Diagnostics

General:

Around 700 million diagnostic tests are carried out by pathologists in the UK each year. Microbiology makes up a subset of these pathological tests.
There are a multitude of settings in which diagnostic testing can be conducted, including:
- Self-testing - Individual tests themselves at home (e.g. pregnancy test, HIV self test) which may result in referral
- Community testing - Individual is tested by a trained healthcare worker in a community setting (e.g. malaria, dengue, etc) for the purpose of triage and occasionally referral
- Clinic testing - Individual is tested by clinical staff (Strep A, Syphilis, etc) which results in diagnosis and treatment
- Lab testing - Individual has, for example, their blood tested in the lab (TB, HBV, HCV, etc) so that they can be adequately managed.

Sensitivity vs Specificity vs Positive/Negative Predictive Value

Sensitivity

The ability of a test to identify true positive results (those who have a condition)
Sensitivity = (True Positives)/(True Positives + False Negatives)
- This is expressed as a percentage

Specificity

The ability to identify the true negative results (those who do not have the condition)
Specificity = (True negatives)/(True Negatives + False Positives)
- This is expressed as a percentage

Positive Predictive Value

The probability of an individual who has tested positive for a condition/pathogen actually having that specific condition/pathogen.
Positive Predictive Value = (True Positives)/(True Positives + False Positives)
- This is expressed as a percentage

Negative Predictive Value

The probability of an individual who tests negative for a condition/pathogen actually not having that specific condition/pathogen
Negative Predictive Value = (True Negatives)/(True Negatives + False Negatives)

Serology

Serology is the analysis of blood serum, plasma, or occasionally saliva/CSF, for the presence of specific antibodies produced by the immune system, especially in regard to foreign and potentially pathogenic organisms.

This method of diagnostic analysis can be used to establish the immunological capacity of an individual, as well as their current response to an infection and/or their epidemiological data.

Immunoassays can be used to determine the respective levels of specific antibodies within an individual by serological means.

Enzyme Immunoassays (EIAs)

How they work:

Utilise antibodies on to identify antigens
Whilst there exist a variety of such immunoassays, they all work in a similar way; their antibodies binding specific enzymes thus allowing their variable regions to interact with the antigens in question. Following such binding enzyme substrates are introduced so that the bound antigens can be analysed.
Most EIAs use chromogenic substances as substrates for these enzymes which, when processed by their respective enzymes, produce a specific colour, thus allowing the antigen bound the enzyme-antibody complex to be identified.

Chemi-Luminescent Immunoassays (CLIA):

Some EIAs, however, utilise fluorogenic substances as their substrate, which gives off a fluorescent light signal when processed by its respective enzyme, which again allows the bound antigen to be detected. This type of EIA is called a Chemi-luminescent immunoassay (CLIA).
Some papers argue this to be a superior method to EIA, as it is of a high sensitivity and can identify smaller concentrations of the molecule in question. Also, there is a direct correlation between luminosity and concentration, thus facilitating an extra step of analysis. These reactions can also be amplified using an 'enhancer' which causes the light to be emitted for an increased duration.

Enzyme-Linked Immunosorbent Assays (ELISA):

Largely utilises the process described in 'how they work' section. Basically, antigens are suspended in within a microtiter plate well, before an antibody-enzyme complex is added. The complex then binds the antigens complementary to the antigen. A chromogenic substance is added thus facilitating a colour change which allows the identification of the target antigens.
Advantage: only uses one antibody. Disadvantage: Poorer signal generated by ELISA testing in comparison to CLIA

Western Blot Assays

Works in a very similar way to EIA. SDS-PAGE is used to separate the proteins based on molecular weight (by applying net negative charge to nullify variable charges of different proteins), before they are transferred to a specific membrane (typically PVDF/Nitrocellulose) for immunostaining.
If it is the whole, unseparated peptide (or recombinant antigens) which are transferred onto the membrane, then this is known as a Line Blot Assay.
Advantage = The researcher is able to visualise the highly specific processes behind the antibody reaction
Disadvantage = The results of this type of testing is widely considered to be more difficult to analyse

Example of Antigen/Antibody Assay: 4th Gen HIV Marker Tests

Around 7-10 following HIV infection, RNA produced by the virus is detectable in the blood of an individual via amplification. Approximately 14 days following the exposure, the antigen 'p24' is identifiable in the blood by what are known as 4th generation tests. Current technologies utilised to diagnose HIV in patients are not effective until around 5-6 weeks following exposure, as they utilise dated methods (Western Blot).

Once an individual has tested positive for p24 antibodies, they must have 2 further tests to confirm the diagnosis, one of which is a 2nd 4th gen test and the other is a HIV line blot assay. The second sample is also utilise to confirm the name, age and date of birth of the individual.

Quantitative Antibody Results Example: HepB

Following three injections of the HepB vaccine, the antibody levels are measured via a blood test:

Anti-HBs >100 IU/mL: Responder
Anti-HBs 10-100 IU/mL: Responder (give additional dose)
Anti-HBs <10 IU/mL: Non-responder to HBV vaccination

MALDI-TOF

Matrix-assisted laser desorption/ionisation-time of flight. Basically it's mass spectrometry. It utilises laser to generate ions from molecules of a greater sizer, causing low levels of fragmentation in the process. It is used typically to identify bacteria and fungi.

Process:

Sample is combined with a matrix compound and transferred to a metal surface.
Sample is then exposed to a pulsing laser, which brings about the sample's ablation and desorption
The sample is then exposed to ablated gases which have been heated to bring about ionisation of the sample molecules.
Following ionisation, the molecules are transferred into a mass spectrometry device for interpretation.

Nucleic Acid Extraction:

Basic Procedure:

Following cell collection, these cells must be lysed, typically using a protein denaturant (guanidine thiocyanate) and a protease (Proteinase K)
The lysed cells are exposed to saline in order to bring about aggregation of cell debris
Centrifugation is then used to separate the debris from the genetic material
DNA purification is conducted via one of a number of method, which include:
- Ethanol precipitation using extremely low-temperature ethanol
- Phenol-chloroform extraction: phenol-mediated protein denaturation, centrifugation to remove debris, then nucelic acid treated with chloroform to get rid of any missed phenol residues.

Commonly used samples for nucleic acid extraction include CSF, plasma, resp sputum, stool, swabs, etc.

Real Time PCR

AKA Quantitative PCR, analyses the amplification of a specific molecule of DNA during PCR instead of after the process has ended. This process is reliant on DNA labelling using fluorogenic substances. There are two common methods:

The application of non-specific fluorescent dyes into the PCR reaction which interact with DNA thus allowing the identification of specific amplicons during the procedure
The labelling of DNA using probes which are specific to a certain DNA sequence (TaqMan probes). These hydrolysis probes consist of an oligonucleotide probe with a fluorophore at one end and a 'quencher' at the other which 'quenches the fluorescence of the fluorophore. As Taq Polymerase replicates the single strand of DNA, as it encounters the TaqMan probe it degrades it, thus releasing the fluorophore and the quencher from the probe. The former is no longer inhibited and thus a fluorescent signal is generated. This signal is recognised by the quantitative PCR thermal cycler.

Reverse Transcriptase PCR

This technique combines the conversion of RNA into complementary DNA via use of a reverse transcriptase before RNase is used to degrade the RNA and PCR is used to amplify specific DNA targets.

Difference between qPCR and rtPCR: RNA has no introns which means that your final PCR product will not show the sequences of those introns, whereas in qPCR you're directly using DNA thus introns are present.

Thus, you can figure out which sections of the DNA are introns by doing both methods and then directly comparing.

Importance of Controls in PCR

No Template Control: This facilitates the identification of PCR contamination and/or primer-dimer development. This type of reaction consists of the PCR constitutents without the template. A positive result in a no template control reaction shows that there has indeed been contamination of the PCR reagents, thus suggesting that this could cause false positive results to arise.

No RT Control: In rtPCR, a PCR test without the reverse transcriptase should be conducted in order to determine whether DNA contamination has occurred. An example of contaminating DNA may be DNA from a virus which has become integrated into the genetic material of the host - this can be removed with DNase before rtPCR is conducted.

No Amplification Control: A further negative control test, the no amplification test removes DNA polymerase (e.g. Taq Polymerase) from the experiment to establish whether the probe (e.g. TaqMan probe) is stable.

Positive Control: The type of control is used to determine the effectiveness of a specific primer-probe set. This can take the form of absolute standards (e.g. genetic material from a recognised cell line) or an already known positive sample. This type of control should have an expected cycle threshold level (Ct Value), which is the amount of cycles needed to push the fluorescent response above established threshold line.

Internal Quality Control: This is a fake sample which is utilised in order to test the sample for PCR inhibiting substances.

Real Example of PCR

Basically, scientists thought that there would be an increased demand on the gastrointestinal microbiology services in London in 2012 owed to the Olympics games and fans/competitors coming from all over the world to compete (thus bringing loads of different bacteria/viruses with them from their respective countries).

As part of the preparation for this influx, scientists developed a universal method for the rapid extraction of nucleic acids from stool samples so that they could be more efficiently processed by diagnostic tools, thus saving time and thus money and allowing the microbiological services to cope better with the predicted rise in demand of their service (which, funnily, never actually happened).

This developed method utilised 'bead beating' to mechanically interrupt the stool samples before using qPCR to analyse them. This method has been subsequently shown to be useful for the isolation and identification of the Cryptosporidium spp. parasite, however has not been yet utilised for enteric viruses, despite having been proven to be 'suitable' for utilisation with such viruses.

	Created by Matthew Coulson over 4 years ago

Honours Degree Microbiology (Core Skills in Microbiology) Note on Diagnostics, created by Matthew Coulson on 30/03/2020.