Diagnostic Methods Infectious Diseases

Science characterizing & measuring antibodies, antigens & other immunological substances in body fluids (primarily serum)

• Diagnose a current or acute infection
• Determine disease susceptibility or immunity

• 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

• Epstein Barr Virus
• Hepatitis A, B, C, D
• Measles
• Rubella
• Mumps
• Arbovirus
• Parvovirus B19
• Respiratory viruses
• HIV
• Hantavirus

• Specific proteins found on the surfaces of bacteria, viruses, fungi, & parasites
• Provoke an immune response
• “THEM”

• Also called immunoglobulins
• Nomenclature: Ig
• Gamma globulin part of serum proteins
• Secreted by clones of primed B cells
• “us”

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

Clumping

Forcing insolubility & settling out of solution

• 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

• 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

• 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

• 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

• 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

• Compare acute & convalescent results
• 4-fold rise in titer of paired sera collected 2-4 weeks apart verifies recent infection & is considered diagnostically significant

• 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

• 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

• 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

• Immunoprecipitation
• Complement Fixation
• Neutralization
• Particle agglutination (PA) / Agglutination inhibition (PAI)
• Immunofluorescence
• Enzyme immunoassay
• Radioimmunoassay

• 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

• 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)

• 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

• 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

• 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

• 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

• 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

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

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.

• 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

Certain other bacteria “partially” stain acid fast, e.g. Legionella, Nocardia, Rhodococcus & parasitic cysts of Cryptosporidium & Isospora

Direct examination of sputum only positive about 60% of time in patients who grow M. tuberculosis

• 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

• 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.