Interpretation by Public Health

1. Simplifying Laboratory
Test Interpretation
Maria del Rosario, MD, MPH
Division of Infectious Disease Epidemiology
WVDHHR/BPH/OEPS
May 2011
1WVDHHR/BPH/OEPS/DIDE

2. 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.
2WVDHHR/BPH/OEPS/DIDE

3. 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.
WVDHHR/BPH/OEPS/DIDE 3

4. Laboratory Tests
 Detection Methods
o Microscopy
o Culture
o Antigen test*
 Identification Methods
o PCR*
o Viral load*
o PFGE
o Genotyping
 Serology
 Antimicrobial susceptibility
 Ancillary tests
*both detect and identify
WVDHHR/BPH/OEPS/DIDE 4

5. Microscopy
Direct examination of a specimen (or may use stains) to
detect the presence of organisms.
WVDHHR/BPH/OEPS/DIDE 5
Gram negative
diplococci
Pros:
• Quick and easy
• Preliminary results
Cons:
• Not specific

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
S. pneumoniae on blood agar plate
WVDHHR/BPH/OEPS/DIDE 6
colony

7. Antigen Test
Use of assay to detect the presence of antigen/s. Some assays are able to
differentiate antigens, some are not able to.
WVDHHR/BPH/OEPS/DIDE 7
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.

8. Result 1
WVDHHR/BPH/OEPS/DIDE 8
Purpose of test
Test result
interpretation

9. Polymerase Chain Reaction (PCR)
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
WVDHHR/BPH/OEPS/DIDE 9
Method used to amplify a specific region of a DNA strand.

10. WVDHHR/BPH/OEPS/DIDE 10
Result 2
Purpose of test
Test result
interpretation

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).
WVDHHR/BPH/OEPS/DIDE 11
The outbreak strain of SalmonellaTyphimurium has been found in ill humans and in food samples during this
outbreak investigation.

12. 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
WVDHHR/BPH/OEPS/DIDE 12

13. 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.
WVDHHR/BPH/OEPS/DIDE 13

14. Basic Anatomy of
Antibody Response to Infection
WVDHHR/BPH/OEPS/DIDE 14

15. WVDHHR/BPH/OEPS/DIDE 15
http://www.stanford.edu/group/virus/parvo/2005/B19.html
Human Parvovirus B-19: Disease and Immune Response

16. 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
WVDHHR/BPH/OEPS/DIDE 16
Antibody Testing

17. WVDHHR/BPH/OEPS/DIDE 17
Result 3
Type of test
Purpose of test
Test result interpretation
IFA

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…
WVDHHR/BPH/OEPS/DIDE 18

19. WVDHHR/BPH/OEPS/DIDE 19
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

20. Result 4
WVDHHR/BPH/OEPS/DIDE 20
Type of test
Purpose of test
Test result interpretation

21. WVDHHR/BPH/OEPS/DIDE 21
HCV RNA HCV RNAHCV RNA HCV RNA

22. Hepatitis C Testing - 1
SEROLOGIC TESTS
• Enzyme Immunoassay (EIA) for Anti-HCV
Positive: past or current infection
Verification of Anti-HCV (+) screening test
1. Reflex supplemental testing*: follow-up with more specific serologic
test, e.g. HCV RIBA or NAT
2. 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
WVDHHR/BPH/OEPS/DIDE 22

23. WVDHHR/BPH/OEPS/DIDE 23

24. VIRAL LOAD TESTS
 Measure HCV RNA (genetic material)
 Detects actively replicating virus
 2 types:
A. 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
WVDHHR/BPH/OEPS/DIDE 24
Hepatitis C Testing - 2

25. GENOTYPING
• HCV Genotype
 6 genotypes, >50 subtypes
 clinical importance: counseling and treatment
 epidemiology
LIVER FUNCTION TEST
• ALT
• SGPT
WVDHHR/BPH/OEPS/DIDE 25
Hepatitis C Testing - 3

26. WVDHHR/BPH/OEPS/DIDE 26

27. WVDHHR/BPH/OEPS/DIDE 27
Result 5a
Type of test
Purpose of test
Test result
interpretation

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.
WVDHHR/BPH/OEPS/DIDE 28

29. Result 5b
WVDHHR/BPH/OEPS/DIDE 29
Type of test
Purpose of test
 Interpretation of
o Test 1
o Test 2
Test 1
Test 2

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
WVDHHR/BPH/OEPS/DIDE 30
MIC Zone of Inhibition

31. WVDHHR/BPH/OEPS/DIDE 31
Sample 6
Type of test
Purpose of test
Test result
interpretation

32. WVDHHR/BPH/OEPS/DIDE 32
Type of test
Purpose of test
Test result
interpretation
Sample 7

33. Ancillary Tests
• CBC and WBC
• CSF cells
• Liver function tests – ALT, AST, bilirubin
WVDHHR/BPH/OEPS/DIDE 33

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?
WVDHHR/BPH/OEPS/DIDE 34

35. 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)
WVDHHR/BPH/OEPS/DIDE 35

36. Thank you
Comments and Questions
WVDHHR/BPH/OEPS/DIDE 36