diagnostic priniciples Flashcards
Assessing the performance of laboratory tests
specifity and sensitivity
sensitivity formula
specifity formula
positive predictive value
higher value at higher prevalences, determines amount of real positive results
For ruling out syphilis
- Rapid plasma reagin (RPR) test
- Venereal Diseases Reference Laboratory(VDRL) test
- These tests have a low false negative rate (very sensitive)
Follow up a positive RPR or VDRL test to confirm syphilis (need a veryspecific test)
Diagnosing infections by microscopy
Direct microscopic examination of clinical specimen
Syphilis microscopy
Syphilis - spirochetes observed in scrapings of lesions
use of staining
- Bacteria in normally sterile body fluid (CSF, pleural fluid, urine)
- Staining properties part of larger effort at diagnosis
- Actual diagnosis (sometimes)
typically will not reveal genus and spp
Gram stain
- Apply the primary stain (crystal violet) to a heat-fixed smear of bacteria.
- Add a trapping agent (or mordant) (Gramsiodine).
- Decolorize with acetone or alcohol. Thick peptidoglycan layer of Gram positives keeps crystal violet-iodine complex trapped within cells. This step must be done quickly (seconds); if too long the crystal violet will be washed from both Gram-negatives and Gram positives).
- Counterstain with safranin to stain decolorized cells.
acid-fast stain (Ziehl-Neelsen method)
- Apply the primary stain (fuschin) + mordant (carbolic acid) (combination is called carbolfuchsin) to a heat-fixed smear of bacteria. Place piece of absorbant paper soaked with carbolfuchsin over the smear and heat the slide to drive the stain+mordant into cells.
- Decolorize with dilute acid in alcohol. The carbolfuchsin will wash out of most cells, but not those with high levels of mycolic acid in their membranes (e.g. mycobacteria)
- Counterstain with methylene blue to stain decolorized cells.
Antibody-based identification
Direct Fluorescent Antibody (DFA) test
Ab compostions and their specifities
monoclonal Ab to epitope would be most specific
bacterial diagnosis with culture
most bacteria vs. some special ones
most bacteria:
culturing involves use of numerous kinds of growth media
can provide preliminary information about biochemical nature of bacterium
additional biochemical tests used following isolation
some bacteria are not routinely cultured:
rickettsias, chlamydiae, and mycoplasmas
identified with special stains, immunologic tests, or molecular methods
approaches to culturing bacteria
Open-ended sampling versus looking for a particular pathogen
types of media
selective, differential, enrichment, and characteristic
selective media
prepared to facilitate the growth of some spp and inhibit others
ex: salmonella-shigella
differential media
incorporation of certain chemicals into the media for diagnostically useful growth or visible change
ex; eosin methylene blue agar
enrichment media
additon of blood/ serum to support fastidious bacteria, use mainly for the isolation of bacteria from body fluids
characteristic media
test bacteria for certain activities, products or requirements
ex; urea broth
choice of media depends on pathogen suspected to be in specimen
Bacteriophage Typing
- only done by CDC and certain labs
- based on specificity of phage surface molecules for host cell surface molecules
- phagovar– bacterium with sensitivity to certain collection of phage types
Measuring the antibody response to infection methods
ELISA and western blot
direct ELISA steps
western blot
Diagnosing infection by detecting microbial macromolecules methods
Detecting microbial antigens, Nucleic acid-based diagnosis of infection, PCR, Microarrays, Next generation sequencing
Latex agglutination test:
latex bead coated with antibody to particular pathogen material (capsular material or surface protein), will agglutinate when exposed to Ag
can have false negatives with no agglutination due to Ag surrounding the beads= prozone, corrected with dilutions
. Enzyme immunoassays for microbial antigen detection.
ELISA for Ag detection
Nucleic acid amplification (polymerase chain reaction (PCR)), basic components?
pathogen DNA, pair of pathogen-specific primers, dNTPs, stable DNA poly
real time PCRs
same as regular PCR but use a probe that is displaced with DNA synthesis causing activation of the fluorescent portion that can be detected
Microarrays
Tests for SARS-CoV-2 Virus (COVID-19)
Real-time reverse transcription-polymerase chain reaction (rRT-PCR) or
sequencing of reverse transcribed SARS-CoV-2 RNA genome regions
Use nasal swab or saliva
Sensitive and specific
Diagnosing HIV infection
Standard tests detect HIV antigen and anti-HIV antibodies
Tests for Zika Virus, positive and negative results?
For symptomatic persons suspected for Zika virus infection
1. Real-time reverse transcription-polymerase chain reaction (rRT-PCR)
Use serum or urine collected within 2 weeks after symptom onset.
A positive rRT-PCR result confirms Zika virus infection and no additional testing is indicated.
Sensitive and specific but subject to limited time window
- A negative rRT-PCR result does not exclude Zika virus infection and serum should be analyzed by IgM antibody (serological) testing.
Sensitive with longer time window but not as specific
cross-reactivity with other flaviviruses
