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LECTURE ON SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES AND TUMOR MARKERS

LECTURE ON SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES AND TUMOR MARKERS. ROBERTO D. PADUA JR., MD, DPSP DEPARTMENT OF PATHOLOGY AND LABORATORY DIAGNOSIS FATIMA COLLEGE OF MEDICINE. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES. SEROLOGY The scientific study of blood sera and their effects

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LECTURE ON SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES AND TUMOR MARKERS

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  1. LECTURE ON SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES AND TUMOR MARKERS ROBERTO D. PADUA JR., MD, DPSP DEPARTMENT OF PATHOLOGY AND LABORATORY DIAGNOSIS FATIMA COLLEGE OF MEDICINE

  2. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SEROLOGY • The scientific study of blood sera and their effects • Subdivision of immunology concerned with in-vitro Ag-Ab reaction • Concerned with the laboratory study of the activities of the components of serum that contribute to immunity

  3. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • IMMUNOLOGY • The study of the molecules, cells, organs and systems responsible for the recognition and disposal of foreign (non-self) material • The study of how the body components respond and interact • The desirable and undesirable consequences of immune interactions • The ways in which the immune system can be manipulated to protect or treat disease

  4. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • IMMUNITY • The ability of an organism to resist infection by means of the presence of circulating antibodies and white blood cells • Distinctive characteristics of the immune system • Specificity • Memory • Mobility • Replicability • cooperativity

  5. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • METHODS OF DETECTION OF ANTIBODIES • Immuno-precipitation Assays = detect antibodies in solution = qualitative indication of the presence of antibodies = end-point is visual flocculation of the antigen and antibody in suspension

  6. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • METHODS OF DETECTION OF ANTIBODIES 2. Complement Fixation = based on the activation or fixation of complement following binding of complement factors to Ag-Ab immune complexes

  7. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • METHODS OF DETECTION OF ANTIBODIES 3. Neutralization = the ineffectivity of an organism or the activity of toxin is neutralized by specific antibody = rarely used for diagnostic purposes = mainly used to detect antibody formation after vaccination

  8. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • METHODS OF DETECTION OF ANTIBODIES 4. Particle Agglutination = relatively simple and fast = capable of detecting lower concentration of antibodies = designed to detect antibodies to viruses, subsequent to interaction or vaccination = utilize Ag coated latex particles, coal particles, bentonite particles or erythrocytes = direct and indirect methods

  9. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • METHODS OF DETECTION OF ANTIBODIES 5. Immunofluorescence = requires use of microscope equipped to provide ultraviolet illumination or an instrument capable of irradiating the assay with UV light and detecting the resultant fluorescence with a fluorometer

  10. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • METHODS OF DETECTION OF ANTIBODIES 6. Enzyme Immunoassay = the most sensitive = usually indirect assay that depends on the use of an antihuman IgG or IgM antibody conjugate = the antibody conjugate (if present) is made to attach to enzyme which catalyzes conversion of the substrate to a colored product which will then be read with the use of a spectrophotometer

  11. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • METHODS OF DETECTION OF ANTIBODIES 7. Radioimmunoassay = high sensitivity

  12. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • Microbial antigen detection provides direct evidence of infection, and is preferred for diagnosis of infection over antibody detection (indirect evidence of infection) • However, not all infectious agents have available antigen assays or culture techniques making the detection of specific antibodies diagnostically useful

  13. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • Infectious Disease Indicators, Non-specific • Acute phase reactants • Limulus lysate assay • Detects trace amounts of endotoxin from all gram (-) bacteria • Presence in CSF = gram (-) bacterial meningitis • Rapid clearance from blood makes serum test unreliable

  14. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • Molecular Biology • Nucleic acid amplification • DNA sequencing and typing • Direct molecular probe (in situ hybridization) • Nucleic acid quantitation

  15. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • Molecular Biology • Uses: • Cases requiring increased sensitivity and specificity of identification • Cases requiring faster report turnaround time • Confirmation of culture • Identification of organisms that are non-viable or cannot be cultured • Identification of fastidious, slow growing organisms • Identification of organisms that are dangerous to culture • Identification of organisms in small numbers or in small volume specimens

  16. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • Molecular Biology • Uses: • Density of amplifiable DNA correlates with microbial density • Monitoring of disease progression or initiation or modification of therapy • Drug susceptibility testing • Differentiation of antigenically similar organisms • Molecular epidemiology and infection control • Disease diagnosis by characterization of genetic materials without direct identification of infectious agent • Determination of virulence of antimicrobial resistance genes

  17. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS • The most commonly acquired spirochete disease in the U.S. • A complex sexually transmitted disease that has a highly variable clinical course • Over 50,000 cases reported in 1990 in the U.S. • Causative agent is Treponema pallidum • No natural reservoir in the environment, requires living host • Spiral shaped and motile due to peri-plasmic flagella • Variable length

  18. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS • Three other pathogens in the group Treponema which are morphologically and anti-genetically similar to T. pallidum, differences are in characteristics of lesions, amount of systemic involvement and course of the disease • T. pertenue (Yaws) • T. endemicum (non-venereal syphilis) • T. carateum (pinta) • T. cuniculi (rabbit syphilis)

  19. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS • Mode of Transmission • Organism is very fragile, destroyed rapidly by heat, cold and drying • Sexual transmission most common, occurs when abraded skin or mucous membranes come in contact with open lesion • Can be transmitted to fetus • Rare transmission from needle stick and blood transfusion

  20. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS - - Stages of the Disease • Primary stage = primary lesion is chancre = the lesion heals spontaneously after 1-5 weeks = swab of chancre smeared on slide, examined under dark-field microscope, spirochetes will be present = 30% become serologically positive one week after appearance of chancre, 90% positive after three weeks

  21. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS - - Stages of the Disease 2. Secondary Stage = occurs 6-8 weeks after initial chancre, becomes systemic, patient highly infectious = characterized by localized or diffuse mucocutaneous lesions, often with generalized lymphadenopathy = primary chancre may still be present = secondary lesions subside in about 2-6 weeks = serology tests nearly 100% positive

  22. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS - - Stages of the Disease 3. Latent Stage = stage of infection in which organisms persists in the body of the infected person without causing symptoms or signs = this stage may last for years = one-third of untreated latent stage individuals develop signs of tertiary syphilis = after 4 years it is rarely communicable sexually but can be passed from mother to fetus

  23. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS - - Stages of the Disease 4. Tertiary Stage = occurs anywhere from months to years after secondary stage, typically between 10 to 30 years = gummatous syphilis = cardiovascular syphilis = neurosyphilis

  24. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS • Congenital Syphilis • Transmitted from mother to fetus • Fetus affected during the second or third trimester • 40% result in syphilitic stillbirth • Live-born infants show no signs during first few weeks = 60-90% develop clear or hemorrhagic rhinitis = skin eruptions (rash) especially around mouth, palms of hands and soles of feet = general lymphadenopathy, hepatosplenomegaly, jaundice, anemia, painful limbs & bone abnormality

  25. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS - - DIAGNOSIS • Evaluation based on 3 factors • Clinical findings • Demonstration of spirochetes in clinical specimen • Present of antibodies in blood or CSF = more than one test should be performed = no serological test can distinguish between other treponemal infections

  26. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS - - DIAGNOSIS • Laboratory Testing • Direct examination of clinical specimen by dark-field microscopy or fluorescent antibody testing of sample • Non-specific or non-treponemal serological test to detect reagin, utilized as screening test only, not diagnostic = Reagin is an antibody formed against cardiolipin = Found in sera of patients with syphilis as well as other diseases = Non-treponemal tests become positive 1-4 weeks after appearance of primary chancre, in secondary stage may have false positive due to prozone, in tertiary 25% are negative, after successful treatment will become non-reactive after 1 to 2 years

  27. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • SYPHILIS - - DIAGNOSIS • Laboratory Testing C. Specific Treponemal antibody tests are used as a confirmatory test for a positive reagin test

  28. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN TEST • Venereal Disease Research Laboratory=VDRL = Flocculation test, antigen consists of very fine particles that precipitate out in the presence of reagin = Utilizes antigen consists of cardiolipin, cholesterol and lecithin = serum must be heated to 56 C for 30 minnutes to remove anti-complimentary activity which may cause false positive = reported as Non-reactive, weakly reactive and reactive = used primarily to screen CSF

  29. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN TEST 2. Rapid Plasma Reagin – RPR = general screening test = can not be performed on CSF = the VDRL cardiolipin antigen is modified with choline chloride to make it more stable and is attached to charcoal particles to allow macroscopic reading, the antigen comes prepared and is very stable = serum or plasma may be used for testing, serum is not heated = results are read macroscopically = appears to be more sensitive than the VDRL

  30. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • NON-TREPONEMAL SEROLOGICAL TESTS – REAGIN TEST 3. Other tests which use modified VDRL Ag A. USR – unheated serum reagin test = modified VDRL Ag, uses choline chloride/EDTA = microscopic flocculation test B. RST – reagin screen test = modified VDRL Ag with Sudan Black = Sudan Black makes flocculation reaction macroscopically visible

  31. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • SPECIFIC TREPONEMAL TESTS • Treponema pallidum Immobilization Test – TPI = live T. pallidum become immobilized by antibody in serum of infected persons = cumbersome and expensive, no longer used in U.S.

  32. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • SPECIFIC TREPONEMAL TESTS 2. Treponema pallidum Hemagglutination – TPHA = adapted to microtechniques (MHA-TP) = tanned sheep RBC’s are coated with T. pallidum antigen from Nichol’s strain = positive result is agglutination of RBC’s

  33. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • SPECIFIC TREPONEMAL TESTS 3. Fluorescent treponemal antibody absorption test (FTA-ABS) = one of the most used confirmatory test = diluted, heat inactivated serum added to Reiter’s strain of T. pallidum to remove cross reactivity due to other Treponemes = slides are coated with Nichol’s strain of T. pallidum and add absorbed patient serum = slides are washed and incubated with Ab bound to a fluorescent tag = after washing again the slides are examined for fluorescence = requires experienced personnel to read = highly sensitive and specific, but time consuming to perform

  34. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • SPECIFIC TREPONEMAL TESTS 4. ELISA = tubes coated with T. pallidum antigen = antibody in serum attaches to antigen = following washing, add an anti-antibody tagged with enzyme alkaline phosphatase = detectable color changes occur

  35. Sensitivity and Specificity of Serologic Tests for Untreated Syphilis at Different Stages

  36. Serologic Test for Syphilis in Various Conditions

  37. Algorithm for Positive Serologic Test for Syphilis

  38. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • PROBLEM AREAS • Biologic False Positives (BFP) A. Collagen diseases such as arthritis, LE, etc., sometimes result in increased amount of reagin B. Certain infections : IM, malaria, leprosy C. Other treponemal infections

  39. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • PROBLEM AREAS 2. False negatives A. Very early in disease or latent, inactive stage B. Immunosuppressed patients C. Consumption of alcohol prior to testing (temporary)

  40. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • PROBLEM AREAS 3. Congenital syphilis A. Non-treponemal tests on cord blood or baby serum detect IgG antibody, maybe of maternal origin B. Detection of IgM lacks sensitivity C. Western blot has demonstrated high sensitivity and specificity D. Recommended that all mothers be tested

  41. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • PROBLEM AREAS 4. Cerebrospinal Fluid tests A. Used to determine if Treponemes have invaded the CNS B. VDRL utilized to confirm neurosyphilis C. Lacks sensitivity

  42. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = SYPHILIS • CORRELATION OF TREATMENT WITH TEST RESULTS • Treatment at the primary stage, serology tests become non-reactive after 6 months • Treatment at secondary stage, tests usually non-reactive after 12-18 months • If treatment is not initiated until 10 or more years, the reagin tests probably positive for life

  43. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES • LYME’S DISEASE = Disease first recognized in 1977 in Lyme, Connecticut = Causative organism is Borrelia burgdorferi = Can be cultured but it is very difficult = Organism has been isolated from blood, CSF, skin lesions and joint fluid = Can be transmitted perinatally, causing intrauterine death = Vector of transmission is the Ixodes tick = Must remain attached a minimum of 24-48 hours for transmission to occur

  44. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = LYME’S DISEASE • STAGES OF THE DISEASE • Localized rash – erythema chronicum migrans • Dissemination to multiple organ system = occurs by way of the bloodstream = may occur weeks to months after infection = migratory pain may occur in the joints, tendons and bones = neurologic  Bell’s palsy, peripheral neuropathy, aseptic meningitis = cardiac include carditis and arrythmia 3. Chronic disseminated = characterized by chronic arthritis = affects the large joints, especially the knee

  45. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = LYME’S DISEASE • Diagnostic criteria • Isolation of organism from clinical specimen or • Diagnostic titers of IgG and IgM in serum or CSF or • Significant change in serum titers of IgG or IgM in paired acute and convalescent sera

  46. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = LYME’S DISEASE • LABORATORY DIAGNOSIS • Diagnosed clinically, confirmed serologically • Antibodies to antigens of B. burgdorferi can be detected by latex agglutination, IFA, ELISA, and Western Blot • Serological tests are often falsely negative during early weeks. • Specific IgM Abs usually appear 2- 4 weeks after erythema migrans, peak after 3-6 weeks of illness, decline to normal after 4-6 months • IgG titers appears more slowly (4-8 weeks after the rash), peak after 4-6 months, may remain high for months or years • Western Blot is most sensitive • IFA and ELISA are more commonly performed due to ease of procedure, but are subject to false positives due to either spirochete diseases and some autoimmune diseases

  47. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = STREPTOCOCCAL INFECTION • STREPTOCOCCAL SEROLOGY • Streptococci are gram (+), beta-hemolytic, spherical, ovoid, or lancet-shaped organisms which are catalase negative and seen in pairs or chains • Divided into groups or serotypes based on cell wall components  Streptococcus pyogenes belongs to Lancefield group A and it is believed the M protein is the chief virulent factor of this group • Numerous exo-antigens are produced and excreted as the cell metabolizes (Streptolysin O, DNase, Hyaluronidase, Nicotinamide, Adenine dinucleotidase (NADase), Streptokinase) • Culture and rapid screening tests detect early infection • Sequelae include Rheumatic Fever and Acute GN

  48. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = STREPTOCOCCAL INFECTION • GROUP A STREPTOCOCCAL INFECTION • Two major sites of infection : upper respiratory tract and skin • Upper respiratory tract = sore throat, tonsillar exudate • Skin = pyoderma or impetigo • Suppurative complications = erysipelas, scarlet fever, septic arthritis, meningitis • Non-suppurative complications = RF or Post-streptococcal GN

  49. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = STREPTOCOCCAL INFECTION • GROUP A STREPTOCOCCAL INFECTION • Rheumatic Fever = only certain serotypes of S. pyogenes is involved = develops as sequelae in 2-3% untreated upper respiratory infections = symptoms occur about 18 days after sore throat = Group A streptococcus share antigenic determinants with host tissue, especially heart and even joints = inflammation of mitral valve most serious = 30-60% of patients may suffer permanent disability

  50. SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = STREPTOCOCCAL INFECTION • GROUP A STREPTOCOCCAL INFECTION B. Post-Streptococcal Glomerulonephritis = follows Streptococcal infection of skin or pharynx = occurs about 10 days following initial infection = characterized by damage to glomeruli of the kidneys = renal function impaired due to reduction in glomerular filtration rate, results in edema and HPN = renal failure not typical = one theory is damage caused by antigen-antibody complexes depositing in kidneys = complement is activated resulting in low levels

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