Diagnostic Methods Infectious Diseases
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Science characterizing & measuring antibodies, antigens & other immunological substances in body fluids (primarily serum)
2 Uses of Serology in Infectious Disease
- Diagnose a current or acute infection
- Determine disease susceptibility or immunity
Why choose serology?
- Time: culture may take days to weeks
- Specimen: difficult or painful to obtain
- Availability: culture impossible
- Sensitivity/Specificity: in some instances, lower with culture
- Serological assays are particularly useful in the diagnosis of viruses, because viruses are small (Gram stain not applicable), & must use cell culture techniques, rather than traditional culture media
Viruses: Serologic Diagnosis
- Epstein Barr Virus
- Hepatitis A, B, C, D
- Parvovirus B19
- Respiratory viruses
- Specific proteins found on the surfaces of bacteria, viruses, fungi, & parasites
- Provoke an immune response
- Also called immunoglobulins
- Nomenclature: Ig
- Gamma globulin part of serum proteins
- Secreted by clones of primed B cells
Antibodies inactivate antigens by 4 main methods: Complement Fixation, Neutralization, Agglutination, Precipitation
Proteins attach to antigen surface & cause holes to form, which results in cell lysis
Binding to specific sites to prevent attachment
Forcing insolubility & settling out of solution
5 Antibody Classes
- IgA: Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents colonization by pathogens. Also found in saliva, tears, and breast milk.
- IgD: Functions mainly as an antigen receptor on B cells that have not been exposed to antigens. It has been shown to activate basophils and mast cells to produce antimicrobial factors.
- IgE: Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms.
- IgG: In its four forms, provides the majority of antibody-based immunity against invading pathogens. The only antibody capable of crossing the placenta to give passive immunity to fetus.
- IgM: Expressed on the surface of B cells and in a secreted form with very high avidity. Eliminates pathogens in the early stages of B cell mediated (humoral) immunity before there is sufficient IgG.
- 3 are formed in response to infection: IgM, IgG, IgA
IgM After Infection
- Appears in serum in 1-2 weeks
- Persists for 2-3 months
- Consistent with current or recent infection
- Largest size
- Does not cross the placenta
IgG After Infection
- Appears 2-3 weeks after infection
- May persist for life
- May represent somewhat recent infection or immunity
- Most common circulating antibody (by far)
- Only antibody that crosses the placenta
IgA After Infection
- Antibodies formed in response to infections of cells with secretory activity (cells lining the respiratory, GI and GU tracts)
- Secretory IgA is released at the infection site, then goes to bloodstream
- Not frequently measured
- Amount of antibody at a particular dilution of patient serum
- Results expressed as titer: 1:2, 1:4, 1:8, 1:16, 1:32, etc.
- Higher dilution is consistent with higher level of antibody in patient serum
Acute & Convalescent Antibody Titers
- Used to assess IgG levels at 2 phases
- Acute phase: first serum sample collected after exposure or symptom onset, may be asymptomatic
- Convalescent phase: serum sample collected 2-4 weeks later
- Simultaneous assay of both sera is most meaningful
Interpreting IgG Results in Diagnosing Acute Infection
- Compare acute & convalescent results
- 4-fold rise in titer of paired sera collected 2-4 weeks apart verifies recent infection & is considered diagnostically significant
Interpreting Antibody Results
- For some infections, a single serum result above a certain value may be significant & diagnostic of infection
- Some require additional testing to confirm a diagnosis
Serologic Diagnosis of Infections
- Advantages: Specimen (usually serum) is easy to obtain, Serologic tests are widely available, Ease of specimen transport, Serologic Diagnosis of Infections
- Disadvantages: IgG tests require acute & convalescent sera in some disease states, IgM tests can have false-positive & false-negative results, 2-3 week delay in diagnosis in infections with short incubation period
Epidemiologic vs Clinical Relevance
- Public Health Issues / Outbreak: Influenza as example; individual patient management unchanged, but serology guides vaccination for next flu season
- Treatment as Standard of Care: confirming diagnosis may not change treatment plan; treat based on history & clinical suspicion
- Protection against infectious disease by the immune response
- Generated by Immunization or Previous infection
- Lack of adequate immunity
- Possible reasons: No prior infection, No prior immunization, Waning immunity, Incompetent or compromised immune system
- Interpretation of serology results may depend on type of test/assay used
- Separation of IgM from IgG
- Concern with IgM antibody assays
- False-negative IgM result if patient has both IgM & IgG to same antigen
- False-positive IgM result if Rheumatoid Factor present, or cross-reaction Abs to other viral infections or autoimmune dz
- To prevent, perform extra step in lab to separate IgM & IgG prior to IgM assay
Antibody Detection Methods
- Complement Fixation
- Particle agglutination (PA) / Agglutination inhibition (PAI)
- Enzyme immunoassay
- Formation of insoluble complexes too large to remain in suspension
- Soluble antigens are mixed with a specific antibody
- Not commonly used in ID
- Is used to detect M-spike for multiple myeloma.
- Not frequently used.
- Inactivation (fixation) of complement after binding of complement factors to antigen-antibody immune complexes
- If bound, complement is not available to induce lysis of sheep RBCs
- Slow assay
- Examples: used to dx fungal infections, VZV
- Infectivity of a microorganism or activity of a toxin is neutralized by specific antibody
- Rarely used for diagnostic purposes
- Used for screening populations of patients from immunity or past exposure
- Example: used to test efficacy of new vaccines against viruses
Particle Agglutination / Particle Agglutination Inhibition
- PA: antigen-coated particles are mixed with patient serum dilutions then examined for macroscopic PA
- PAI: competition between antibody-coated particles and serum antibodies for a limited number of Ag-binding sites
- Simple & fast assay
- Examples: fungal infections, ABO blood typing
- Apply a dilution of patient serum to a slide containing infectious agent or cells expressing antigens of the infectious agent, then add anti-human IgG or IgM Ab conjugate, then examine microscopically or with UV light fluorescence
- Very sensitive, not very specific
- Examples: bacterial infections (Borrelia, Rickettsia, Ehrlichia), HSV, CMV, EBV, Parvovirus
- EIA, ELISA
- Indirect assay, uses an anti-human IgG or IgM antibody-enzyme conjugate
- Endpoint is color development read by spectrophotometer or eye
- Very sensitive antibody detection assay
- Commonly used to detect viruses.
- Very sensitive antibody detection assay
- Assay principle same as EIA, EXCEPT; Endpoint reading requires gamma or beta scintillation counter; Special handling & disposal of radioactive waste is required
- Specimens: serum; (occasionally other specimens, such as spinal fluid, urine or oral mucosal transudate)
Alert Values in Serology
- Lab will alert you immediately of positive results.
- Influenza & RSV EIA (in season)
- Positive HIV antibody & Western Blot
- Positive results called from CDC or NC State Laboratory of Public Health
- Includes syphilis tests such as RPR, VDRL and FTA-AVS, HIV
- Initiation of treatment prior to establishment of a firm diagnosis.
- Always obtain culture (if applicable) prior to initiation of antibiotics
- Evidence Based Medicine…know your pathogen & antibiotic susceptibility
Indigenous bacteria that reside symbiotically (protective) on human skin or mucous membranes.
Sites within human body that are considered sterile or “free” of microbes (blood, brain, muscle tissue).
- Infectious agent that resides outside host
- multiple sources in environment…water, soil, foods & other animals (also humans)
- Infectious agent resides within host’s indigenous flora (becomes pathogenic)
- eg. HIV+ patient who is immunocompromised
Eye: Aqueous/Vitreous Humor
- Culture for bacterial suspect…don’t forget viral, parasitic, fungal & AFB possibilities
- Swabs appropriate for suspicion of bacterial conjunctivitis
- Fluid in sterile container for other infections
Ear: Bacterial, Viral
- Otitis Externa (swimmer’s ear): Specimen required, aerobic swab
- Otitis Media: Usually treated empirically unless chronic infection (usually assoc. with anaerobes)
- If chronic, then tympanocentesis or mastoid bone (if obtained peri-operatively)
- Use anaerobic culture medium
Eyes, Ears & Sinuses
- Sinuses: Normally sterile
- Preceding Viral Infection most frequent
- Normal Respiratory pathogens predominate (following viral URI)
- Usually a clinical diagnosis unless severe
- Severe sinusitis cultures obtained by maxillary sinus aspiration (avoid normal flora contamination) anaerobic media
Upper Respiratory Tract
- Throat: Pharyngitis & Tonsillitis
- Most cases occur in combination with viral infections (influenzae, Epstein-Barr, or CMV)
- Primary bacterial agent is Group A Streptococcus.
- Treat to avoid sequelae of Rheumatic Fever or Glomerulonephritis
- Throat swab (aerobic) is obtained
Inflammation of tracheo-bronchial tree
Associated with viral URI almost always, exception is peds
Whooping Cough (Bordetella pertussis)
Requires special collection with wire NP (nasopharyngeal) swab & specific media in lab (or PCR test)
- Rarely Rx antibiotics
- Acute: Viral Upper Respiratory Tract Infection, so culture is not appropriate.
- Chronic: Affects 10 to 25% adults (smokers, exposures, depressed immune system). Mostly viral.
- Resist the temptation to treat with antibiotics unless absolutely indicated.
- Inflammation of lower respiratory tract involving the lung airways.
- Viral, Bacterial (Typical & Atypical), Fungal, AFB & Parasitic (immuno-compromised patients with HIV).
- Specimens: Sputum, Endotracheal Aspirates, Bronchoscopy wash/brush
- Gram Stain denotes specimen quality
Inflammation of GI tract caused by viral, parasitic, or bacterial infection or bacterial toxin.
- May require serial (2 or 3 specs) due to intermittent organism shedding
- Specimens may require additives or preservatives for certain specialized testing
- Infections classified as Exogenous (STIs) or Endogenous (imbalance of normal genital flora)
- Female Lower Tract: vulva, vagina & cervix.
- Female Upper Tract: uterus, fallopian tubes, ovaries & abdominal cavity.
- Male Lower Tract: urethra; Upper Tract epididymis, prostate or testicles.
- Transport swabs often require special media
- Urinary Tract Infections (UTIs) include Lower Tract (bladder & urethra) or Upper Tract (ureters & kidneys).
- Female anatomy favors infection
- Primarily bacteria (rarely AFB) parasitic due to genital tract contamination.
- Specs: Clean catch, catheters (indwelling or straight) supra-pubic aspirate
- Immediately refrigerate sterile containers.
- Culture inoculation is calibrated to allow quantification to define clinical significance. Complicated schematic.
- >100,000 CFU/ml of 1 or 2 organisms
- >10,000 CFU/ml of 1 pathogen (males)
- >3 orgs = mixed flora/contamination
- Depends on anatomic site & Superficial vs. Deep anatomy
- Remember Normal Flora for site that is cultured. Swab? (aer & ana) or media.
- Always sterile
- Tissue: sterility depends on location
- Tissue ALWAYS better than a swab!
- Bacteria in Bloodstream
- Transient: Incidental or Spontaneous (e.g. Dental Proc, Urine Cath, I&D of Abscess, Indwelling devices (IV, Cardiac, GI)
- Continuous: organisms consistently released (septic shock, endocarditis)
- Intermittent: Bacteria released about 45 minutes prior to febrile episode (e.g. undrained abscess)
Bacteremia causes circulatory changes that reduce tissue perfusion & result in multi-organ failure
Infection of endocardium (membrane lining of heart chambers & valves) *bacteria + “defective” valve
Web of bacteria, platelets, fibrin & inflammatory cells entrapped on heart valve seed bacteria into bloodstream @ slow, continuous rate.
- Pathogens from all 4 major groups: Bacteria (aerobic, anaerobic & fastidious organisms), fungi, parasites and viruses.
- Important to obtain 2 to 3 “sets” of Blood Cultures with fever spike 38.5 °C
- Aseptic technique to avoid contamination!
- All Body Fluids should be sterile
- Most Body Fluids collected by sterile needle aspiration of fluid
- Follow guidelines of transport media & volume of fluid necessary for specific tests based on laboratory protocol.
- Know what you are looking for & cover all your bases!
- 4 Routes of Infection
- Hematogenous spread (most common)
- Extension of infected site (otitis media)
- Anatomic defect in CNS structure (trauma or surgery)
- Direct intraneural (least common-rabies)
Inflammation of the brain parenchyma (typically viral)
- Concomitant meningitis & encephalitis (lower WBC counts than meningitis…lymphocytes)
- Often, Viral Encephalitis & Aseptic Meningitis clinically indistinguishable
- Infection within the subarachnoid space.
- 2 Categories
- Purulent: CSF contains large # of WBC’s (neutrophils)…cause is typically bacterial
- Aseptic: CSF contains large # of lympho-cytes or other mononuclear cells…usually viral & self limiting
- CSF collected by aseptic lumbar puncture…3 to 4 sterile tubes delivered immediately to lab for multiple tests.
- Appropriate handling is imperative!
- Bacterial, Fungal, AFB & sometimes parasitic studies requested in Micro.
- Gram Stain Stat, India ink vs. serology, AFB stain, wet prep for amoebas rarely
- MIC-Minimal Inhibitory Concentration
- Lowest concentration of drug that inhibits visual growth of bacteria
- Most common automated method for antibiotic susceptibility testing
- Useful in guiding antibiotic prescribing
- Resistance is “community” specific
- MICs are standardized by national agency by bacteria & achievable serum levels
- Interpretation of Susceptibility Results
- S = Susceptible – appropriate for Rx
- I = Intermediate - gray zone (e.g. certain antibiotics are “concentrated” in urine)
- R = Resistant – serum levels to “kill” aren’t achievable or due to resistance mechanisms
- Useful for bacterial cultures to distinguish gram positive & gram nega-tive infections rapidly. Remember to distinguish what is pathogenic from “normal flora” in non-sterile specimens.
- Gram stains may guide antibiotic therapy (i.e. some drugs are directed @ G+ vs. G-)
- Gram Stain - Bacteria NOT visualized
- Organisms that exist almost exclusively within host cells (obligate intracellular), e.g. Chlamydia
- Organisms that lack a cell wall, e.g. Myco-plasma & ureaplasma
- Organisms that are of insufficient dimension to be resolved by light microscopy, e.g. Spirochetes
Sputum Gram Stain
To ascertain quality of specimen (ie culture or reject)
- Primarily to detect bacteria (G- & G+), based on “classic” morphology, may “tentatively” ID bug
- Fungi/Yeasts & elements sometimes visualized
- WBCs & Epithelial cells, mucus & cellular debris also visualized.
Acid Fast Stain
- Specific for subset of bacteria whose cell walls contain long-chain fatty (mycolic) acids, which render cells resistant to decolorization with acid alcohol e.g. Mycobacterium
- Gram stain may show faintly stained “beaded” G+ rods for Mycobacterium
Acid Fast Stain
Certain other bacteria “partially” stain acid fast, e.g. Legionella, Nocardia, Rhodococcus & parasitic cysts of Cryptosporidium & Isospora
Acid Fast Stain
Direct examination of sputum only positive about 60% of time in patients who grow M. tuberculosis
India Ink Prep
- Historical staining technique for Cryptococcus neoformans (fungal infection, performed on CSF)
- Latex agglutination more sensitive & specific (India Ink <50% accurate)
- Some C. neoformans species lack capsule & thus have a false negative India Ink
- Microbiology Stains
KOH (Potassium Hydroxide) Prep
- Utilized for rapid detection of fungi by dissolving host proteins & enhancing visualization of fungal elements
- The “Traditional” Method…calcofluor white fluorescent stain is replacing KOH in some labs
- Primarily used for direct visualization of Trichomonas vaginalis (common parasitic STI malodorous, greenish vaginal discharge)
- Wet prep is still primary ID method found in most labs, serological testing is rarely used.
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