System of detection of pathogens 1

Question 1

Define what a pathogen is

Answer 1

A pathogen is an organism that causes disease.

1) Viruses:

• These are infectious particles consisting of a nucleic acid core (either DNA or RNA) surrounded by a protein coat
• Intracellular obligate parasites; rely on host cells to replicate

2) Bacteria:

• These are single-celled organisms that can exist independently of a host

3) Fungi:

• This group includes organisms like yeasts and moulds

4) Protozoa:

• These are single-celled eukaryotic organisms, many of which are parasitic and cause disease in their host

5) Helminths:

• parasitic worms

Opportunistic Pathogens: harmless under normal conditions and only become pathogenic under certain circumstances, such as when the immune system is compromised, or they gain access to parts of the body they would normally not inhabit

Question 2

Explain why not all samples that are positive are actually diagnostic of active disease

Answer 2

1) Past infection:

• Some diagnostic tests detect antibodies, which are proteins the immune system produces in response to an infection
• However, these antibodies often remain in the body long after the infection has been cleared
• Thus, a positive test result could indicate a past infection rather than an active one

2) Latent infection:

• Some pathogens, like herpesviruses or tuberculosis, can establish a latent infection
• This is a state where the pathogen remains in the body without causing symptoms but can become reactivated under certain conditions like stress thus not active

3) Carrier State:

• In some cases, an individual may carry a pathogen without developing symptoms of the disease
• This is often the case with bacteria like Streptococcus or Staphylococcus, which can be part of the normal human microbiota but can cause disease under certain circumstances

4) Non-specific test:

• Some tests are not specific to one pathogen and can yield positive results if similar pathogens are present
• This is often the case with serological tests, which detect antibodies that can cross-react with different pathogens

5) False positive:

• This can be due to technical errors or non-specific reactions in the test

Question 3

Explain why a test result depends on the quality of the sample acquired and how is handled/processed

Answer 3

1) Sample collection:

• If a sample is not collected correctly, it might not contain enough of the pathogen for detection
• For instance, a swab taken from the wrong location or not deep enough might not yield a representative sample
• Similarly, blood samples should be drawn in the correct amount and from the appropriate source (arterial, venous, or capillary)

2) Contamination:

• Improper collection and handling procedures can introduce contaminants into the sample
• These contaminants could either compete with the pathogen, reducing its detectability or could falsely indicate the presence of a pathogen

3) Sample integrity:

• Improper handling could degrade the sample, reducing the ability to detect the pathogen
• For example, some pathogens or biomarkers are sensitive to temperature, light, or mechanical stress and may degrade if the sample is not stored and transported under appropriate conditions

4) Timing:

• The timing of sample collection is also crucial. Some pathogens or disease markers are best detected at certain times during the course of the disease
• For instance, the level of certain antibodies might be too low to detect early in infection but may rise later on

5) Processing:

• Once in the lab, the sample must be processed correctly for the test to work
• This may include steps such as centrifugation to separate cells from plasma, lysis of cells to release intracellular pathogens or amplification of pathogen genetic material
• If these steps are not performed correctly, it could reduce the ability to detect the pathogen

Question 4

Describe different techniques that allow for the detection of pathogens using microscopy

Answer 4

1) Light Microscopy:

• Gram stain and acid-fast stain are often performed on bacterial cultures, allowing for the identification of bacterial cell morphology and gram reaction
• For parasitic infections, wet mount preparations can be examined for the presence of parasites or their eggs

2) Fluorescence Microscopy:

• This technique uses high-intensity light to excite fluorescent dyes or naturally fluorescent components of a sample
• Fluorescence microscopy can be highly specific and sensitive when used with antibody-based stains that target specific pathogens
• For example, fluorescent antibodies can be used to detect rabies virus in brain tissue

3) Electron Microscopy:

• This technique uses a beam of electrons instead of light to create an image
• Electron microscopy has a much higher resolution than light microscopy and can be used to visualise tiny structures such as viruses or bacterial subcellular structures
• However, it is more costly and time-consuming than other forms of microscopy

4) Confocal Microscopy:

• This technique uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal
• the major contributor to image degradation in standard fluorescence microscopy
• The result is an image with better resolution, less background, and less photobleaching than those acquired with widefield microscopy

5) Atomic Force Microscopy (AFM):

• AFM can achieve resolution similar to that of electron microscopy, but unlike electron microscopy, AFM does not require any special sample treatment that might damage the sample or prevent imaging under natural conditions

Question 5

Define different the characteristics of different bacteriological culture media and techniques that allow for the identification of pathogens

Answer 5

Culture Media:

1) Nutrient Agar:

• This is a basic culture medium used in laboratories
• It contains beef extract, peptone, and agar, which together provide the necessary nutrients (carbon, nitrogen, salts) for a wide range of microorganisms

2) Blood Agar:

• An enriched medium where red blood cells have been added
• Bacteria can be differentiated based on their hemolytic properties
• Alpha hemolysis creates a greenish halo around the colony
• beta hemolysis creates a clear halo
• gamma hemolysis shows no colour change

3) MacConkey Agar:

• This medium includes lactose and bile salts
• lactose fermenters produce red colonies due to a pH indicator in the media
• non-lactose fermenters produce colourless or pale colonies
• Bile salts inhibit non-enteric (intestinal) bacteria

4) Chocolate Agar:

• This medium is heated blood agar
• used primarily for growing fastidious respiratory bacteria like Haemophilus influenzae or Neisseria species
• which require additional growth factors (X and V factors) released from red blood cells when they are lysed by heating

Bacterial Identification Techniques:

1) Gram Staining:

• used to classify bacterial species into gram-positive and gram-negative
• based on peptidoglycan in the cell wall
• Gram-positive have thicker peptidoglycan in their cell wall so they retain crystal violet stain + iodine treatment more after wash out with decolourisation agent (alcohol) so they appear purple whereas gram-negative will be colourless and thus take in the counterstain safranin, appearing pink

2) Biochemical Tests:

• The physiological reactions of the microorganism are examined using various substrates
• Catalse: E.coli = +ve and Clostridium perfringens = -ve
• Indole test: Can cleave indole from tryptophan: E.coli = +ve and Clostridium perfringens = -ve

3) Antibiotic Sensitivity:

• It involves the placement of antibiotic-impregnated paper disks on a bacterial lawn grown on Mueller-Hinton agar
• The antibiotic diffuses, creating a gradient, and the zones of inhibition (areas without bacterial growth) are measured to determine susceptibility or resistance

Question 6

Describe and explain the concept of systematic bacteriology

Answer 6

Systematic bacteriology is the scientific study of bacteria that is focused on their classification, identification, and description,

1) Classification:

• Bacteria are classified according to their characteristics
• These can include factors such as their morphology (shape, size, staining characteristics)
• genetic makeup
• metabolic capabilities (what they can metabolize and how)
• environmental preferences (temperature, oxygen, pH)

2) Nomenclature:

• Binomial nomenclature, genus name and species name

3) Identification:

• Gram staining
• biochemical tests (which examine the metabolic capabilities of the bacteria)
• molecular techniques (such as PCR or sequencing which allow for identification based on the bacterial DNA)

4) Description:

• This involves describing the characteristics of the bacteria, such as its morphology, staining characteristics, metabolic capabilities, and where it’s typically found

Question 7

Contrast cell culture and immunofluorescence methods that allow us directly identify viruses with those, like serology, that allow us to detect their antigens

Answer 7

Cell culture and immunofluorescence methods are direct methods for identifying viruses

while serological testing allows for the indirect identification of viral antigens or the host’s immune response to viruses

1) Cell Culture:

• This is a traditional method for detecting viruses
• It involves inoculating appropriate cells with the specimen
• If the virus is present, it will infect the cells and cause changes or damage (cytopathic effect), which can be observed under a microscope
• time-consuming and requires living cells and suitable conditions for virus growth
• CON: Not all viruses can grow in culture, and the absence of cytopathic effect does not rule out the presence of a virus

2) Immunofluorescence:

• This method involves using antibodies labelled with fluorescent dyes to detect viral antigens in a sample, The fluorescence can be detected under a microscope
• Direct immunofluorescence can detect viruses in infected cells, while indirect immunofluorescence can detect specific antibodies in the patient’s serum that bind to viral antigens
• faster than cell culture, but it requires specific reagents and expertise to interpret the results

Serology:

• This involves testing a patient’s serum for antibodies against specific viruses, indicating a current or past infection
• Serology can also detect viral antigens
• include ELISA, radioimmunoassay (RIA), immunofluorescence, and Western blotting
• highly sensitive and specific, but they are indirect tests – they detect the immune response to the virus rather than the virus itself
• The presence of antibodies doesn’t always indicate an active infection, as antibodies can remain for a long time after the infection has resolved
• Also, it takes time for the body to produce antibodies after infection, so serology may not detect early infections