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

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Presentation on theme: "Simplifying Laboratory Test Interpretation Maria del Rosario, MD, MPH Division of Infectious Disease Epidemiology WVDHHR/BPH/OEPS May 2011 1WVDHHR/BPH/OEPS/DIDE."— Presentation transcript:

1 Simplifying Laboratory Test Interpretation Maria del Rosario, MD, MPH Division of Infectious Disease Epidemiology WVDHHR/BPH/OEPS May WVDHHR/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/DIDE3

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/DIDE4

5 Microscopy Direct examination of a specimen (or may use stains) to detect the presence of organisms. WVDHHR/BPH/OEPS/DIDE5 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/DIDE6 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/DIDE7 TechniquePrinciple AgglutinationKnown 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/DIDE8  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/DIDE9 Method used to amplify a specific region of a DNA strand.

10 WVDHHR/BPH/OEPS/DIDE10 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/DIDE11 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/DIDE12

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/DIDE13

14 Basic Anatomy of Antibody Response to Infection WVDHHR/BPH/OEPS/DIDE14

15 WVDHHR/BPH/OEPS/DIDE15 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/DIDE16 Antibody Testing

17 WVDHHR/BPH/OEPS/DIDE17 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/DIDE18

19 WVDHHR/BPH/OEPS/DIDE19 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/DIDE20  Type of test  Purpose of test  Test result interpretation

21 WVDHHR/BPH/OEPS/DIDE21 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/DIDE22

23 WVDHHR/BPH/OEPS/DIDE23

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/DIDE24 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/DIDE25 Hepatitis C Testing - 3

26 WVDHHR/BPH/OEPS/DIDE26

27 WVDHHR/BPH/OEPS/DIDE27 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/DIDE28

29 Result 5b WVDHHR/BPH/OEPS/DIDE29  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/DIDE30 MIC Zone of Inhibition

31 WVDHHR/BPH/OEPS/DIDE31 Sample 6  Type of test  Purpose of test  Test result interpretation

32 WVDHHR/BPH/OEPS/DIDE32  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/DIDE33

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/DIDE34

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/DIDE35

36 Thank you Comments and Questions WVDHHR/BPH/OEPS/DIDE36


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