Simplifying Laboratory Test Interpretation Maria del Rosario, MD, MPH Division of Infectious Disease...

Simplifying Laboratory

Test Interpretation

Maria del Rosario, MD, MPHDivision of Infectious Disease Epidemiology

WVDHHR/BPH/OEPS

May 2011

1WVDHHR/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.

2WVDHHR/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.

WVDHHR/BPH/OEPS/DIDE 3

Laboratory Tests Detection Methods

o Microscopyo Cultureo Antigen test*

Identification Methodso PCR*o Viral load*o PFGEo Genotyping

Serology

Antimicrobial susceptibility

Ancillary tests*both detect and identify


MicroscopyDirect examination of a specimen (or may use stains) to detect the presence of organisms.


Gram negative diplococci

Pros: • Quick and easy• Preliminary results

Cons:• Not specific

CultureThe 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

organismsS. pneumoniae on blood agar plate


colony

Antigen TestUse 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.

Result 1


Purpose of testTest result interpretation

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


Method used to amplify a specific region of a DNA strand.


Result 2Purpose of testTest result interpretation

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.

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


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.


Basic Anatomy of Antibody Response to Infection


WVDHHR/BPH/OEPS/DIDE 15http://www.stanford.edu/group/virus/parvo/2005/B19.html

Human Parvovirus B-19: Disease and Immune Response

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


Antibody Testing


Result 3

Type of testPurpose of testTest result interpretation

IFA

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…



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

Result 4




HCV RNA HCV RNAHCV RNA HCV RNA

Hepatitis C Testing - 1SEROLOGIC TESTS

• Enzyme Immunoassay (EIA) for Anti-HCVPositive: past or current infectionVerification 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-2Some RIBA-positive patients are HCV RNA-negative


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


Hepatitis C Testing - 2

GENOTYPING• HCV Genotype

6 genotypes, >50 subtypes clinical importance: counseling and treatment epidemiology

LIVER FUNCTION TEST• ALT• SGPT


Hepatitis C Testing - 3


Result 5aType of testPurpose of testTest result interpretation

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.


Result 5b


Type of testPurpose of test Interpretation of

o Test 1 o Test 2

Test 1

Test 2

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


MIC Zone of Inhibition


Sample 6




Sample 7

Ancillary Tests• CBC and WBC• CSF cells

• Liver function tests – ALT, AST, bilirubinWVDHHR/BPH/OEPS/DIDE 33

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?


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)


Thank you

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