen DNA (B) Signup and view all the answers

Which component does NOT play a role in the ELISA testing procedure?

Bacterial culture (C) Signup and view all the answers

Which staining technique is specifically used for identifying systemic protozoal infections?

Giemsa stain (B) Signup and view all the answers

How does the specificity of monoclonal antibodies compare to polyclonal antibodies in diagnostic microscopy?

Monoclonal antibodies are more specific. (A) Signup and view all the answers

Which method utilizes lysis-centrifugation for selecting cultures?

Direct inoculation of blood into nutrient broth (C) Signup and view all the answers

What type of microscopy uses fluorophores conjugated to primary antibodies?

Direct immunofluorescence (A) Signup and view all the answers

Which type of culture media is commonly used to identify specific bacterial cultures?

Selective media (A) Signup and view all the answers

What is a primary advantage of using antimicrobial sensitivity testing?

It assesses the effectiveness of antimicrobial agents. (D) Signup and view all the answers

Which of the following techniques is most useful for diagnosing systemic fungal infections?

Silver stain (C) Signup and view all the answers

What does Western blot primarily measure in the context of infections?

Antibody response (A) Signup and view all the answers

Study Notes

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.

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Description

Explore the key principles of laboratory diagnostics, focusing on rapid diagnostic tests (RDTs) and their impact on patient care. Understand the performance of RDTs in various settings and their role in combating antimicrobial resistance. Learn to assess and apply diagnostic protocols effectively.