1. Simplifying Laboratory Test Interpretation
Maria del Rosario, MD, MPH
Division of Infectious Disease Epidemiology
WVDHHR/BPH/OEPS
May 2011
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2. WVDHHR/BPH/OEPS/DIDE
Objectives
Review laboratory tests commonly encountered in public health surveillance.
Discuss laboratory test reports and practice report interpretation using specific examples.
Disclaimer: This lecture is not intended to replace the advice and recommendations of a healthcare provider.
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3. WVDHHR/BPH/OEPS/DIDE
Definition of Terms
Normally sterile site: sites in the human body that are normally free from organisms or foreign material, e.g. blood, joint, brain, etc.
Unsterile site: sites in the human body that generally harbor microorganisms, e.g. gut, oral cavity, nose, skin, etc
Specimen: a sample of tissue (blood, urine, etc.) that may or may not contain organisms
Isolate: a population of organisms (bacteria) that has been separated from a mixture
Serotype: a group of closely related organisms with distinct characteristics.
Assay: A test to detect or quantify a substance in a sample.
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4. WVDHHR/BPH/OEPS/DIDE
Laboratory Tests
Detection Methods
Microscopy
Culture
Antigen test*
Identification Methods
PCR*
Viral load*
PFGE
Genotyping
Serology
Antimicrobial susceptibility
Ancillary tests
*both detect and identify
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5. Gram negative diplococci
Microscopy
Direct examination of a specimen (or may use stains) to detect the presence of organisms.
Pros:
Quick and easy
Preliminary results
Cons:
Not specific
Gram negative diplococci
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6. Culture
The process of growing and propagating organisms in a media that is conducive for their growth.
Pros:
Confirm the organism
Reproduce the organism and use for additional testing
Cons:
Delay in confirmation
Require viable organism
Difficult for fastidious organisms
colony
S. pneumoniae on blood agar plate
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7. WVDHHR/BPH/OEPS/DIDE
Antigen Test
Use of assay to detect the presence of antigen/s. Some assays are able to differentiate antigens, some are not able to.
Technique
Principle
Agglutination
Known antiserum causes bacteria or other particulate antigens to clump together or agglutinate
Complement fixation
Known antiserum mixed with the test antigen and complement is added. Sheep red blood cells and hemolysins are then added. Positive test: no hemolysis, negative test: hemolysis
Enzyme-linked immunosorbant assay (ELISA) ; Enzyme immunoassay (EIA)
A rapid test where an antibody or antigen is linked to an enzyme as a means of detecting a match between the antibody and antigen.
Fluorescent antibody
Fluorescent dye is attached to known antibodies. When the fluorescent antibody reacts with the antigen, the antigen will fluoresce when viewed with a fluorescent microscope.
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8. WVDHHR/BPH/OEPS/DIDE
Result 1
Purpose of test
Test result interpretation
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9. Polymerase Chain Reaction (PCR)
Method used to amplify a specific region of a DNA strand.
Pros:
Simple process, eliminates tedious work, results available within a day
Does not require a viable organism since only a strand of DNA is needed,
Sensitive test
Cons:
Sensitive – pick up environmental contaminants
Unable to distinguish between certain species
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10. WVDHHR/BPH/OEPS/DIDE
Result 2
Purpose of test
Test result interpretation
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11. Pulsed Field Gel Electrophoresis (PFGE)
A technique to separate large DNA molecules by applying an electric field that periodically changes direction (electrophoresis)…to compare DNA banding patterns (fingerprints).
The outbreak strain of SalmonellaTyphimurium has been found in ill humans and in food samples during this outbreak investigation.
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Serology
Serology: the study of blood serum, with emphasis on testing of antibodies in the serum
Antigen: A substance which stimulates the body to produce antibody; usually a ‘foreign’ substance
Antibody: A protein molecule produced by the body’s immune system in response to a specific antigen. The antibody combines with the antigen and disables it.
Also called Immunoglobulins (e.g. IgG, IgM, IgA, IgE)
Referred to as anti-(name of antigen), e.g. anti-HCV, anti-HAV
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13. WVDHHR/BPH/OEPS/DIDE
Antibodies
IgM: type of antibody produced by the body, usually the first antibody to appear in response to a foreign substance exposure, then eliminates the organism in the early stages of immunity before there is sufficient IgG
IgG: type of antibody that provides the majority of antibody-based immunity against invading organisms. The only antibody that crosses the placenta to provide immunity to the fetus
Titer: the amount of antibodies present in the blood, usually as a result of infection.
Acute titer and Convalescent titer: At the acute stage of disease, serum is tested (acute phase), followed by another blood draw and testing about 3 weeks (convalescent phase) later. IgG levels are compared and a 4-fold increase between acute and convalescent samples usually indicate infection.
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14. Basic Anatomy of Antibody Response to Infection
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15. Human Parvovirus B-19: Disease and Immune Response
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16. WVDHHR/BPH/OEPS/DIDE
Antibody Testing
Pros:
Screening tool
Readily available
Indicates response to antigen (even if antigen is not detectable) – may indicate infection or immunity
Cons:
Paired testing necessary for some diseases - may take a while to get results, impact on patient management
Unable to differentiate between immunity and disease
Sensitivity and specificity:
False-negative result: compromised immune system
False-positive result: liver disease, low disease prevalence
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Result 3
IFA
Type of test
Purpose of test
Test result interpretation
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18. Ehrlichia chaffeensis Infection Laboratory criteria for diagnosis
Supportive: Serological evidence of elevated IgG or IgM antibody reactive with E. chaffeensis antigen by IFA, ELISA, dot-ELISA, or assays in other formats (CDC uses an IFA IgG cutoff of ≥1:64 and does not use IgM test results independently as diagnostic support criteria.), OR … Confirmed: Serological evidence of a fourfold change in immunoglobulin G (IgG)-specific antibody titer to E. chaffeensis antigen by IFA between paired serum samples (one taken in first week of illness and a second 2-4 weeks later), OR Detection of E. chaffeensis DNA …OR Demonstration of ehrlichial antigen…, OR Isolation of E. chaffeensis from a clinical specimen…
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19. WVDHHR/BPH/OEPS/DIDE
Hepatitis A Antibody Tests
Hepatitis A antibody Total
Anti-HAV Total
Antibody to Hepatitis A Virus
HAV Ab Total
- measures both IgM and IgG
Hepatitis A antibody IgM
Anti-HAV, IgM
Antibody to Hepatitis A Virus, IgM
HAVAb, IgM
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20. WVDHHR/BPH/OEPS/DIDE
Result 4
Type of test
Purpose of test
Test result interpretation
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HCV RNA
HCV RNA
HCV RNA
HCV RNA
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Hepatitis C Testing - 1
SEROLOGIC TESTS
Enzyme Immunoassay (EIA) for Anti-HCV
Positive: past or current infection
Verification of Anti-HCV (+) screening test
Reflex supplemental testing*: follow-up with more specific serologic test, e.g. HCV RIBA or NAT
Signal-to-cut-off ratio (s/co): predict supplemental test-positive results ≥95% of the time, s/co dependent on test type
HCV RIBA* (Recombinant Immunoblot Assay)
Detects antibodies to individual HCV antigens and confers increased specificity compared to EIA-2
Some RIBA-positive patients are HCV RNA-negative
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24. WVDHHR/BPH/OEPS/DIDE
Hepatitis C Testing - 2
VIRAL LOAD TESTS
Measure HCV RNA (genetic material)
Detects actively replicating virus
2 types:
Qualitative test - detects presence of HCV RNA virus (result: positive/negative)
Nucleic Acid Test (NAT)* for HCV RNA using RT-PCR
Detects HCV RNA in the blood
Very sensitive
B. Quantitative test – measures the amount of virus in 1 ml of blood, use to assess response to treatment
Branched-chain DNA (bDNA)
Easy and cheap, especially for large number of samples
Only measures viral loads greater than 50 IU/ml
Transcription-mediated Amplification (TMA)
New, easy
Amplifies and detects viral genetic materia;l in the blood
Can measure viral loads as few as 5-10 IU/ml
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Hepatitis C Testing - 3
GENOTYPING
HCV Genotype
6 genotypes, >50 subtypes
clinical importance: counseling and treatment
epidemiology
LIVER FUNCTION TEST
ALT
SGPT
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27. WVDHHR/BPH/OEPS/DIDE
Result 5a
Type of test
Purpose of test
Test result interpretation
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28. Hepatitis C, past or present
Clinical Case Definition
No symptoms are required…
Laboratory criteria for diagnosis
1 or more of following 4 criteria: Anti–HCV positive (repeatedly reactive) EIA verified by at least 1 additional more specific assay, OR
HCV RIBA positive, OR
NAT positive for HCV RNA (including genotype), OR
Anti-HCV screening-test-positive with a signal to cut-off ratio predictive of a true positive as determined for the particular assay and posted by CDC.
Case classification
Confirmed: laboratory confirmed and does not meet the case definition for acute hepatitis C.
Probable: anti-HCV positive (repeat reactive) by EIA and has ALT or SGPT values above the upper limit of normal, but the anti-HCV EIA result has not been verified by an additional more specific assay or the signal to cut-off ratio is unknown.
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29. WVDHHR/BPH/OEPS/DIDE
Result 5b
Type of test
Purpose of test
Interpretation of
Test 1
Test 2
Test 1
Test 2
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30. Antimicrobial Susceptibility
MIC (minimum inhibitory concentration)
lowest concentration of antimicrobials that will inhibit the growth of organisms. MICs are important to confirm resistance of organisms to an antimicrobial agent.
Methods:
Disk diffusion test
E test
Broth dilution test
Zone of Inhibition
MIC
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31. WVDHHR/BPH/OEPS/DIDE
Sample 6
Type of test
Purpose of test
Test result interpretation
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32. WVDHHR/BPH/OEPS/DIDE
Sample 7
Type of test
Purpose of test
Test result interpretation
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33. WVDHHR/BPH/OEPS/DIDE
Ancillary Tests
CBC and WBC
CSF cells
Liver function tests – ALT, AST, bilirubin
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34. Tips when reviewing a laboratory report
Is the organism (or disease) reportable?
When was the specimen obtained in relation to onset of illness?
Was the source from a normally sterile site?
Were antibiotics used prior to specimen collection?
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35. WVDHHR/BPH/OEPS/DIDE
Summary
Basic understanding of a laboratory test is key to maximizing its use.
Laboratory tests have ‘strengths’ and ‘weaknesses’.
Timing is everything!
(between disease onset and specimen collection)
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36. Thank you
Comments and Questions
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