32 Diagnosis of infection and assessment of host defense mechanisms

Question 1

What are the three broad categories that microbe testing falls into?

Answer 1

• identification by isolation/ culture. Beneficial as can perform sensitivities
• identification of specific gene/ product e.g cell wall antigen/ toxins, nucleic acid. Does not allow tetsing senitivity
• detection of antibodies to pathogen. Important when cannot be cultured e.g T pallidum/ viruses, or when culture would be dangerous to lab staff e.g tularaemia. Antibody response delayed, so more useful for retrospectic analysis than acute diagnostics

Question 2

Which sampling sites are normally sterile?

Answer 2

Blood
Bone marrow
Serous fluids
Tissues
Lower respiratory tract
Bladder
Joints

If organism found in sterile site, it is considered infective.

This can be complicated when sites have commensal pathogens

Some specimens from sites that should be sterile (e.g bladder/ sputum lower resp tract), have to pass through orifices which contaminate specimen

Question 3

What are four different applications of light microscopy?

Answer 3

Bright field - wet/ stained preparations

Dark field

Phase contrast

fluorescence - immunoflourescence

Question 4

What are uses for bright field microscopy?

Answer 4

Examine blood for organisms
ova/ cysts/ parasites stool
fungi in skin
protozoa in blood/ tissues

dyes can be used to stain cells, so they can be seen more easily

Question 5

Most important dye is Gram stain, which allows assessment of cell wall/ morphology.

What chemicals are used?

What do colours mean?

Answer 5

Crystal violet
Iodine

Then challenge with acetone.

• Gram positive remain purple
• Gram negative turn colourless, until pink counterstain applied (neutral red/ safranin

Question 6

Certain organisms such as mycobacterium do not take up Gram stain, as has waxy cell wall.

How are they visualised under light microscopy?

Answer 6

Ziehl-Neelsen stain uses heat to drive fuchsin stain into cells.

Acid and alcohol applied, which would normally decolourises other bacteria. Becuase mycobacterium retains colour, they are know as acid-fast, and alcohol-fast (AAFB)

Alternative to use fluorescent dye auramine, which has strong affinity for waxy cell wall of mycobacteria. Then use fluorescence miscroscopy

Question 7

Dark field microscopy modifies condenser so that object appears brightly lit against a dark background, making them appear larger. Living organism can be observed.

what are its uses?

Answer 7

spirochetes -/ leptospires

Question 8

How does fluoresence microscopy work?

Direct

Indirect

Answer 8

direct - antibody labelled with fluorescent dye applied to tissue, binds antigen, and fluorscence detected.

indirect - antibody (not fluorscent) binds to antigen. Fluorescent labelled anti-immunoglobulin applied which binds to antibody, and fluorescence detected

Question 9

Electron microscopy can be used as transmisison/ scanning/ immuno EM. specimen must be cut into thin sections, as even a bacterial cell wall is too thick for electrons.

Electorn beam used, and magnets used to focus beam, in a vacuum

What are uses of EM?

Answer 9

Virus identification.

Requires approx 1 million virus particles per ml to be visible

Question 10

Detection of specific microbial antigens is more rapid. Tests involve detecting antigens by their interactions with specific antibodies, or those that detect toxins

How does antigen detection work?

Useful when patient already been given antibiotics, as would fail to grow in culture.

Answer 10

Specific specimen such as CSF containing haemophilus is mixed with suspesion of latex or RBC coated with specific antibody (H. influenzae antibodies).

Interaction between antigen/ antibody causes immediate agglutination of particles, visible to naked eye

Question 11

What are examples of organisms which can be detected by detecting antigens?

Answer 11

Step pneumoniae CSF/ urine

Hameophilus influenzae type b capsule in CSF/ urine

Neisseria meningitidis capsule in CSF/ urine

Cryptococcus neofromans capsule in CSF/ urine

Strep pyogenes group antigen - throat swab

Question 12

What are examples of toxins that can be detected?

Answer 12

Clostridium botulinum - inject patient’s serum into mice

Clostridium difficile in faeces

Clostridium perfringens faeces

E. coli entertoxin

Question 13

enzyme-linked immunosorbent assay ELISA can measure antigen concentration called direct ELISA. (can also be used for antibody detection)

How does this work in antigen detection?
(direct ELISA)

ELISA is also known as EIA (enzyme immunoassays)

Answer 13

Have standard/known amount of antibody on solid phase. Wash away unbound antibody

Sample added, if antigen present, binds to antibody. Wash away unbound antigen.

• Anti-human enzyme linked antibody added. Unbound antihuman antibody is washed away. Substrate added, if enzyme present, converts to coloured product which is detected

or

• Add immunofluorescent probe, which is either:
• non-competitive assay - binds to Ag/Ab complex
• competitive - binds to unbound Ab

Measure coloured product or fluorescence with fluorescence microscope or a spectrophotometer. Can then infer antigen concentration - can give quantitative result

For example, detecting HIV p24 antigen

Question 14

ELISA can use monoclonal antibodies to detect species/ strains of certain pathogens.

What is it commonly used for?

Answer 14

HBV
HIV
HSV
Rotavirus
RSV

Chlamydia trachomatis

Question 15

enzyme-linked immunosorbent assay ELISA can measure antibody concentration called indirect ELISA. (can also be used for antigen detection)

How does this work in antibody detection?
(indirect ELISA)

Answer 15

Have standard/known amount of antigen on solid phase. Wash any unbound antigen

Add unknown antibody, which binds to antigen. Unbound antibody is then washed away.

• Anti-human enzyme linked antibody added. Unbound antihuman antibody is washed away. Substrate added, if enzyme present, converts to coloured product which is detected

or

• Add immunofluorescent probe, which is either:
• non-competitive assay - binds to Ag/Ab complex
• competitive - binds to unbound Ag

Measure coloured product or fluorescence with fluorescence microscope or a spectrophotometer.

The secondary antibody allows us to quantify how much antigen-specific antibody is present in the patient’s serum by the intensity of the color produced. - can give quantitative result

For example, detect HIV antibody

Question 16

ELISA can be used to confirm infection such as HIV antibodies.

Why can we not confirm with one single ELISA?

Answer 16

There is always concern about cross-reactivity with antibodies directed against some other antigen, which can lead to false-positive results. Thus, we cannot definitively diagnose an HIV infection (or any other type of infection) based on a single indirect ELISA assay.

We must confirm any suspected positive test, which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR (RT-PCR) or a nucleic acid antigen test

Question 17

What is a lateral flow test?

How is it performed?

Answer 17

Immunochromatographic assay - large volume fluid passed through filter, leaving on remaining imunoglobulin. Useful for very small volume antibodies

Fluids such as urine are applied to an absorbent pad on the test strip. The fluid flows by capillary action and moves through a stripe of beads with antibodies attached to their surfaces.

The fluid in the sample actually hydrates the reagents, which are present in a dried state in the stripe.

Antibody-coated beads made of latex or tiny gold particles will bind antigens in the test fluid.

The antibody-antigen complexes then flow over a second stripe that has immobilized antibody against the antigen; this stripe will retain the beads that have bound antigen.

A third control stripe binds any beads.

A red color (from gold particles) or blue (from latex beads) developing at the test line indicates a positive test. If the color only develops at the control line, the test is negative

Question 18

What are examples of tests which use lateral flow technique?

Answer 18

Rapid POC tests

Malaria
Streptococcal antigen
Beta-HCG
HIV - GEENIUS

Question 19

What are benefits/ draw backs of lateral flow POC test

Answer 19

Not quantitative
Need confirmatory test/ follow up with health professional
False- positive

Rapid
Easily performed

Question 20

Why is antibody detection a powerful tool for diagnosis?

Answer 20

Ab are highly specific, due to nature of antigen binding site on variable region. So presence can make us confident about exposure to pathogen

Antibodies last life-time, so can be sure person has had infection, just not when

Question 21

What is antibody cross-reactivity?

How does this affect sensitivity/ specificity?

Answer 21

some antigens are similar to each other

so an antibody can bind to target antigen, and also the other antigen, thereby given a false positive

Any tests involving antibodies have higher rate of false-positivity, as low specificty. Antibody tests often used as screening tests, and if positive, further confirmatory test performed. e.g check HCV IgG, if positive, check HCV RNA

Also risk of false-negatives, if patient does produce antibodies due to immunodeficiency

Question 22

How are antibodies obtained for diagnostic purposes?

Answer 22

Animal infected with antigen - produces immune response - including plasma cells/ memory B

Re-exposure to antigen - further Ab produced. This can be polyclonal, as multiple types of antibodies may be produced, which each target different part of antigen

Then harvested form serum. Purified to remove any other antibodies present which are not target

Question 23

Antibodies produced from animals are polyclonal - ie attack different epitopes on antigen.

Why might we want just monoclonal antibodies?

What are drawbacks to producing these?

Answer 23

Monoclonal has higher sensitivity and specificity.

Expensive
Time-consuming

Question 24

Polyclonal antibodies produced in vivo by live animals.

How are monoclonal antibodies produced?

Answer 24

produced in vitro using tissue-culture techniques

mAbs are produced by immunizing an animal, often a mouse, multiple times with a specific antigen.

B cells from the spleen of the immunized animal are then removed. Since normal B cells are unable to proliferate forever, they are fused with immortal, cancerous B cells called myeloma cells, to yield hybridoma cells.

All of the cells are then placed in a selective medium that allows only the hybridomas to grow; unfused myeloma cells cannot grow, and any unfused B cells die off.

The hybridomas, which are capable of growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those producing the desired mAb are grown in tissue culture; the culture medium is harvested periodically and mAbs are purified from the medium.

Mouse B cell mAb variable region then ligated to human Ab constant region to create hybrid. This step is required, because if just administered mouse antibodies, human host would quickly produce neutralising antibodies

ZMapp is human mAb which has been trialled to treat ebola

Question 25

What is precipitin reaction?

Answer 25

Test tube (in vitro) assays can help provide visual clues to antigen-ab complex formation.

Ag-Ab complex is called a precipitin.

Occurs as two Ab arms bind to epitopes, and bind to other Ab forming lattice.

Occurs more often in polyclonal Ab, as higher chance of binding to different epitopes

Question 26

What is precipitin ring test?

Answer 26

used to determine the relative amount of antigen-specific antibody in a sample of serum.

set of test tubes is prepared by adding an antigen solution to the bottom of each tube. Each tube receives the same volume of solution, and the concentration of antigens is constant (e.g., 1 mg/mL).

Next, glycerol is added to the antigen solution in each test tube, followed by a serial dilution of the antiserum. The glycerol prevents mixing of the antiserum with the antigen solution, allowing antigen-antibody binding to take place only at the interface of the two solutions.

The result is a visible ring of precipitin in the tubes that have an antigen-antibody ratio within the equivalence zone. This highest dilution with a visible ring is used to determine the titer of the antibodies.

e.g if ring visible at 1/16 dilution, suggests higher biological Ab activity that is ring only visible at 1/4 dilution

Question 27

Ouchterlony assay is similar to precipitin ring test, but is easier to perform.

How is it performed?

Answer 27

When agar is highly purified, it produces a clear, colorless gel.

Holes are punched in the gel to form wells, and antigen and antisera are added to neighboring wells.

Proteins are able to diffuse through the gel, and precipitin arcs form between the wells at the zone of equivalence.

Because the precipitin lattice is too large to diffuse through the gel, the arcs are firmly locked in place and easy to see. Looks like rings around the wells.

Provides quick answer whether antibodies present, but not much else.

Question 28

An advance on Ouchterlony assay is the radial immunodiffusion (RID assay)

How is it different?

Answer 28

Can precisely quantify antigen concentration rather than to compare different antigens

antiserum is added to agar which is poured into a small petri dish

Wells are cut in the cooled agar, and antigen is then added to the wells and allowed to diffuse.

As the antigen and antibody interact, they form a zone of precipitation. The square of the diameter of the zone of precipitation is directly proportional to the concentration of antigen.

By measuring the zones of precipitation produced by samples of known concentration, we can prepare a standard curve for determining the concentration of an unknown solution.

The RID assay is a also useful test for determining the concentration of many serum proteins such as the C3 and C4 complement proteins, among others.

Question 29

Another form of precipitin reaction, is a flocculation assay.

How is this performed?

Answer 29

A flocculation assay is similar to a precipitin reaction except that it involves insoluble antigens such as lipids

A flocculant is similar to a precipitin in that there is a visible lattice of antigen and antibody, but because lipids are insoluble in aqueous solution, they cannot precipitate.

Instead of precipitation, flocculation (foaming) is observed in the test tube fluid.

Uses for VDRL test - cardiolipin

Question 30

Original syphilis test was Wassermann test, which relied on detection of antiphospholipid antibodies that are nonspecific to T. pallidum. This had high rate of false positives due to cross-reactivity. VDRL is modified version of this.

VDRL test is form of flocculation test for syphilis. How is it performed?

Answer 30

patient serum or cerebral spinal fluid is placed on a slide with a mixture of cardiolipin (an antigenic phospholipid found in the mitochondrial membrane of host and cell wall of various pathogens), lecithin, and cholesterol.

The lecithin and cholesterol stabilize the reaction and diminish false positives.

Anti-treponemal antibodies from an infected patient’s serum will bind cardiolipin and form a flocculant.

Although the VDRL test is more specific than the original Wassermann assay, false positives may still occur in patients with autoimmune diseases that cause extensive cell damage (e.g., systemic lupus erythematosus).

Example of non-treponemal test. Measures anti-cardiolipin antibodies directed at lipid molecules, which are released from cells damaged by T pallidum

Question 31

Plaque reduction neutralisation test is used to quantify the titer of antibody to virus. Currently it is considered to be the “gold standard” for detecting and measuring antibodies that can neutralise the viruses that cause many diseases. However, much more time consuming than ELISA.

How is this performed?

Answer 31

When viruses infect cells, they often cause damage (cytopathic effects) that may include lysis of the host cells. Cytopathic effects can be visualized by growing host cells in a petri dish, covering the cells with a thin layer of agar, and then adding virus

The virus will diffuse very slowly through the agar. A virus will enter a host cell, proliferate (causing cell damage), be released from the dead host cell, and then move to neighboring cells. As more and more cells die, plaques of dead cells will form

During the course of a viral infection, the patient will mount an antibody response to the virus, and we can quantify those antibodies using a plaque reduction assay. To perform the assay, a serial dilution is carried out on a serum sample. Each dilution is then mixed with a standardized amount of the suspect virus. Any virus-specific antibodies in the serum will neutralize some of the virus.

The suspensions are then added to host cells in culture to allow any nonneutralized virus to infect the cells and form plaques after several days.

The concentration of serum to reduce the number of plaques by 50% compared to the serum free virus gives the measure of how much antibody is present or how effective it is. This measurement is denoted as the PRNT50 value

The presence of antibodies in the patient’s serum does not tell us whether the patient is currently infected or was infected in the past. Current infections can be identified by waiting two weeks and testing another serum sample. A four-fold increase in neutralizing titer in this second sample indicates a new infection.

Question 32

immunoelectrophoresis is a test primarily used to assess abundance of various protein levels.

How is this performed?

Useful to test for myeloma, but first step in western blot

Answer 32

firstly polyacrylamide electrophoresis (PAGE) is performed.

Due to its negatively charged backbone, DNA is strongly attracted to a positive electrode.

Samples are loaded into sample wells on the side of the gel closest to the negative electrode, then drawn through the molecular sieve of the agarose matrix toward the positive electrode.

The agarose matrix impedes the movement of larger molecules through the gel, whereas smaller molecules pass through more readily.

Thus, the distance of migration is inversely correlated to the size of the DNA fragment, with smaller fragments traveling a longer distance through the gel.

Sizes of DNA fragments within a sample can be estimated by comparison to fragments of known size in a DNA ladder also run on the same gel.

Immunoelectrophoresis is against selected serum proteins which are added to troughs running parallel to the electrophoresis track, forming precipitin arcs similar to those seen in an Ouchterlony assay. This allows the identification of abnormal immunoglobulin proteins in the sample.

Question 33

After performing protein gel electrophoresis, specific proteins can be identified in the gel using antibodies. This technique is known as the western blot.

how is this performed?

Answer 33

Following separation of proteins by PAGE, the protein antigens in the gel are transferred to and immobilized on a nitrocellulose membrane.

This membrane can then be exposed to a primary antibody produced to specifically bind to the protein of interest.

A second antibody equipped with a molecular beacon will then bind to the first. These secondary antibodies are coupled to another molecule such as an enzyme or a fluorophore (a molecule that fluoresces when excited by light). When using antibodies coupled to enzymes, a chromogenic substrate for the enzyme is added. This substrate is usually colorless but will develop color in the presence of the antibody. Polyclonal antibodies used, as will bind to more epitopes.

The fluorescence or substrate coloring identifies the location of the specific protein in the membrane to which the antibodies are bound (Figure 9).

Question 34

What is the basis of complement-mediated assays?

Answer 34

One of the key functions of antibodies is the activation (fixation) of complement.

When antibody binds to bacteria, for example, certain complement proteins recognize the bound antibody and activate the complement cascade.

In response, other complement proteins bind to the bacteria where some serve as opsonins to increase the efficiency of phagocytosis and others create holes in gram-negative bacterial cell membranes, causing lysis.

This lytic activity can be used to detect the presence of antibodies against specific antigens in the serum.

RBCs are good mediators to assess for complement-mediated lysis, as haemolysis increases brightly coloured pigment which can be measured. This is basis of complement fixation test

Question 35

When are complement-fixation tests useful?

Answer 35

Used to detect organisms which are difficult to culture

Viruses
Fungi
chlamydia

Question 36

What are steps of complement fixation test?

Answer 36

antigen from a pathogen is added to patient serum.

If antibodies to the antigen are present, the antibody will bind the antigen and fix all the available complement.

When red blood cells and antibodies against red blood cells are subsequently added to the mix, there will be no complement left to lyse the red cells.

Thus, if the solution remains clear, the test is positive.

If there are no antipathogen antibodies in the patient’s serum, the added antibodies will activate the complement to lyse the red cells, yielding a negative test

Question 37

In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination.

Can be used to test for antibodies against bacteria/ RBC

How is this performed?

Answer 37

To identify antibodies in a patient’s serum, the antigen of interest is attached to latex beads.

When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly (visible); this indicates the presence of the antibody

This technique is most often used when looking for IgM antibodies, because their structure provides maximum cross-linking.

To identify antigen, monoclonal antibodies are attached to latex beads, and similar process occurs.

Question 38

What are agglutination tests useful for?

What are the drawbacks?

Answer 38

Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria.

For example, the Widal test, used for the diagnosis of typhoid fever, looks for agglutination of Salmonella enterica subspecies typhi in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not as accurate as tests that involve culturing of the bacteria.

Streptolysin O Ab titre. Have latex beads with Strep Ag. Add serum. If Ab present, will bind to antibody. Dilute serum until it fails to prevent toxin from lysing RBC

False positives due to antibody cross-reaction

Patients do not produce antibodies in detectable levels for first week following infection

Question 39

Haemagglutination is aglgutination of RBC.

Commonly used in Coombs test for haemolytic anaemia.

What are uses in detecting infections?

Answer 39

Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells.

For example, influenza viruses have two different types of viral spikes called neuraminidase (N) and hemagglutinin (H), the latter named for its ability to agglutinate red blood cells (see Viruses). Thus, we can use red blood cells to detect the presence of influenza virus by direct hemagglutination assays (HA), in which the virus causes visible agglutination of red blood cells.

The mumps and rubella viruses can also be detected using HA.

Question 40

Flow cytometry is an automatic cell-counting system, which detects fluorescing cells as they pass through narrow tube one cell at a time.

Useful for HIV CD4 count, urine culture

How is it performed?

Answer 40

To check CD4 count - the analysis begins by incubating a mixed-cell population (e.g., white blood cells from a donor) with a fluorescently labeled mAb specific for a subpopulation of cells (e.g., anti-CD4).

The cells are then introduced to the flow cytometer through a narrow capillary that forces the cells to pass in single file.

A laser is used to activate the fluorogen. The fluorescent light radiates out, which is then detected by automated cell-counting system

Question 41

PCR is useful for organisms which cannot be cultured e.g

• viruses - HIV/ HBV/ HCV/ HSV/ VZV/ CMV/ enterovirus/ parechovirus
• fungi/ PCP
• bacteria - mycoplasma, chlamydia, neisseria, TB

It can be used to amplify specific DNA sequence, to produce millions of copies within a few hours

How is real-time PCR (RT-PCR) different?

Answer 41

Uses same basic method and reagents

Addition of fluorescently labelled sequence-specific probes allow reaction to be monitored in real time

amount of fluorescence is directly proportional to amount of amplified product produced

If we amplify our sample alongside a known pre-quantified DNA standard, this allows us to estimate the copy number of nucleic acid in our sample. e.g if standard sample has same fluorescence as our sample, we can work out nucleic acid levels in original sample

Certain samples such as stool have high viral load, and does not need amplification

Question 42

What reagents are required for PCR?

Answer 42

Sample nucleic acid

Buffer

Primers - can be nested primers. Usually 18-22 base pairs. This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature.

Nucleotides

Probe (one of) -

• fluorescently labelled nucleotide, which binds downstream of primer. e.g SYBR green probe
• Taqman probe with quencher. Quencher inhibits fluorescenes from Taqman. After DNA polymerase more across its path, quencher degradaes, and fluorescence can be detected

Polymerase

Question 43

Extraction provides nucleic acid in pre-amplification step. Which then requires amplification.

What are steps of PCR?

Answer 43

Denaturing - heat 95deg - strands separate

Annealing - cool 55 deg - add primers

Extension - heat 72 deg - new complementary strand synthesised by DNA polymerase, which adds new nucleotides to 3’ end

Electrophoresis to detect product - amplified DNA forms distinct bands which can be visualised under UV light, or fluorescence detected from probes

Repeat 25-30 cycles will produce over a billion copies

Question 44

Primers are short chains of oligonucleotides which provide specificity of PCR.

How does it do this?

Answer 44

Primers bind to target region on specific pathogen, and so any other contaminate material, will not be amplified.

Nested primers initially bind to longer target region. Secondary primers then added which bind to target sequence within length of first pair. This improves sensitivity and specificity of PCR

Question 45

Cultures of bacteria/ fungi can be performed in liquid media (broth) or solid media.

What are drawbacks of liquid media?

Different organisms have different growth requirements, there is not one culture media which is universal. We pick specific growth media depending on what we are looking for. This will promote suspected organism, whilst additionally inhibiting other organisms

Answer 45

More than one species can grow, which can confuse diagnosis

Question 46

Certain organisms such as virusues, chlamydia, rickettsia can only live intracellularly. Can be detected by growing in cell culture, and obersiving for cytopathic effects.

This is time consuming.

which tests have replaced this?

Answer 46

Antigen detection
Antibody detection
PCR

Question 47

Bacteria which are grown on plates, what characteristics are they classifed by?

Answer 47

• Gram stain
• Morphology
• Ability to grow aerobic/ anaerobic
• Growth requirements - simple/ fastidious
• Produce certain enzymes
• Ability to use substrates for growth e.g glucose, lactose, sucrose
• Basis of antigens reacting with antisera

Question 48

Fungi can take up to two weeks to grow in culture.

How are they identified by microscopy?

Answer 48

colour

morphology

biochemical tests

Question 49

How are protozoa/ helminth infections diagnosed?

Answer 49

Microscopy - often visible worm/ eggs

PCR becoming more common for entamoeba, giardia, cryptosporidium

Question 50

Mass spectrometry using MALDI-TOF
(matrix-assisted laser desorption ionisation time of flight)

is being used more commonly.

How is this performed?

Answer 50

Matrix contains sample

Matrix heated rapidly by laser, this causes vaporisation, which creates charged ions

Charged ions of various sizes are generated. Smaller ions move quickly to detector, followed by heavier ions which move slower through drift space.

Consequently, the time of ion flight differs according to the mass-to-charge ratio (m/z) value of the ion. The method of mass spectrometry that exploits this phenomenon is called Time of Flight Mass Spectrometry

Question 51

When assessing possible immunodeficiency, what tests are performed?

Answer 51

Immunoglobulins/ Functional Ab
Complement
HIV/ HTLV
blood film
Urine dip - proteinuria losing antibodies

Nitroblue tetrazolium - add to blood, forms complexes with heparin/ fibrinogen, then phagocytosed by neutrophils. Activated by addition of exogenous endotoxin. Dye complex taken into stimulated neutrophils, forming blue subtstrate. Useful for assessing phagocytic activity

Question 52

How does mantoux test work?

Answer 52

This assess effector T cells

Intradermal challenge of antigen gives rise to erythema and induration, peaking at 48 hour

Question 53

Extraction provides nucleic acid in pre-amplification step. Which then requires amplification.

What is process of extraction?

Answer 53

Cells lysed using bugger containing guanidium thiocynate, and proteinase K, which helps free nucleic acid.

Add silica, which binds to both DNA/ RNA, and acts as magetic material

L6 buffer can be used to remove proteinatious products (we dont want) from solution. This usually a DNAase/ RNAase depending on what we are wanting to preserve

Sample centrifuged. Supernatant containing DNA/ RNA eluted to clean tube, which can then go on to PCR

Some machines such as Panther can perform extraction and PCR on same platform

Question 54

PCR can be used to amplify RNA or mRNA.

How is RT-PCR different to regular PCR?

Answer 54

Reverse transcriptase is used to convert RNA to complementary DNA in extra step

Some platforms allow you to add reverse transcriptase to DNA polymerase and other reagents, and perform both processes together

PCR process same as original

HIV/ HCV/ measles/ influenza

Question 55

In real-time PCR, melting point is important.

What is it?

Answer 55

Melting point (Tm), is temperature at which 50% of the DNA is single strand, and 50% double stranded.

Accurate prediction of Tm identifies duplexes that are likely to form at specific temperatures, allowing you to determine appropriate thermal cycling parameters.

During the annealing phase of PCR, the reaction temperature needs to be sufficiently low to allow both forward and reverse primers to bind to the template, but not so low as to enable the formation of undesired, non-specific duplexes or intramolecular hairpins, both of which reduce reaction efficiency.

Both primers in PCR should be chosen to have a similar Tm. IDT recommends selecting an annealing temperature 5–7°C below the lowest primer Tm.

Primers with melting temperatures in the range of 52-58 oC generally produce the best results. Primers with melting temperatures above 65oC have a tendency for secondary annealing.

Question 56

What are limitations of PCR?

Answer 56

Any form of contamination of the sample by even trace amounts of DNA can produce misleading results

Primers used for PCR can anneal non-specifically to sequences that are similar, but not completely identical to target DNA.

DNA polymerase is error prone, and can incorporate incorrect nucleotides can be incorporated into the PCR sequence (at low rate)

In order to design primers for PCR, some prior sequence data is needed. Therefore, PCR can only be used to identify the presence or absence of a known pathogen or gene.

Question 57

What is multiplex RT-PCR?

Answer 57

In a singleplex relative quantification experiment, only one gene—either the gene of interest or the control—is amplified in each well. So if need multiple tests on sample, sample must be divided.

In multiplexing, you can reduce the amount of sample required for a qPCR reaction by measuring the expression of more than one gene in a reaction. The process is as sensitive and accurate as single-gene amplification (or singleplexing), but more technically complex.

Add primers which bind at different parts of nucleic acid, and do not overlap. Add probes with different fluorescence, which can be differentiated by analyser

Question 58

What are advantages of multiplex RT-PCR?

Answer 58

Conserve sample

Multiple tests on one sample - use same reagents

Cost reduction

Time reduction as tests performed together

Fewer pipetting errors

Question 59

What are disadvantages of multiplex RT-PCR?

Answer 59

Multiple simultaneous reactions in competition for same limited pool of reagents. One gene may be more abundant than others in sample, and will start amplification earlier in run than less abundant genes. It may therefore use up most of nucleotides/ other reagents before amplifcation can start on other genes. Can limit specific primers for each gene, to reduce this

Requires primers which bind to nucleic acid, but do not overlap.

Probes must be fluorescently distinct, so can distinguish between each gene presence

Question 60

What is importance of internal control, when performing PCR?

Answer 60

Internal controls are used as indicator of perfect nucleic acid extraction, quality of samples, quality of PCR. Can use negative and positive controls

Negative controls can be molecular water, which should remain negative. This helps ensure there is no cross-contamination of tests, and helps exclude false positives.

Positive control required to ensure extraction and amplification performed correctly. For example, need covid positive sample, to test that extraction and amplification was performed correctly - known as exogenous control. This helps exclude false negatives

We also need to test for human DNA, which helps confirm extraction/ amplification worked correctly known as endogenous internal control. Such as form a cheek swab, you would expect human and viral nucleic acid. This helps exclude false negatives

Question 61

What are four broad categories for virus detection?

Answer 61

Virus isolation in cell culture - slow and is not used often

Ag detection

Ab detection - serology

Nucleic acid detection

Question 62

Viral diagnostic screening test, what characteristics should it have?

Answer 62

Low chost
High throughput/ capacity
High sensitivity/ specificity

Question 63

HIV viral load testing.

At what level is it determined undetectable?

Answer 63

<40 copies per ml

Make sure test is suitable to be sensitive enough to detect this

Question 64

What is signal-to-cutoff ratio (S/CO) when analysing assay results?

Answer 64

obtained by measuring the signal strength of sample and the signal strength of an internal cutoff.

Samples with an S/Co ratio of ≥1.0 are positive, if using test where signal is proportional to input. e.g HAV IgM, HBV IgM, HIV Ab. Would expect Ab to increase in proportion to antigen

Samples with an S/Co <1.0 are negative, if using competitive/ blocking assay, e.g HBcAb total. As would expect signal to be inversely proportional to input. e.g if HbcAb not present, then S/Co ratio will increase, and result would be negative

Question 65

What are applications of molecular diagnostics?

Answer 65

Diagnose disease

Monitor disease progression/ response treatment

Assess genotype, and predict resistance/ susceptibility - DNA sequencing

Question 66

with regards to molecular diagnostics, what is meant by uni-directional workflow?

Answer 66

Specimens/ equipment in pre-amp, is kept separate from “clean” amplification room. To prevent contamination, which may give false results.

Question 67

After PCR performed, how do we analyse the result?

Answer 67

To assess if significant amount of fluorescence generated in PCR reaction, we must assess if amplification plot cross the threshold line at Ct (crossing threshold)

Question 68

DNA sequencing is new important technique for diagnosing viral infections.

Sanger invented technique in 1977, and used to sequence huamn genome 2003.

How is Sanger sequencing performed?

Answer 68

relies on dideoxynucleotides (ddNTPs),

when these bases bind to the growing DNA sequence, they terminate replication as they cannot bind other bases. T

add your primers to a solution containing the genetic information to be sequenced, then divide up the solution into four PCR reactions.

Each reaction contains a with dNTP mix with one of the four nucleotides substituted with a ddNTP (A, T, G, and C ddNTP groups).

At the end of the PCR, each of your four reactions will yield PCR products of various lengths because replication is randomly terminated. By running the samples on a gel with 4 lanes, you can piece together the sequence as each sequence has been replicated from the same original material.

Question 69

What is the process of next-generation sequencing (NGS)?

Answer 69

Fluorescently labelled chain-terminating ddNTPs are added to the PCR reaction mix.

By the Sanger sequencing method, PCR products of various lengths are created, and then separated according to their size.

Size is measured by the PCR product’s overall negative charge. The more negative the charge, the longer the fragment.

As the fragments are pulled toward the positive electrode of a capillary they pass a laser beam that triggers a flash of light from the fluorochrome attached to the ddNTP that is characteristic of the base type (for example, green for A, yellow for T, blue for G, red for C).

In this way, the genome is carefully read.

This is much faster/ cheaper compared to first generation sequencing.

Question 70

What is Stanford University database for?

Answer 70

Stanford University HIV drug resistance database is public access whereby labs upload HIV genomes.

This can be analysed, to assess for resistance patterns, and help predict drug susceptibility/ resistance based on protease, reverse transcriptase, integrase sequences

Question 71

What are benefits and drawbacks of next-generation sequencing?

Answer 71

High throughput
High sensitivity - can detect species Sanger cannot
Can detect previously unknown pathogens
Can be used for whole genome of large organisms

It is more time consuming, and difficult to set up

Question 72

Diagnostic lab can use in-house tests, or CE accredited tests.

What are advantages of in-house testing?

Answer 72

Can respond to genomic sequence variation of pathogens rapidly, by changing test. Other manufacturers will continue to sell same product

Question 73

Diagnostic lab can use in-house tests, or CE accredited tests.

What are advantages of CE accredited tests?

Answer 73

Performance will be known to certain standard

Kits usually user friendly

Less prone to contamination if instructions followed