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Simplifying Laboratory Test Interpretation

Simplifying Laboratory Test Interpretation . Maria del Rosario, MD, MPH Division of Infectious Disease Epidemiology WVDHHR/BPH/OEPS May 2011. Objectives. Review laboratory tests commonly encountered in public health surveillance.

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Simplifying Laboratory Test Interpretation

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

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

  5. 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 WVDHHR/BPH/OEPS/DIDE

  6. Culture The process of growing and propagating organisms in a media that is conducive for their growth. S. pneumoniae on blood agar plate Pros: • Confirm the organism • Reproduce the organism and use for additional testing Cons: • Delay in confirmation • Require viable organism • Difficult for fastidious organisms colony WVDHHR/BPH/OEPS/DIDE

  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

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

  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 WVDHHR/BPH/OEPS/DIDE

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

  11. Pulsed Field Gel Electrophoresis (PFGE) The outbreak strain of SalmonellaTyphimurium has been found in ill humans and in food samples during this outbreak investigation. 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

  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

  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

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

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

  16. 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 WVDHHR/BPH/OEPS/DIDE

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

  18. Ehrlichiachaffeensis 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

  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 WVDHHR/BPH/OEPS/DIDE

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

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

  22. 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 WVDHHR/BPH/OEPS/DIDE

  23. WVDHHR/BPH/OEPS/DIDE

  24. 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 WVDHHR/BPH/OEPS/DIDE

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

  26. WVDHHR/BPH/OEPS/DIDE

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

  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

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

  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 WVDHHR/BPH/OEPS/DIDE

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

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

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

  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

  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

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

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