diagnostics Flashcards
importance of clinical diagnostics
guide care of patients, determine appropriate treatment of infections, determine the risk of spread of infectious organisms to other patients, public, or clinic workers
specimen types
blood tissues scraps/swabs/ impressions transudate/exudate urine/misc fluids feces vomitus/sputum
guidelines for collecting specimens
- use aseptic technique– no contamination
- collect samples specific for flora (avoid environmental/ normal flora contamination)
- specimens should be taken before antibiotics/treatments are started and during acute phase of disease
acute phase
-symptoms are very pronounced, typically a strong reaction of immune system and active replication/multiplication of pathogen
most common specimens for bacterial infections– dogs
skin (dermatitis, pyoderma) ear urine wounds blood
most common specimens for bacterial infections– horses
nasal
wound
most common specimens for bacterial infections– cats
wounds
urine
ear
skin (dermatitis, pyoderma)
most common specimens for bacterial infections– dairy cows
milk
most common specimens for bacterial infection– food producing animals
post mortem tissue/organ
collection technique– dog pyoderma
LIMITS CONTAMINATION FROM NORMAL FLORA
remove fluid specimen from intact pustule & avoid contamination from bacteria on healthy skin
collection technique– cystocentesis
LIMITS CONTAMINATION FROM NORMAL FLORA
remove urine directly from bladder with sterile syringe
-avoids contamination from lower urinary tract– cystocentesis is preferred specimen for UTI diagnostics
specimens for detection of parasite infections
- feces
- vomit
- sputum
- blood
- muscle biopsy
- skin scraping
- urine
common specimens for viral infections
swabs: nasal/tracheal/sputum/eye feces blood specimen type depends upon suspected viral infection and patient symptoms also from tissue and organs
consequences of incorrect specimen collection and mishandling
incorrect/unsuccessful diagnosis due to specimen contamination/degradation/ inappropriate collection, infection of other patients/the public/ workers
handling of specimens
specimens should be handled in regard to their temp requirements, oxygen requirements, and also stored to preserve them, timing may be a key factor in the specimen’s survival
diagnostic tests– phenotypic methods
- microscopic examination (view under a microscope)
- culture/biochemical tests: grow and isolate pathogens
diagnostic tests– immunological and serological methods
- serological test blood specimen
- immunological- based diagnostics: binding of specific antibodies or antigens
molecular diagnostics
identification of markers in genome or proteome
parasite diagnosis by phenotypic tests
-direct examination from blood smear, skin scraping, other specimens can visualize +/- stain
concentration techniques
- filtration or centrifugation methods
- flotation/sedimentation techniques from feces, vomit, sputum
- Baermann test for larval identification
success of parasite diagnosis by phenotypic techniques depends upon several factors
- stage of infection (life-cycle stage of parasite), parasite type/sex, amount of parasite egg shedding
- animal age and species
- specific technique procedure
- severity of infection/parasite type (amount of egg production varies)
there are many FALSE NEGATIVES and sometimes FALSE POSITIVES, repeat tests
cytology– use microscopes for:
- cell morphology
- bacteria (+/-) morphology
- parasite (+/-) morphology
- can’t see viruses
- simple stain- 1 dye used- may not stain all components/ cells
- differential stain- more than one dye used, multistep, can distinguish between different types of cells and structures
advantages of cytology
- determine cell/tissue morphology
- cellular association of bacteria/parasites/fungi
- morphology (shape of bacteria, fungi, parasites)
- provides impression of disease stage/severity
- immediate analysis
disadvantages of cytology
- mild/chronic infection may not be readily detected
- not all samples are appropriate for cytology of bacterial infections (i.e. fecal)
H & E stain
- common tissue stain used for wide range of normal and abnormal cells and tissues
- different structures= different affinities for dyes
Romanowsky stain (Wright’s/ Giesma/ Diff-Quick)
- used to sample abnormalities (traditionally blood), bacterial and parasite infections
- Diff quick= fast and can stain multiple specimen types
Gram stain
-differentiates between Gram +/ Gram - bacteria
to culture or to not culture… that is the question
- type of infection: bacterial/fungal, virus/parasite are not cultured
- time (typically 2-10 days for result)
- expensive
- can be unidentifiable
environmental requirements bacteria
- temp: common range 20-42 C
- pH: acidophiles (<7), neutrophiles (7), alkaliphiles (>7)
- atmospheric composition- O2 and CO2
nutritional differences bacteria
- require carbon and nitrogen- differences in rqmt
- require nitrogen, phosphate, sulfate, potassium, magnesium, calcium, iron
- trace elements, purines/pyrimidines and vitamins
agar (solid)
- nutrient media- general growth
- selective media- growth of suspected pathogen type
- differential media- most are selective media and helpful in bacterial id
broth (liquid)
- nutrient broth
- enrichment broth- increase number of a specific bacterial type, inhibit growth of others
biochemical testing
- enzymes
- fermentation
helpful in pathogen ID
non-selective media
- basic nutrient media: TSA, LB agar, MH agar
- enriched nutrient media: blood agar, brain heart infusion agar, chocolate agar, lysed blood agar
fastidious bacteria
require specific nutrients and culture conditions
oxygen requirements
- anaerobic bacteria require growth in the absence of oxygen
- capnophiles require growth with carbon dioxide
selective medias
- for gram +: Phenylethyl alcohol agar
- for fungi- SabDex agar (low pH)
- for gram - :eosin methylene blue agar
- antibiotics in agar- resistant organisms will grow
differential media
- blood agar- not selective
- MacConkey Agar- gram -, lactose fermentation
- Mannitol salt agar- gram +, mannitol fermentation
- CLED agar- urinary bacteriology, lactose fermentation
enzyme production
- catalase: breaks down hydrogen peroxide
- coagulase: causes fibrin in blood to clot
- urease: hydrolyses urea
- indole: converts tryptophan into indole
- different bacteria produce different enzymes– used for species id
ID test strips/plates
- economizes space, time, and raw materials
- usually generates a unique code of results for identification
- remel rapID, sensititre ID plates, BD crystal
urine paddles: UTI culture paddles
- provides semi-quantitative colony count
- presumptive ID of many common pathogens
- paddle has two sides:
- one is EMB media for gram- neg bacteria
- one is non selective CLED media
- incubate at 37 C for 18-24 hrs for id
immunochemical tests
- detect pathogen- specific antibodies or antigens
- antigen is molecule that triggers host immune response
- antigens from pathogens can be
- the whole pathogen itself (only a small part on the surface is actually the antigen)
- molecule produced by pathogen
- pathogen molecules presented on surface of host cells
- immunochemical tests exploit principles of pathogen-specific immune response to detect and ID pathogens
Common specimens for antibodies
IgG: blood (common), tissue fluids
IgM: blood (common antibodies detected)
specimens for anigens
-primarily area of infection where pathogen replicates or antigen is present
indicators of active/recent infection
- pathogen detection (phenotypic methods or antigen)
- present or recent clinical symptoms of infection
- amount or titer of antibodies- number of antibodies circulating will decrease with time
because pathogen-specific antibodies can be detected in absence of infection may need additional diagnostics – may need to retest
ELISA (enzyme- linked immunosorbent assay)
- specific antigen or antibody detection
- detects immune response to virus, parasites, bacteria or fungi
- immune system makes unique antibodies for each pathogen encountered
- high sensitivity and specificity
- quantitative: amount of antigen or antibody present
basic ELISA assay steps
-standard plate set up
-either:
A. add serum from patient, if antibodies bind to that antigen, antibody will not be washed away
B. add specimen from patient, if antigens bind to antibodies: antigen will not be washed away
immunological based diagnostics- lateral flow immuniochromatographic assay (variation of ELISA)
- analyte flows by capillary action
- antigen binds to conjugate antibody
- conjugate antibody- antigen complex= captured by antigen specific antibodies, if antibodies bind it is positive for antibodies
- second control line for conjugate antibodies
IDEXX snap tests
- variation of lateral-flow immunochromatopgraphic assay
- detection of antigen or antibody from specimens
- works with conjugate and antibody complex
- conjugate contains enzyme that interacts with substrate molecules to give color on snap test for positive result
- can measure several infectious disease in one snap test
immunofluorescence and immunohistochemistry
- direct or indirect (uses second antibody)
- specimen: tissue or cell smear
1. primary antibody specific to antigen, binds to antigen
2. secondary antibody is specific to Fc portion of primary antibody, binds to primary antibody
3. secondary antibody is immunoflorescent– multiple secondary antibodies can bind to primary antibody, which amplifies the signal
immunological-based diagnostics
- specific antigen or antibody detection: detects immune response to virus, parasites, bacteria
- agglutinations: particles clump together (antibody and its antigen interaction)
- indirect (most common): antigen or antibody coated on beads
advantages of immunochemical tests
- ID pathogen when pathogen cannot be cultured
- most have high sensitivity
- most have high specificity
- mid to high-volume testing possible
disadvantages of immunochemical tests
- detection of antibody may not indicate active infection (titer)
- antibody detection from specimen: very early in infection may not be detected (can re-test)
- possible that antibodies may detect more than one pathogen
molecular diagnostics for infectious disease
- techniques or tests that determine pathogen ID by characteristic genetic or protein material
- uses pathogen-specific genetic sequences to ID pathogen
- can use pathogen-specific protein profile to ID pathogen
MALDI-TOF: bacterial identification
- type of mass spectrometry
- detects parts of pathogen (mostly ribosomal peptides by their mass and charge)
- each pathogen has signature pattern of fragments
- software analyzes pattern- matches pattern to its database
- takes bacterial or fungal “fingerprint”
advantages- MALDI-TOF
rapid ID, high throughput, can ID bacteria and fungi
disadvantages- MALDI-TOF
- isolated pathogen analysis
- ID is limited to reference spectra in database
- high initial cost
multiplex PCR
- detection of nucleic acid from multiple virus, bacteria, fungi, parasite species in single sample
- either PCR based or uses tagged oligonucleotide probes in digested sample
real-time PCR
- pathogen-specific sequence amplification of nucleic acid and measurement
- can be individual or multiplex
- quantitative for pathogen
- used highly for virus ID and amount of virus in specimen (quantitative)
advantages: Molecular diagnostics
- faster than culture based methods
- nucleic acid-based techniques are highly sensitive and can detect low level of pathogen
- accurate
- high volume testing possible
disadvantages: molecular diagnostics
- expensive: initial investment for equipment
- expensive reagents
- more specialized personnel to run machines
- yes/no answers
- possible false negatives/positives
on site analysis
- cytology
- point of care tests: urine paddles, parasite direct ID, IDEXX SNAP tests
diagnostic lab
- specimen culture
- antimicrobial susceptibility testing
- molecular testing and advanced immunochemical testing
good diagnostic lab…
- provides guidance for optimal specimen management (tells you how to get it to them)
- performs immunochemical and molecular methods for ID and antimicrobial susceptibility testing
- implements transparent and continuous quality assurance methods
- is accredited by national reference laboratories
- has availability of skilled microbiologists, parasitologists, and virologists for case-based expert advice and data interpretation
some potential reasons for no diagnosis
1incorrect sample collection/handling (contamination) or pathogen not present in sample
- incorrect/inappropriate diagnostic test for pathogen/stage of infection
- can be bad timing
- may need to reassess patient
