Diagnostics Flashcards
Diagnostic techniques
Direct examination
Culture
Immunological methods
Molecular analysis
Molecular diagnosis
DNA, RNA and proteins can be used to help identify the pathogenic agents Sensitivity (true positive rate) Specificity (true negative rate) Reduction in dependency on culture Safe
Molecular Diagnosis Examples
Electrophoresis Restriction fragment length polymorphism Hybridization and probe Nuclear acid amplification-target Protein detection- Western blot, proteomics
Electrophoresis
Separated in an electrophoretic field
Negatively charges molecules go to the positive end
Mobility
-size: the smaller, the faster
-structure: supercoiled>linear>nicked circles (DNA)
Restriction fragment length polymorphism
Analyzing differences among homologous DNA sequences by restriction enzymes
Restriction enzymes: cut DNA at the specific recognition nucleotide sequences (sequence specific)
Restriction Enzymes
Sticky End
Blunt end
Hybridization
Denatured, single-stranded DNA i.e, probe binds to a complementary single-stranded sequence
Dot, in situ, Southern, Northenr, microarray
Probe
Fragment of nucleic acids
Labeled using radioisotope, enzyme, or chemiluminescence
Detecting complementary sequences in the samples
High degree of specificity
varies in size
Nucleic acid amplification
Target amplification- enzyme-mediated process to synthesize copies of targeted nucleic acid Polymerase chain reaction (PCR) isothermal amplification, such as LAMP High sensitivity False positive
PCR primers
Single-stranded DNA fragments, complementary to sequences flanking the region to be amplified
The distance between the primer binding sites determines the size of the PCR product
Determine the specificity
Features of primers
Types: random and specific
Primers: product size, annealing temp and specificity
Nucleotide composition
PCR variations
Reverse-transcriptase PCR
Nested PCR
Multiplex PCR
Quantitative or real-time PCR
Real time PCR
Probe or dye to generate a fluorescent signal from the product
Signal in real time allows quantification of starting material
Signal- an exponential curve with a lag phase, log phase, and a stationary phase
Lag phase is inversely proportional to the amount of starting material
LAMP
Pros: no thermal cycler needed Quick- 1h sensitivity > PCR Visible results Cons: design of primer sets complicated
Western Blot
separate proteins on Page gel
Transfer
probe with antibody to detect signals
detects proteins
importance of clinical microbial diagnostics
Guide care of patients
Determine appropriate treatments for infectious diseases
Determine the risk of pathogen transmission - public heath and surveillance
Molecular testing
HT sequencing
Serology
Diagnostic process
Patient specimen collection in house analysis specimen shipment laboratory analysis data reporting and interpretation diagnosis and treatment
Physical Characteristics of infectious diseases
Blue tongue in sheep: suspected blue tongue disease
Cloudy, bad smelling urine, blood in urine, frequent urination: suspected UTI
Lethargic puppy with diarrhea anorectic, dull coat, big belly: suspected intestinal parasites
Pathognomic sings
Diagnostic sign marking the presence of a particular disease
Silver dollar plaques: edematous cutaneous patches=daurine
Specimens to collect
Tissue Scraps/swaps/impression Transudate/exudates Urine/misc fluids Feces Vomitus/sputum blood
Transudate
Watery, clear fluid pushed through capillary due to high pressure
Low protein content
few cells
Exudate
Cloudy- higher protein content and many contain some white and red cells
Inflammation based vascular permeability that is increased
What to collect
Type of specimen to collect depends upon several factors
Clinical symptoms: type of infecting pathogen, location of infection
Duration of infection
Diagnostic test to be performed/available
Common specimens for bacterial infections
Dogs and cats
Skin scrap, impressions, hair ear swab urine wound swab and fluids blood
Common specimens for bacterial infections
Horses
nasal swabs and fluids
wound swabs and fluids
Common specimens for bacterial infections
Food-producing animals
post-mortem tissue and organs
milk
Common specimens for parasitic infections
Feces Vomit sputum blood urine skin scrap muscle biopsy post mortem samples
Common specimens for viral infections
feces blood nasal, tracheal, eye swabs sputum post mortem samples
When to collect
Before starting any treatments or therapies
During the acute phase
Multiple time point collection
Detection of agent
Detection of the immune response
How to collect
Aseptic sampling techniques:
Reduce and/or avoid contamination with normal host microbiota
Avoid environmental contamination
Reduce risk on secondary infections following sampling
collection from intact pustule or cystocentesis
Cystocentesis
Remove urine directly from bladder with sterile syringe through abdominal wall
Preferred specimen collection for UTI diagnositcs
Specimen storage and transport
Correct handling and transport depends upon pathogen type, specimen type and diagnostic tests to be performed
Temperature?
Moisture?
Additive to preserve specimen and reduce contaminant growth
Proper specimen storage and transport should always aim to avoid contamination and care for biosaftey
Principles of pathogen-specific immune response
Antigen= molecule that can trigger a host immune response
-the pathogen itself (surface molecule)
-a molecule produced by the pathogen
-pathogen molecules presented on the surface of host cells
Antibodies=bind specific antigen
Why immunological methods
determine infectious agent as quickly as possible because they pose a threat to other animals or humans
Organisms that take too long for culturing
Organisms that are difficult to culture
unculturable organisms in artificial media
Sensitivity
The ability of the test to detect even very minute quantities of antigen or antibody
Specificty
The ability of the test to detect reactions between homologous Ag and Ab and with no other (minimal false positive reactions)
What to collect: serology
to detect antibodies
Blood
Tissue fluids
What to collect: serotyping
To detect antigens
Area of infection where pathogen replicates
Area of infection where antigen is present
Timing is key for accurate testing
Collect in acute phase: Ag and possibly Ab detection
Collect again (10-14 days after infection) for Ab
Indicators for an active or recent infection: pathogen detection, clinical symptoms
Presence of antibody alone may not indicate an active infection
Absence of antibody may not mean absence of pathogen
Antibody titer
Measurement of antibody quantity
Number of circulating antibodies will decrease over time
Elisa
Enzyme linked immunosorbent assay
Detects Ab response to viruses, parasites, bacteria or fungi
High sensitivity and specificity
Quantitative: indication of amount of Ag or Ab present
Direct Elisa
Antigen is bound to the solid phase, washed to remove unbound molecules and directly incubated with a conjugated antibody. After substrate addition, resulting signal is quantified
- quantitative or qualitative Ag detection in the sample
- antibody screening
- epitope mapping (since only Ab is involved)
- immunohistochemical staining of tissues and cells
Indirect elisa
Addition of a labled secondary antibody for detection on the basis of direct ELISA. The secondary antibody serves to enhance the signal of the primary antibody, which makes the test more sensitive than direct ELISA. Detect specific antibodies in serum samples
Increases signal
Sandwich ELISA
This direct or indirect assay requires a compatible antibody pair that recognize different epitopes on the same antigen. Designed for soluble antigens or at low concentration
Lateral-flow immunoassay
Diagnostic device used to confirm presence or absence of a target molecule. A variation of ELISA that is mostly qualitative or semi-quantitative User friendly, easy to interpret One-step analysis Low cost versatility of formats rapid
IDEXX SNAP
SNAP pet-side test is a variation of the lateral-flow immunoassays and uses bidirectional flow. The test includes an integrated wash step to remove debris (minimize false positives) and amplifies the result (increases sensitivity)
Different snap tests have been developed for detection of specific antigen or antibodies
Immunofluorescence
Direct or indirect (uses second antibody) assay for tissue or cell smears
-localization of pathogen (arrangement with host tissue cells)
-autoimmune diseases, viral diseases (antibody testing for heartworm)
High sensitivity and specificity
Direct Immunofluorescence
Pros: rapid single-step staining
Can use multiple antibodies from same host
Cons: no signal amplification from secondary
Each primary must be labeled individually
Indirect Immunofluorescence
Pros: secondary amplifies signal
A few labeled secondaries can detect many primaries
Cons: two-step staining
Requires antibodies from different hosts
Combined Immunofluorescence
Pros: Amplify signal for weaker targets with secondaries
Stain with multiple primaries from same species
Cons: multi-step staining
Agglutination test
Visible clumping of particulate (insoluble) Ag with its specific Ab forming visible lattice
Pros: latex test (indirect method) are most common and require no additional instruments
-easy to manufacture and use, cheap, reaction clearly visible
Cons: low sensitivity and specificity and affected by vaccine-induced Ab
Phenotypic characteristics
Observable characteristics of microorganisms
Morphology
Biochemical reactions
Concentration techniques
To increase the concentration of pathogen material
Commonly used for parasite diagnosis
-filtration or centrifugation techniques
-flotation/sedimentation techniques for feces, vomit, sputum
-baermann test for larval identification
Fecal flotation: fresh feces
detect parasites inhabiting the GI tract, liver and bile ducts
Gross examination
Fecal flotation
Based on the differences in specific gravity between the parasites eggs, larvae, cysts or oocysts and the majority of fecal debris
- qualitative examination: presence of parasite
- determine egg types
- determine existence and general level of infectionL eggs per field view
McMaster egg counting slide
Determine general egg types
Determine level of infection: eggs per gram of feces
Baermann technique
A method for the extraction of nematode larvae from fresh feces
warm water stimulates larvae in the sample to move out of it and gravity pulls them to the bottom of the container
Microscope identification
Pros: determine cell/tissue morphology
-cellular association of pathogens
-pathogen morphology
-impression of the disease stage and severity
-rapid and immediate analysis
Cons: mild/chronic infection may not readily detect
not all specimens can be used
Staining techniques: simple stain
Simple stain: one dye
-identification of morphology and cellular arrangement
Drawback: may not stain all components of the cell- difficult interpretation
Differential stain
More than one dye
Identification of morphology and cellular arrangement
Distinguish between different cell types and structures
Drawback: multistep method requires more time, reagents, and expertise
H and E
Common tissue stain used in histopathology to identify a wide range of normal and abnormal cells and tissues. Can identify bacteria, fungi, parasitic and viral infections
Key concept: different cellular structures have different affinities for the dyes dur to chemical composition
Hematoxylin
Basic stain (basic stain) stains acidic or negatively charges components– purple
Eosin
Acidic stain: stains basic or positively charges components stains also extracellular components- red/pink
viral inclusion bodies
Nuclear or cytoplasmic aggregates mostly made of proteins, that respresent the site of viral replication
Gram-stain
Common stain to differentiate between gram-positive and gram-negative bacteria based on the physical and chemical properties of their cell walls (peptidoglycan)
Gram-positive purple
Gram-negative pink
Acid fast stain
Bacteria
stains organisms with impenetrable cell wall
Capsule stain
Bacteria
Negative staining technique=contrast a translucent, darker colored background (using nigrosine or congo red) with stained cells but unstained capsule
Endospore stain
Bacteria
Spores are dyed by heating malachite green dye
Flagella stain
Bacteria
flagella are thickened with moredant
Lactophenol cotton blue stain
Parasite and fungal
Cotton blue stains chitin present in cell wall of fungi
Gomori methenamine silver
Parasite and fungal
Dark brown staining of fungal cell wall, surrounding tissue green
Periodic acid schiff
Parasite and fungal
Mucin stain
Leishman stain
Parasite and fungal
Staining blood smears- better contrast between violet nuclei and neutrophil granules
Wheatley’s trichrome stain
detection of protozoa
Diff-quick
Commonly used in histological staining for rapid staining (mainly cytoplasmic details)
Fixative (fast green in methanol)
Sample should be airdried prior to dipping into solutions
Culture methods
Different bacteria have different environmental and nutritional requirements Info used to: ensure pathogen growth from out specimen, enrich the number of pathogens for further study, determine pethogens identity
Drawbacks: time consuming, supplies, expertise, unculturable bacteria
Fastidious Bacteria environmental requirements
Temperature (common range 20-42)
pH
Atmospheric composition
Fastidious Bacteria nutritional requirements
Large amounts of C and N from different sources
Phosphate, sulfate, potassium, magnesium, calcium, iron
Trace elements, vitamins, purines/pyrimidines
Great-plate count anomoly
The term “unculturable” is used to describe bacteria that are not grown on artificial media till date (we do not have sufficient biological info to culture these in vitro)
Agar
Solid
Nutrient media: general growth
Selective media: growth of suspected agent
Differential media: most are selective and aid in ID
Broth
Liquid
Nutrient broth
Enrichment broth: increase number of specific bacteria and limit others
Culture media
Bacteria grow on solid media as colonies
Streak plate method to obtain pure cultures
Consider oxygen requiremnts
Bacterial colony
Visible mass of bacteria all originating from a single mother cell. Thus a bacterial colony constitutes a clone of bacteria all genetically alike
Basic nutrient media
Trypticase Soy Agar (TSA) Luria bertani (LB) agar Mueller hinton (MH) agar
Enriched nutrient media
blood agar
brain heart infusion (BHI) agar
chocolare agar
lysed blood agar
Phenylethyl alcohol agar
Selective for gram positive organisms
Sabouraud dextrose agar
selective for fungi
Eosin methylene blue agar
selective for gram negative organisms
Blood agar
Hemolysis
Macconkey agar
selective for gram negative lactose fermentation (Pink colonies)
Mannitol salt agar
gram positive Mannitol fermentation (yellow colonies)
CLED agar
for urinary bacteriology
Cysteine lactose electrolyte deficient
supports growth of common urinary pathogens
lactose fermentation
enzyme production
Catalase: breaks down hydrogen peroxide
Coagulase: causes fibrin in blood to clot
Urease: hydrolyses urea
Tryptophanase: ability to convert tryptophane to indole
Carbon source utilization
Simmons’ citrate agar test: differentiate gram-negative bacteria on the basis of citrate utilization as carbon source
Use of citrate and metabolization of ammonium salt–amonia production–increase pH– color change
Carbohydrate fermentation
lactose, glucose, sucrose
xylose, maltose, arabinose
commercialized in API system test strips
UTI culture paddles
semi-quantitative colony count
Presumptive id of many common uropathogens
Paddle with 2 sides, incubate 37 for 18-24h
EMB: selectove for gram negative
non selective CLED medium
Interpretation of results with guidlines
colony density–degree of infection
growth on EMB–gram negative
color on CLED–lactose fermentation
Flexicult Vet Urinary test
semi-quantitative colony count
Presumptive id of many common uropathogens
antibiotic susceptibility information of bacteria
Start test within 30 min of urine collection
incubate test at 37 for 18-24h
UTI infection? patient with symptoms and more than 100 pthogen/ml
No growth–bacteria suceptible to antibiotic
Choose good diagnostic lab
Provides guidance for optimal specimen management (selection, collection, storage, and transport of clinical samples)
Performs immunochemical and molecular methods for pathogen identification
performs antimicrobial susceptibility testing
implements transparent and continuous quality assurance measures
Is accredited by national reference laboratories
Has availability of skilled microbiologists, parasitologist, virologists, and pathologists for case based expert advice and data interpretation
standardization of test methods
Data interpretation
Diagnostic tests with yes no results: snap test multiplex PCR Agglutination tests MALDI-TOF MS
with quantitative results urine tests with cut off valves ELISA qPCR Antimicrobial susceptibility tests
False-positives
A diagnostic test is postive for pathogen or pathogen specific antibodies but the patient is not infected with that pathogen
False-negatives
A diagnostic test is negative for a pathogen or pathogen specific antibodies but the patient is infected with that pathogen
