Serological Diagnosis of Infectious Diseases PDF

SEROLOGICAL 17 DIAGNOSIS OF INFECTIOUS DISEASES Group A Streptococci (S. pyogenes) Primary cause of bacterial pharyngitis and impetigo. Untreated GAS may result in sequalae: ⚬ Acute glomerulonephritis ⚬ Rheumatic heart disease Lateral flow immunochromatographic assays Strep A a...

SEROLOGICAL 17 DIAGNOSIS OF INFECTIOUS DISEASES Group A Streptococci (S. pyogenes) Primary cause of bacterial pharyngitis and impetigo. Untreated GAS may result in sequalae: ⚬ Acute glomerulonephritis ⚬ Rheumatic heart disease Lateral flow immunochromatographic assays Strep A antigen extracted from a throat swab reacts with an enzyme-labeled antibody on a test membrane Detection of Streptococcal antibodies Most diagnostically important antibodies: ⚬ Anti-streptolysin O (ASO) ⚬ Anti-DNAse B ⚬ Anti-NADase ⚬ Anti-hyaluronidase(AHase)  Streptozyme test Use to detect all diagnostically important streptococcal antibody. A form of slide agglutination screening test. Sheep RBCs are coated with streptolysin, streptokinase, hyaluronidase, DNAse, and NADase. Positive result: Hemagglutination  Anti-streptolysin O (ASO) testing ASO indicates recent streptococcal infection in patient suspected of having acute rheumatic fever or poststreptococcal glomerulonephritis following a throat infection. Classic test: ⚬ Based on the ability of antibodies in the patient's serum to neutralize the hemolytic activity of streptolysin O. ⚬ Result: The titer was reported as the reciprocal of the highest dilution demonstrating no hemolysis. ⚬ Titer can be reported as: ￭ Todd units (if streptolysin reagent standard is used) ￭ International units (if WHO international standard is used) ⚬ A single ASO titer is considered moderately elevated if the titer is at least: ￭ 240 Todd units for ADULT ￭ 320 Todd units for CHILD  Nephelometric method Currently performed test for ASO. Anti-DNAse B testing Clinically useful in patients suspected of having glomerulonephritis preceded by streptococcal skin infections. Helicobacter pylori CLOtest Detects urease activity in gastric mucosal biopsy. ⚬ Positive result = hot pink ⚬ Negative = gel will remain yellow Detection of H. pylori antibodies Serology is the primary screening method for H. pylori. Antibodies produced: IgG, IgA, and IgM Most serology test for this organism detects the IgG class. Techniques: ELISA, immunoblots, and LFA. Antibody produced are against CagA and VacA Mycoplasma pneumoniae M. pneumoniae- specific antibody detection ELISA can detect either IgG or IgM IgM indicates recent infection. IgG indicates reinfection Cold agglutinins Capable of clumping RBCs at 4 degree Celsius. A titer of 1:64 or greater, along with clinical presentation of the patient, is suggestive of M. pneumonia infection. Rickettsial infection Indirect Immunofluorescent assay Gold standard for the serological diagnosis of Rocky Mountain Spotted Fever (due to R. rickettsii) Uses to demonstrate a significant fourfold rise in antibody titers. Weil Felix test Detects Rickettsial antibodies. Based on cross-reactivity of the patient's antibodies with polysaccharide antigens present on Proteus. Sources of antigens: ⚬ Proteus vulgaris: OX-19 and OX-2 ⚬ Proteus mirabilis: OX-K  results rickettsial infection OX-19 (+) RMSF (R. rickettsii) OX-2 (+) Epidemic typhus (R. OX-K (-) prowazekii) Endemic typhus (R. typhi) OX-19 (-) Scrub typhus (0. OX-2 (-) tsutsugamushi) OX-K (+) OX-19 (-) Rickettsialpox (R. akari) OX-2 (-) Q fever (C. burnetti) OX-K(-) Trench fever (B. quintana) Syphillis Caused by Treponema pallidum subspecies pallidum. Mode of transmission: Sexual transmission, congenital, and parenteral (needles or blood). Serological tests: ⚬ Can be classified as either: ￭ Non-treponemal test ￭ Treponemal test  Non-treponemal test: Detects reagin (antibody against cardiolipin) Antigen complex consisting of cardiolipin, lecithin, and cholesterol is used in the reaction. Most widely used nontreponemal test: ⚬ Venereal Disease Research Laboratory (VDRL) ⚬ Rapid plasma reagin (RPR) VDRL and RPR are based on flocculation. Non-treponemal test: VDRL Serum specimen to be tested are heated at 56C for 30 minutes to inactivate complement. Results is read MICROSCOPICALLY A positive VDRL result obtained using spinal fluid is indicative of neurosyphilis RPR Modified VDRL. Reagent contains charcoal allowing for MACROSCOPIC reading. treponemal test: FTA-ABS Uses Nichols strain of T. pallidum Confirmatory test; specific, sensitive TP-PA Uses gel particles sensitized with T. pallidum sonicate (formerly uses sensitized sheep RBCs) Not as sensitive as FTA-ABS Lyme disease Caused by Borrelia burgdorferi. Lyme disease laboratory diagnosis is difficult because IgM antibody is not detectable by laboratory tests until 3-6 weeks after a tick bite, and IgG antibody develops later. Screening test: IFA or EIA test. Supplemental confirmatory testing: Western blot Western blot IgM positive if 2 of the following bands is present: 23 (OspC), 39, and 41 (flagellin). IgG positive if 5 out of 10 band is present. Fungal Serology Candida species Current recommendations include the combined detection of mannan and anti-mannan antibodies for the specific identification of Candida species in serum patients.  Cryptococcus neoformans Detection of cryptococcal polysaccharide antigen in serum and CSF can be performed by LA and enzyme immunoassays (EIA). False positive results are thought to be caused by rheumatoid factor. ⚬ Pretreatment with heat and Pronase, or 2-mercaptoethanol reduces false-positive ⚬ reactions.  Aspergillus species The detection of galactomannan in serum by EIA has increased the ability to diagnose invasive aspergillosis. Alternative antigen: ß-D-glucan Histoplasma capsulatum Complement fixation (CF) and precipitation have been the most common test used for detection of Histoplasma antibodies. Precipitin band testing looks for the presence of H and M antigen. viral Serology Hepatitis viruses mode of transmission Hepatitis A virus Fecal-oral route Hepatitis E virus Fecal-oral route Hepatitis B virus Parenteral route Hepatitis C virus Parenteral route Hepatitis D virus Parenteral route 1. Hepatitis A virus Belongs to the Picornaviridae family. Does not progress to chronic state. serology IgM anti-HAV (+) Acute hepatitis A IgM anti-HAV (-) Immunity via natural Total anti-HAV (+) infection or vaccination Molecular Detecting HAV in HAV RNA via RT- clinical, PCR food, or water samples 2. Hepatitis E virus Belongs to the Hepeviridae family. Can produce fulminant liver failure in pregnant women. serology IgM anti- Acute hepatitis E HEV (+) Detect patients in the later stages of infection, IgG anti-HAV determine past exposure, (+) and identify seroprevalence of the infection in a population 3. Hepatitis B virus Belongs to the Hepadnaviridae (DNA virus) family. serology HBsAg Active hepatitis B infection First marker to appear. HBeAg Active hepatitis B with high degree of infectivity Present during period of active replication. IgM anti- Current or recent acute hepatitis B HBc Useful in detecting infection in cases of hepatitis B in the core window period. Total anti- Current or past hepatitis B HBc Anti-HBe Recovery from hepatitis B Anti-HBs Immunity to hepatitis B interpretation HBsAg Total anti-HBc IM anti-HBc Anti-HBs Never infected NEG NEG NEG NEG Early acute POS NEG NEG NEG infection Acute infection POS POS POS NEG Acute resolving NEG POS POS POS/NEG infection Chronic POS POS NEG NEG infection Immune due to NEG POS NEG POS past infection Immune due to NEG NEG NEG POS vaccination 3. Hepatitis D virus Also known as delta hepatitis. Only member within the Deltavirus family. Only present in coexistence with HBV. Confection HDV is transmitted simultaneously with HBV. Superinfecti HDV is acquired by patients on who are already chronic HBV carriers. serology IgM anti- Acute or chronic DV infection HDV IgG anti- Recovery from HDV or chronic HDV HDV HDV RNA Active HDV, effectiveness of treatment 4. Hepatitis C virus Most frequent cause of chronic liver infection RNA virus belonging to the Flaviviridae family. Transmitted mainly by exposure to contaminated blood. serology Anti-HCV Current or past CV infection Current CV infection; HCV RNA effectiveness of treatment; determine HCV genotype 5. Epstein-Barr virus Causative agent of infectious mononucleosis, lymphoproliferative disorders, and several malignancies. Most common mode of transmission is via intimate contact with salivary secretions. hematology Lymphocytosis of greater than 50% of total leukocytes. At least 20% atypical lymphocytes. serology IM heterophile antibodies are IgM in nature. Disappear in most patients by 3 months after the onset of symptoms.  Davidson Differential test Patient serum is first absorbed with guinea pig kidney cells and beef erythrocyte Antibody Guinea piq Beef kidney cells RBCs Forssman antigen absorbed not absorbed antibodies Serum sickness absorbed absorbed antibodies IM antibodies not absorbed absorbed Forssman No agglutination with Guinea pig kidney cell absorbed Ab serum Agglutination with beef RBC absorbed serum Agglutination with Guinea pig kidney cell absorbed IM Ab serum No agglutination with beef RBC absorbed serum  EBV-specific antibody testing IgM anti-VCA (+) Acute primary infection Anti-EA-D (+) IgG anti-VCA (+) Convalescent or past infection Anti-EBNA (+) Anti-EBNA (+) and Reactivation increased anti-EA 6. Varicella-Zoster Virus Causes varicella (chickenpox) and herpes zoster (shingles). FAMA (Fluorescent Ab to membrane Ag) Most sensitive and reliable serological test for detecting VZV antibodies Reference method for VZV antibodies Requires live, virus infected cells. ELISA Most common method for VZV antibodies. Can be automated, provides objective result, and does not require viral culture. 7. Human Immunodeficiency Virus (HIV) Belongs to the Retroviridae family. Etiologic agent of acquired immunodeficiency virus (AIDS) HIV transmission occurs through one of three major routes: ⚬ Intimate sexual contact (predominant) ⚬ Contact with blood and other body fluids ⚬ Perinatally (from infected mother to infant) Host cells are CD4+ T-helper lymphocytes. Hallmark feature of HIV infection is a decline in the number of CD Th cells. Testing methods Detecting HIV antibodies Detecting HIV antigens Detecting HIV nucleic acid  Screening tests: Confirmatory test: ELISA Western blot 4th generation automated Ag/Ab PCR assays HIV-1,2 differentiation assays Rapid antibody test western blot Also known as immunoblot. Detects HIV antibodies For the test to be valid, negative control should produce no bands! A negative test result for the patient sample is reported if either no bands are present or if none of the bands present correspond to the molecular weights of any known viral proteins. Positive: Two of three HIV protein bands (p24,gp41, gp120/gp160)  CD4 T-cell enumeration Nucleic acid Tests Normal CD4 Th cell count ranges from 450 to 1,500 cells/uL. Early HIV detection AIDS (stage 3 HIV) = below 200 Documenting infant HIV infection cells/UL. Viral load testing Normal CD4:CD8 T cell ratio = 2:1 AIDS CD4:CD8 ratio= 1:2