ch. 20 - laboratory methods Flashcards
Why take the time to identify an infectious agent?
- many bacteria are resistant to certain antibiotics
- antibiotic-resistant bacteria and viruses are spreading across the world
- specific pathogens are associated with secondary disease complications
- tracking the spread of disease can lead to its source
importance of clinical microbiology and specimen collection
- risk of sequelae with some bacterial infections
- Streptococcus pyogenes and rheumatic fever
- Staphylococcus sp. and bacterial endocarditis
- antimicrobial susceptibility should be known before antibiotics are prescribed
-empirical antibiotic treatment is often started before lab testing is completed
-antibiotic resistance - epidemiological surveillance
sequelae: a pathological condition resulting from a prior disease, injury, or attack
Specimen collection sites (humans)
- blood
- cerebrospinal fluid
- pleural fluid
- synovial fluid
- peritoneal fluid
- any tissues from internal organs
sample collection from body sites: blood cultures
- collected by venipuncture into bottles
- bacterial growth induces fluorescence
sample collection from body sites: Cerebrospinal fluid (CSF)
- collected by lumbar puncture
- direct microscopy and culture
sample collection from body sites: Pleural, synovial, and peritoneal fluid
- collected by needle aspiration
- direct microscopy and culture
Classification
placing organisms in groups of related species
- lists of characteristics of known organisms
Identification
matching characteristics of an “unknown” organism to lists of known organisms
- clinical lab identification
biochemical algorithms to identify bacteria: acid-fast bacteria
Lowenstein-Jensen medium enables growth of mycobacterial species,
- some of which grow extremely slowly
- the colonies look like bread crumbs
biochemical algorithms to identify bacteria: Gram-negative enteric bacteria
uses test strip that contains 10-20 different biochemical tests for differentiating strains within that microbial group
- color of reaction gives information
e.g. API 20NE test strip
problem-solving algorithms to identify bacteria: dichotomous keys
method of identification
- groups of organisms are divided into two categories repeatedly
taxonomic key is characteristic of a dichotomous key
- dichotomous key has paired statements in the form of “either-or”
- followed by statements to go to another pair of statements or the identification of the bacteria
automation in clinical laboratories
use machines to give accurate, rapid, and inexpensive identification of microorganisms isolated from clinical specimens
e.g.
- continuous-monitoring blood culture systems,
- automated microbial identification,
- automated antimicrobial susceptibility testing system
pathogen identification by genetic fingerprinting: Restriction fragment length polymorphism (RFLP)
form of DNA fingerprinting
- DNA is digested with restriction enzymes, run on a gel, and stained with fluorescent or radioactive probe
RFLP analysis - DNA fingerprinting steps for M. tuberculosis isolates
- restriction endonuclease cleave chromosomal DNA at restriction sites
- some DNA fragments contain insertion sequence IS6110 (marked yellow)
- exclusively Mycobacterium tuberculosis complex - gel electrophoresis separates fragments by size
- fragments containing yellow hybridize to a specific radioactive probe
- becomes visible
- a characteristic banding pattern (fingerprint) appears for each isolate
- drug resistant
- evaluation of circulating strains
- effective transmission control
pathogen identification by genetic fingerprinting: Polymerase Chain Reaction (PCR)
- most widely used molecular method in the clinical laboratory
- thermocyclers are needed to carry out PCR identification of known pathogens
- DNA primers can be made for specific pathogens
-multiple sets of DNA primers can identify individual genes from a pathogen for more specific typing - useful for pathogens that are hard to grow or slow to grow
-mycobacterium tuberculosis can take weeks to grow on standard media
-detecting its DNA in the specimen yields a rapid diagnosis
-specimen has very small amount of pathogen, difficult to detect
-PCR amplifies M. tuberculosis nucleic acid, turning one copy of DNA into billions of copies
RFLP analysis: Clostridium botulinum example
C. botulinum is divided into types based on the neurotoxin genes they possess
- 9 lanes
lane 1: DNA size markers
lanes 2-5: individual strains of C. botulinum
lanes 6-8: mixtures of strains
- lanes 6-8 are your clinical isolates
Thermocycler
instrument that precisely and rapidly cycles the temperatures required for the melting, annealing, and polymerizing steps of PCR
qRT-PCR cycle graph
- initiation phase
- starts at baseline level (no template) and fluorescence begins to increase towards the threshold as cycles occur - exponential phase
- fluorescence begins to increase exponentially
- during this phase fluorescence levels hit threshold values at Ct (threshold cycle) - plateau phase
- this is where the reaction ends and the fluorescence is stabilized
- fluorescence is at highest levels of the entire graph
note:
- this is the best choice for viral load quantification
- amount of fluorescence released = DNA amplified
qRT-PCR cycle: Ct
Ct: threshold cycle - tells you at which cycle you can start detecting fluorescent signals
- the intersection point between the amplification curve and the threshold line
- threshold level is represented in the graph by a horizontal line
the higher the initial DNA amount
- the lesser number of cycles are needed (low Ct values) to reach the threshold
pathogen identification by genetic fingerprinting: quantitative reverse transcriptase PCR (qRT-PCR)
variation of PCR used to obtain DNA copies of a specific viral mRNA molecule
- begins with the conversion of viral mRNA to cDNA by enzyme reverse transcriptase
used routinely for the high-throughput diagnosis of viral pathogens
the more cDNA present to begin with
- the fewer amplification cycles are needed to register a fluorescence increase over background
quantitative reverse transcriptase PCR steps
part 1: conversion of mRNA to cDNA by reverse transcription
1. a virus specific DNA primer is added to an RNA prep
2. reverse transcriptase (RT) copies first cDNA strand
3. RT digests and displaces mRNA and copies second strand of cDNA
4. the result is double-stranded cDNA
part 2: PCR analysis
1. the reporter probe contains a fluorescent (green) dye and a quenching dye (red) so no florescence is emitted
2. target DNA (cDNA) is denatured at 95 degrees C
3. temperature is lowered to 55 degrees C, the reporter probe anneals downstream of a DNA primer, still no fluorescence
4. temperature is raised to 72 degrees C, Taq polymerase extends upstream DNA and degrades reporter
- the release of the fluorescent dye from the vicinity of the quencher allows fluorescence, which is measured
Antigen (Ag)
foreign agents to the body that provoke an immune response
- i.e. a macromolecule that reacts with components of the immune system
- an antigen may contain several motifs that are recognized by immune cells
- viruses, parasites, bacteria, fungi, chemicals
Antibody (Ab)
immune responders produced by B cells
- bind antigens for which they are specific
antibody classes
the five immunoglobulin classes:
1. IgG monomer
2. IgM pentamer
3. secretory IgA dimer
4. IgD monomer
5. IgE monomer
antibody classes: IgG
structure: monomer
crosses placenta: yes
- only one able to
function:
1. neutralization
2. agglutination
3. complement activation
- activates immune system responses
4. opsonization
5. antibody-dependent cell-mediated cyotoxicity
antibody classes: IgM
structure: pentamer
crosses placenta: no
function:
1. neutralization
2. agglutination
3. complement activation
- triggers immune system responses
4. the monomer form serves as the B-cell receptor
- i.e. IgM monomer is the receptor for antigens on B-cells → antigen binds → antibodies are formed
- IgM monomer similar function to IgD monomer
antibody classes: IgA
structure: dimer
- has a secretory component
crosses placenta: no
function:
1. neutralization
2. trapping of pathogens in mucus
- secretory component allows for this
antibody classes: IgD
structure: monomer
crosses placenta: no
function:
1. B-cell receptor
IgD (& IgM monomer) is the receptor for antigens on B-cells → antigen binds → antibodies are formed
antibody classes: IgE
structure: monomer
crosses placenta: no
function:
1. activation of basophils and mast cells against parasites and allergens
- basically for allergies only
epitope
the specific region on an antigen where an antibody binds to
- a single antigen can have multiple epitopes for different, specific antibodies
part of antibody specificity
Immunoprecipitation
uses antibodies to precipitate antigen out of solution, need to form complex
ratios of antigen and antibody dependence
1. antigen excess or antibody excess: no complex possible
2. equivalence: complex possible
(antigenic sites equals antigen-binding sites)
immunoprecipitation: formation of complexes
complexes are formed between antigens and antigen-binding sites
situation 1: more antigens than antigen-binding sites
- no complex is possible
situation 2: antigen numbers equal the number of antigen-binding sites
- complexes are possible
situation 3: more antigen-binding sites than antigens
- no complex possible
Radial immunodiffusion assay (RIA)
immunoprecipitation test that can be used to measure the concentration of an antigen or antibody class
- in a patient’s serum
- antigen is placed in a well in agarose that contains antibody to that antigen
- diffuses out through the agarose and creates a zone of equivalence/precipitation
zone of equivalence
- point that antigen is equal to embedded antibody
Zone of precipitation
point that antigen is equal to embedded antibody
- varies with different concentrations of antigen
part of radial immunodiffusion assay (RIA)
types of antibody-antigen assays
- agglutination
- hemagglutination
both have direct and indirect mechanisms
Agglutination
agglutination or aggregation involves the cross-linking of pathogens by antibodies to create large aggregates
- antibodies, especially IgM pentamer antibodies, agglutinate bacteria by binding to epitopes on two or more bacteria simultaneously
two types of tests
1. agglutination test for antibodies
2. agglutination test for antigens
agglutination: types of mechanisms
direct
- antibody is used to clump bacterial cells or other large structures
- e.g. serotyping bacteria
indirect
- latex beads are coupled with antigen or antibody to look for antibody or antigen, respectively in patient serum
- e.g. confirming the presence of rheumatoid factor (IgM binding Ig) in patient serum
Hemagglutination
agglutination of red blood cells
- involves the clumping of red blood cells
- reactions involve red blood cell surface antigens and their complementary antibodies
red blood cells that only have the A antigen on their surface are called type A red blood cells
- Rh factor: Rh positive - red blood cells with D antigen on their surface
→ if the D antigen is not present, the red blood cells are Rh-negative
blood type: antigens on RBCs and antibodies in plasma
blood type A-negative
- A antigen
- anti-B
- anti-D (if exposed)
blood type A-positive
- A antigen
- D antigen
- anti-B
blood type B-negative
- B antigen
- anti-A
- anti-D (if exposed)
blood type B-positive
- B antigen
- D antigen
- anti-A
blood type AB-negative
- A and B antigens
- anti-D (if exposed)
blood type AB-positive
- A and B antigens
- D antigen
- no antibodies
blood type O-negative
- no antigens
- anti-A
- anti-B
- anti D (if exposed)
blood type O-positive
- Rh antigen
- anti-A
- anti-B
hemagglutination: types of mechanisms
direct
- some bacteria and viruses cross-link RBCs and clump them together
- also tests for RBC binding antibody (IgG)
- e.g. diagnosing influenza, mumps, and measles
direct Coombs’ test (DAT)
- detects nonagglutinating antibodies OR complement proteins on RBC in vivo
- e.g. checking for maternal antibodies binding to neonatal blood cells
indirect Coombs’ test (IAT)
- screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum in vitro
- e.g. performing pretransfusion blood testing
viral hemagglutination inhibition
- uses antibodies from a patient to inhibit viral agglutination
- e.g. diagnosing various viral diseases by the presence of patient antibodies against the virus
blood typing and cross-matching
- detects ABO, Rh, and minor antigens in the blood
- matches donor blood to recipient immune requirements
Coombs test
tests for hemagglutination
* checks for antibodies that attack red blood cells (IgG) in your blood
* this test may be used to screen your blood before a procedure, such as a blood transfusion
* or, it may be used to find out if you have certain conditions, such as autoimmune hemolytic anemia
Coombs reagent
detects whether RBC binding antibody (IgG) is present on RBC membranes
* antiserum containing antihuman IgG antibodies
* detects the presence of IgG bound to red blood cells
* links the IgG attached to neighboring red blood cells and thus promoted agglutination
Direct antiglobulin (Direct Coombs) test
uses hemaglutination
* test commonly done if/when baby is born with jaundice
* used to determine whether the child’s red blood cells have been bound by the mother’s antibodies
* antibodies lead blood cell lysis and the subsequent jaundice
indirect Coombs test
looks for antibodies that are floating in the blood
* if there are antibodies present in bloodstream that could attach to red blood cells
its used as a screening process to see how you’ll react to a blood transfusion
routinely given as part of prenatal testing
* screen pregnant women for antibodies that may cause hemolytic disease of the newborn
Rh incompatibility
Rh antigens are chemical groups on RBCs
- a mother who is Rh-negative can react to a fetus that inherited the Rh+ gene from the father
occurs when the mother’s blood type is Rh negative and her fetus’ blood type is Rh positive
- Antibodies from an Rh negative mother may enter the blood stream of her unborn Rh positive infant,
- damaging the red blood cells (RBCs)
Rh incompatibility steps
- the child has at least a 50% chance of inheriting the Rh+ factor
- if the father is Rh+ and the mother Rh- - during the first pregnancy, the Rh+ fetus is safe
- the Rh- mother has not yet generated any anti-Rh antibodies - during birth, Rh+ blood cells from the fetus can enter the maternal circulation
- once sensitized to the Rh+ antigen, the Rh- mother generates anti-Rh antibodies -
in a second pregnancy, Rh+ cells from the fetal circulation can occasionally enter the maternal circulation
- stimulates the mother’s memory B cells to make more anti-Rh antibodies
- the IgG antibodies can cross the placenta and attack fetal RBC’s causing them to lyse
ABO blood group system
the four major blood groups are
* A, B, AB, and O
incompatibility occurs
* specific antibodies in serum bind to antigen on foreign red blood cells (RBCs)
incompatibility example:
* individual with B blood type
* carries anti-A antibodies
* anti-A antibodies attack transfused RBCs with A antigen
* cell lysis
major human blood types and donor compatibility
blood type antigen A
* serum antibodies: anti-B
* acceptable donor blood types: A, O
* unacceptable donor blood types: B, AB
blood type antigen B
* serum antibodies: anti-A
* acceptable donor blood types: B, O
* unacceptable donor blood types: A, AB
blood type antigen AB
* serum antibodies: neither A nor B
* acceptable donor blood types: A, B, AB, or O
* unacceptable donor blood types: none
blood type antigen O
* serum antibodies: anti-A and anti-B
* acceptable donor blood types: O
* unacceptable donor blood types: A, B, AB
universal recipients
blood group AB and Rh+ (AB+)
- can receive blood from donors of any blood type
universal donors
blood type O and Rh- (O-)
- can donate blood to recipients of any blood type
neutralization
binding of specific antibodies to antigens found on bacteria, viruses, and toxins
- prevents them from attaching to target cells
e.g. neutralization of the diphtheria toxin produced by the pathogen Corynebacterium diphtheriae
- antibodies from host attach to the diphtheria toxin on the virus making them unable to attach to target cells
neutralizing substance
antitoxin-specific antibody
- a specific antibody produced by a host as it responds to a bacterial exotoxin
- or to its corresponding toxoid (inactivated toxin)
- the antitoxin combines with the exotoxin to neutralize it
viral hemagglutination inhibition assay
used to detect antibodies to a virus
- these viruses will normally cause hemagglutination when mixed with red blood cells
if antibodies to the virus are present in patient serum:
- they neutralize the virus and inhibit hemagglutination
- agglutination between antiviral antibody and virus, RBC unaffected
enzyme-linked immunosorbent assay (ELISA)
enzyme-linked antibody, - can detect antibody or antigen
- converts chromogenic (colorless) substrate to a colorful product
→ enzyme will convert the substrate to a colored product
three types:
1. direct
2. indirect
3. sandwich
direct ELISA
- patient antigen (Ag) on bottom of well
- enzyme conjugated primary antibody added
1&2 forms complex and substrate released → color change
indirect ELISA
- antigen (Ag) at bottom of well
- primary antibody from patient serum added
- secondary antibody conjugate detect antibody-antigen complex
- enzyme releases substrate, changing color
titer
measurement that expresses the antibody or antigen concentration in a solution
e.g. sandwich ELISA can detect serum quantity (titer) of IgM that binds to a specific antigen (ex: dengue virus)
sandwich ELISA (capture assay)
- capture antibody
- antigen from patient serum sample
- secondary antibody
- enzyme-conjugated antibody
a sandwich ELISA can detect the serum quantity (titer) of an IgM antibody that binds to a specific antigen
- it can also detect an antigen in a patient’s blood
sandwich ELISA: steps to detect serum quantity (titer) of IgM antibody
looking for titer of IgM antibody that binds to a specific antigen
1. bottom of plate well is coated with antibody against human IgM
2. IgM captured from patient serum
3. known antigen (dengue virus)
- sticks only to the captured anti-dengue IgM from the patient
4. antibody-enzyme conjugate
example with ebola:
1. albumin and Ebola antigen together in a section of an ELISA plate
↓ rinse off excess and add patient serum
2. human anti-Ebola antibody from patient serum binds to Ebola antigen
↓ wash off unbound serum and add conjugated antibody
3. rabbit anti-human IgG antibody with attached (conjugated) enzyme
↓ wash off unbound conjugated antibody and add substrate
4. rate of conversion of substrate to colored product is proportional to the amount of anti-Ebola antibody present in the patient’s serum
sandwich ELISA: detecting antigen in blood steps
- plate is coated with an an anti-hepatitis capture antibody (made from a rabbit)
- serum sample is added, any hepatitis antigen in serum binds to antibody
- secondary antibody (IgG) added and binds to antigen (IgG is made from a mouse)
- enzyme-linked detection antibody added and binds to secondary antibody
- substrate added
note:
- capture and secondary antibodies must be from different animals (e.g. rabbit and mouse)
- so that the detection antibody (anti-mouse) will bind only to secondary antibody
ELISA plate reader
measures absorbance
- absorbance is the amount of colored product formed
primary immune response
initial exposure to a pathogen or vaccine triggers a primary response
- specificity and memory are achieved
slower than secondary immune response
- also lower concentration of antibody
Hepatitis B: primary response lasts around 1-6 weeks
- during this IgM levels are high, no IgG
- ELISA and the immune responses are used to differentiate between acute and chronic infections of Hepatitis B
secondary immune response
follows the secondary exposure to antigen
- secondary response is specific to the pathogen in question
- e.g. varicella-zoster virus will not provide protection against other viral diseases (e.g. measles)
faster than the primary response
- provides a much higher concentration of antibody as well
Hepatitis B: secondary response around 6-9 weeks
- during this, IgG levels increase exponentially and are high
- low levels of IgM and there is a small peak around week 7
- ELISA and the immune responses are used to differentiate between acute and chronic infections of Hepatitis B
types of immunofluorescence microscopy tests
- direct immunofluorescence test
- indirect immunofluorescence test
direct immunofluorescence test
reveals pathogenic organisms in tissue
- uses fluorescently labeled anti-pathogen antibody to label bacteria from patient samples
- if the pathogen is in the tissue, it will fluoresce (visualize w microscopy)
e.g. T. pallidum on patient tissue
- fluorescently tagged anti-treponeme Ab added
indirect immunofluorescence test
reveals pathogen-specific antibodies in serum
- used to detect antigen-specific antibodies by allowing them to bind to antigen fixed to a surface
- then illuminates complexes with a secondary antibody-fluorogen conjugate
e.g. T. pallidum only on plate
- anti-treponeme Ab (patient) added
- then fluorescently tagged anti-human Ab added
fluorescent antibody (FA) techniques
direct fluorescent antibody (DFA)
- uses fluorogen-antibody conjugates to label bacteria from patient samples
- e.g. visualizing Legionella pneumophila from a throat swab
indirect fluorescent antibody (IFA)
- detects disease-specific antibodies in patient serum
- e.g. diagnosing syphilis, detecting antinuclear antibodies (ANA) for lupus and other autoimmune diseases
phage typing
test for determining to which phages a bacterium is susceptible
- highly specialized, in that they usually infect only members of a particular species or even particular strains within a species
a drop of each different phage type to be used in the test is then placed on the bacteria
- wherever the phages are able to infect and lyse the bacterial cells, there are clearings in the bacterial growth (plaques)
plaques, or areas of lysis were produced by bacteriophages
- indicates that the strain was sensitive to infection by these phages
*used to distinguish S. enterica serotypes and Staphylococcus aureus types
fatty acid profiles
bacteria synthesize a wide variety of fatty acids
- fatty acids are mostly constant for a particular species
commercial systems have been designed to separate cellular fatty acids
- this is done to compare them to fatty acid profiles of known organisms
fatty acid profiles called FAME-fatty acid methyl ester are widely used in clinical and public health laboratories
point of care laboratory tests
used directly at the site of patient care
- the result is obtained quickly in order to make appropriate treatment decisions
important to make sure POC tests are accurate and reliable
- good POC tests have high specificity and high sensitivity
POC test: sensitivity
refers to how small of a sample a test can detect
- sample containing less than that amount of antigen: false, negative reaction
good POC tests have high sensitivity
POC test: specificity
refers to how well a test can distinguish positives and negatives
- antigen from the other species could produce a false-positive reaction
good POC tests have high specificity
immunochromatographic assays (ICT)
many tests involve immunochromatographic assays (ICT)
- newer tests have higher specificity and sensitivity but require more advanced instrumentation
immunochromatographic assay (ICT) steps
- extracting the relevant antigen from a clinical specimen and placing a few drops of the extract on a test strip containing rabbit antibodies to the antigen
- antigen: C-ps derived from S. pneumoniae - antigen-antibody complexes that form move by capillary action to the upper level of the strip
- as they move up the strip they are captured by a line of more anti-antigen antibodies embedded in the strip, forming a sandwich - rabbit antibodies not bound to the antigen pass through the test line
- but they are then captured by goat anti-rabbit IgG antibodies on a second line
- once again forming a red line, as a control showing that the strip components are working
in summary:
1. drop of specimen
- C-ps from S. pneumoniae and other components
2. passes through layer of antipneumnococcal C-ps colloidal gold-labeled rabbit polyclonal antibody
3. passes through test line
- C-ps complex with antibody
4. passes through layer of immunoconjugates
5. passes through control line to the end
immunoblotting (western blot)
used to detect the presence of a specific protein in the cell extracts
- proteins are separated by size on a gel
- antibodies are used to detect the proteins of interest
immunoblotting (western blot) steps
- proteins from different samples are separated by SDS-PAGE
- each band is a different protein
- the bands shown are seen only after being stained with Coomassie blue - the gel is placed against a PVDF membrane
- an electrical field moves the buffer and proteins through the gel and onto the membrane, where the proteins stick - membrane is probed with primary antibody
- visualized with secondary antibody
