my notes on Compare and contrast different general approaches for laboratory diagnosis of viral infections.

my notes on Compare and contrast different general approaches for laboratory diagnosis of viral infections.

Microscopy • Time-consuming • Not high throughput • Poor sensitivity • Poor specificity • Subjective • Training • When justifiable? • What samples? • What stains?

Microscopy Unstained preparations: “Wet prep” – Amoeba – Trichomonas vaginalis • Dark-ground illumination for syphilis – Motile spirochaetes
Microscopy Stained preparations- • Gram-stain • Acridine orange • Acid-fast stain – Ziehl-Neelsen • Fluorescence – Direct, e.g. auramine – Immunofluorescence

Factors limiting usefulness of bacteriological investigations • wrong sample – e.g. saliva instead of sputum • delay in transport / inappropriate storage – e.g. CSF • overgrowth by contaminants – e.g. blood cultures • insufficient sample / sampling error – e.g. in mycobacterial disease • patient has received antibiotics

Advantages of Solid Media • isolation of single clonal colonies – get bacterium in pure culture • identify by colonial morphology • quantification by colonyforming units • Organism available for further tests such as susceptibility testing

Disadvantages of Cultivation • Sensitivity – Only 0.1% microbes are cultivable! • Specificity – Overgrowth – Selective medium • Relevance – Multiple isolates • Conditions – Atmosphere – Temperature – Time • Slow • Infection risk

dentification of Bacteria • Morphology • Growth requirements • Biochemistry • Enzymes • Antigens • Molecular typing • Maldi-Tof
Non-cultural diagnostic methods • Antigen detection (Serology) – e.g. latex agglutination, direct immunofluorescence • Antibody detection (Serology) – e. g. agglutination tests, ELISA, indirect immunofluorescence, lateral flow assays, gamma interferon assays • Molecular methods – Polymerase Chain Reaction (PCR) – Hybridisation (microarrays, luminex) – Whole genome sequencing

Susceptibility tests • on solid media – disc diffusion technique • in liquid media – minimum inhibitory concentration (MIC) test • Breakpoint methods • E-test
Antimicrobial agents impregnated into filter paper disc – Control on same plate – Control on different plate • Break points • MIC’s • MBC’s


Susceptibility Testing Problems • Organism in tissues – drug penetration • Microbial biofilms • Mixed cultures • Adverse affect on normal flora • Microbial agent interactions • Selective toxicity • Too slow!

*!!REMEMBER TO STAY POSITIVE LIKE A PROTON!!**

Serology my introduction

serology Introduction 

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES 
•The scientific study of blood sera and their effects 
•Subdivision of immunology concerned with in-vitro Ag-Ab reaction 
•Concerned with the laboratory study of the activities of the component of serum that contribute to immunity. 

METHODS OF DETECTION OF ANTIBODIES Immuno-precipitation Assays = detect antibodies in solution = qualitative indication of the presence of antibodies = end-point is visual flocculation of the antigen and antibody in suspension

METHODS OF DETECTION OF ANTIBODIES Immunofluorescence = requires use of microscope equipped to provide ultraviolet illumination or an instrument capable of irradiating the assay with UV light and detecting resulting fluorescence with a fluorometer

IMMUNOFLUORESCENCE 
These are Ag-Ab reactions in which Ab or anti-human immunoglobulin is labelled with fluorescein. 
Fluorescein is a dye which emits greenish fluorescence under UV light. 
There are two ways for this test; 
Direct immunofluorescence (detect antigen with labelled antibody),
Indirect immunofluorescence (detect antibody with labelled anti-human immunoglobulin).

METHODS OF DETECTION OF ANTIBODIES Enzyme Immunoassay = the most sensitive = usually indirect assay that depends on the use of an antihuman IgG or IgM antibody conjugate = the antibody conjugate (if present) is made to attach to enzyme which catalyzes conversion of the substrate to a colored product which will then be read with the use of a spectrophotometer

ENZYME-LINKED IMMUNO SORBENT ASSAY (ELISA) 
This technique is ; 
Very sensitive 
 Does not require specialized equipment
  Avoid the hazards of radioactivity. 
The method depends on conjugation of an enzyme to either Ag or Ab, then substrate is added as a quantitative measure of enzyme activity.

ELISA  Indirect ELISA: 
 In this test an enzyme- labelled anti-human Ig is used to detect the presence of specific Abs in patients’ sera. 
 Known Ag is fixed by adsorption onto a plastic surface. 
 The serum sample is added (if specific Ab is present, it will bind the fixed Ag).
 Wash 
 Add the enzyme-labelled antihuman Ig 
 wash the excess 
 add the substrate, then quantitatively measure for the degree of colour change.

TO IMPROVE SPECIFICITY – WESTERN OR IMMUNOBLOTTING
 Separation of antigens by gel electrophoresis 
 Antigens blotted onto nitrocellulose
 Antigens printed directly onto nitrocellulose 
 Strips of separated antigens on nitrocellulose reacted with serum
 Washed
 React with anti-human conjugate 
 Washed
 Colour detected with substrate

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = HIV • Laboratory diagnosis of HIV infection Western Blot Testing = interpretation of result 
 no bands, negative 
 in order to be interpreted as positive a minimum of 3 bands directed against the following antigens must be present : p24, p31, gp41 or gp120/160 
 CDC criteria require 2 bands of the following : p24, gp41 or gp120/160

ROLE OF SEROLOGY IN MICROBIAL DIAGNOSTICS 
 Generally considered as the back-up when cultivation is not appropriate
 Organisms too slow to grow 
 Organism cannot be grown  Organism to hazardous to grow
 Imprecise compared to isolation
 Antigens may cross-react with those of other organisms
 Antigens may be shared between organisms (lateral gene transfer)


GOOD THINGS ABOUT SEROLOGY 
 Cheap 
 Easy to automate 
 Good for population screening 
 Can be used for several specimen types 
 Serum 
 CSF
 Synovial fluid 
 Milk 
 Can be used for retrospective diagnosis 
 Sensitive if used 10-14 days post-infection

BAD THINGS ABOUT SEROLOGY
 Poor for acute infection 
 Often require repeat samples 
 Poor if the infection is endemic 
 Poor for determination of reinfection or relapse 
 Specificity variable (depending upon antigen) 
 Vaccination can complicate results 
 Early treatment can interfere with development of positive response 
 Risk of dealing with blood samples (bloodborne pathogens)

**!!REMEMBER TO STAY POSITIVE LIKE A PROTON!!**

What are Zoonoses?

Zoonoses: Infections acquired from animals. By direct contact. Indirect contact by vector. Indirect contact through environmental contamination.

Zoonosis refers to diseases that can be passed from animals to humans. They are sometimes called zoonotic diseases. Animals can carry harmful germs, such as bacteria, fungi, parasites, and viruses. These are then shared with humans and cause illness
Animals can carry harmful germs, such as bacteria, fungi, parasites, and viruses. 

These are then shared with humans and cause illness. Zoonotic diseases range from mild to severe, and some can even be fatal.

Before the introduction of new hygiene regulations around 100 years ago, zoonotic diseases such as bovine tuberculosis, bubonic plague, and glanders caused millions of deaths. They are still a major problem in developing countries.

Rabies
Rabies is a disease that affects the nervous system of mammals. It is usually caused by a virus and is transmitted if an infected animal bites a person or other animal.
Rabies is almost always fatal once symptoms appear. However, rabies vaccines exist and are commonly available.

Lyme Disease
Lyme disease and Rocky Mountain spotted fever
Lyme disease is transmitted through tick bites. Symptoms can range from mild to severe, but it can be treated using antibiotics.
Dengue, malaria, and chikungunya
These are mosquito-borne diseases and are more common in certain areas, such as the Caribbean.
Symptoms include fever, vomiting, and headaches. It is vital to treat these conditions as soon as possible, as they can be fatal.

Salmonella infection
Salmonella is often caused by handling reptiles or amphibians that carry Salmonella, or by handling baby chicks or ducks.
The illness usually lasts for between 4 and 7 days, and symptoms include diarrhea, fever, and abdominal cramps. People can usually recover without medical treatment, although conservative measures are recommended.
E. coli infection
This infection is often caused by touching infected animals or handling contaminated food. Cows also have E. coli germs on their udders.

**!!REMEMBER TO STAY POSITIVE LIKE A PROTON!!**

How microorganisms enter a host and how they damage host cells with exotoxins?

How microorganisms enter a host and how they damage host cells with exotoxins?

To cause disease most pathogens must: 
 gain access to the host 
 gain access to the tissue
 evade host defences 
 damage the host tissue 

• Some microbes cause disease without invading the body • Portals of entry - gaining entrance to the human body (and other hosts) through several avenues
Adhesins/ligands bind to receptors on host cells
The host cell cytoskeleton provides the mechanism: • Actin is used:  to penetrate host cells  to move through and between host cells
Invasins – Salmonella alters host actin to enter a host cell
Use actin to move from one cell to the next – Listeria
Cadherin: bridges membrane junctions
Exotoxins 

• Antibodies: 
 provide immunity to exotoxins • Inactivated exotoxins: 
 can no longer cause disease 
 can still stimulate the body to produce exotoxins 

• These altered exotoxins are called toxoids 
 toxoids (as vaccines) stimulate antitoxin production (immunity) 
 Vaccines – stimulate antitoxin production
Exotoxins 

• Are named on the basis of many characteristics: 
 neurotoxins - nerve cells 
 cardiotoxins - heart cells
 hepatotoxins - liver cells 
 leukotoxins - leukocytes 
 enterotoxins - the lining of the gastrointestinal tract 
 cytotoxins - a wide variety of cells diphtheria toxin (cause of diphtheria) botulinum toxin (Clostridium botulinum) vibrio enterotoxin (Vibrio cholerae)

**REMEMBER TO STAY POSITIVE LIKE A PROTON!!**

viral infections

viral infections 

A virus (from the Latin virus meaning toxin or poison) is a sub-microscopic infectious agent that is unable to grow or reproduce outside a host cell.”
Acute virus infection:
Following host entry the virus multiplies and spreads rapidily. and contact with the virus activates host immune responses. Hosts immune mechanisms overcome and eliminate virus. The hosts retains immunological virus and virus survival dependent on rapid dissemination and susceptible hosts aviabliliy.

Characteristics of actute virus disease:
-normally rapid onset.
-symptoms often corresponds with peak viral multiplication.
-often fever and mygalia (from activation of immune response)
-vary in servity from one individual to another.
-Resolve rapidly once the innate immune response develops.

Acute viral infections are serve public health problems.
-usually associated with epidemics
-short incubation period
-delay in indentifibile symptoms until the virus has already spread.
-Acute infections occur in crowded places e.g schools, militariy, nursing homes.

EXAMPLE OF ACUTE VIRAL INFECTION:
RHINOVIRUSES (INFLUENZA)
-common cold often caused by rhinoviruses more than 110 serological types. killed by gastric acid and replicated at 33'C.
-Bind to ICAM-1 on epithelial cell surface and are not easily removed by muco-ciliary secretions.
-enter host cells by translocation
-Lyse host cells after replication and spread to neighbouring cells leading to progressive and extensive damage.
Pathogenesis of the common cold:
-debris from lysed cells provokes a strong inflammatory response
-increased secretion of fluids from lamina propria leads to runny nose and sneezing.
-Phagocytes migrate to to site of infection.
-Release of inflammatory mediators leads to blocked nose and mild fever
-Symptoms are restricted to the upper part of the respiratory tract as these viruses multiply optimal at 33'C and less at 37'C.

Immunity to rhinoviruses
-Rhinoviruses recognises by immune system specific mucosal response -IgA ect rather than humoral response
-immune response quickly clears the infection leading to rapid recovery. (couple of days)
-Immune memory of the infection is retained but is not long lasting as for systematic virus infection.
-frequent reoccurrence of the common cold is due to the larger number >100 of serological distinct strains of rhinoviruses.
EXAMPLE OF ACUTE VIRAL INFECTION: ENTEROVIRUSES.  (MEASLES)

**REMEMBER TO STAY POSITIVE LIKE A PROTON!!**  

positive and negative T cell selection in the thymus.

positive and negative T cell selection in the thymus

 A lymphoid organ is situated in the neck of vertebrates which produces T-lymphocytes for 
the immune system. The human thymus becomes much smaller at the approach of puberty.  

Cortex – immature thymocytes, epithelial cells and scattered macrophages.
Medulla- mature thymocytes, epithelial cells, macrophages and dendritic cells.
T Cell development  in the thymus: 
Step 1: T- cell progenitors develop in the bone marrow and migrate to the thymus-  T cell precursors rearranges its t cells receptor genes in the thymus. 

Step 2: positive and negative selection in the thymus- Immature T cells that recognise self MHC receive signals for survival. Those that interact strongly with self antigen are removed from the repertoire. 

Step 3: Mature T cells migrate to the peripheral lymphoid organs- Mature T cells encounter foreign antigens in the peripheral lymphoid organs are activated. 

Step 4: Activated T cells migrate  to sites of infection- Activated T cells proliferate and eliminate infection. 

T cells move through a number of different developmental stages classified by expression of CD4 and CD8 on their cell surface: They are:
• DN (CD4-8-)
• DP (CD4+8+)
• CD4SP or CD8SP




T cell precursors proliferate extensively in the thymus, but most die there. 95% of thymocytes die by apoptosis before reaching the medulla. Some die due to NEGLECT. 
NEGLECT – no TCR-MHC interaction = NO SURVIVAL SIGNAL
• Positive selection of thymocytes: development of the self MHC-restricted T cell repertoire.  The MHC type of the thymic stroma selects a repertoire of mature T cells that can recognize foreign antigens presented by the same MHC type- self restriction. 
• Only thymocytes whose TCR interact with self-peptide: self-MHC complexes weakly can survive and mature.
• Those that do not recognize the MHC molecules are killed by apoptosis (death by neglect) 
Positive selection is mediated by MHC:peptide on thymic epithelial cells 
Peptides role in positive selection: 
• Peptides presented in the thymus are self –peptides derived from widely expressed 
• Self-peptides presented cause stabilisation of MHC on epithelial cells 
• Self-peptides presented on epithelial cells influence the specificity of the T cells that are selected
• Some self peptides are better than other at positive selection
• Different peptides result in different T cell repertoires
Positive selection coordinates the expression of CD4 or CD8 with the specificity of the T-cell receptor and the potential effector functions of the T cell. This is known as lineage commitment

The Double positive (DP) thymocytes
• DPs initially express low levels of abTCR 
• 10-30% of TCRs recognise cortical epithelial cells expressing ‘self peptide’ presented by ‘self MHC’ – POSITIVE SELECTION
• Positive selection acts on a repertoire of T-cell receptors with inherent specificity for self MHC molecules.
Negative selection removes thymocytes that are capable of strongly binding with "self" MHC peptides. Thymocytes that survive positive selection migrate towards the boundary of the cortex and medulla in the thymus.The remaining cells exit the thymus as immature naïve T cells.

Negative selection of thymocytes: Central Tolerance
• T cells that react strongly (high avidity/strong interaction) with ubiquitous self antigens are deleted in the thymus or become regulatory T cells
• Can occur at the DP or SP stages 
• Can be induced by bone marrow derived antigen-presenting cells in the cortex and medulla such as DCs and macrophages
• thymic medullary epithelial cells can also induce negative selection 

Negative selection is driven by bone marrow derived antigen-presenting cells (DCs and macs) and medullary epithelial cells.

**!!REMEMBER TO STAY POSITIVE LIKE A PROTON!!**

INTERVIEW QUESTIONS

INTERVIEW QUESTIONS 

1.Why do you think it is important for Thames Water to share scientific knowledge among its workforce?

·         Knowledge sharing important because it encourages more connection and collaboration between the workforce.

·         get to know everyone and work in happy environment and be more productive

·         Make everyone knowledgeable about scientific advancements in the field.

·         E.g. from studying the Thames water corporate responsibility and sustainability report suggest that it is important that we share scientific knowledge to help spread awareness of the implications and understanding of how to protect public water supplies and the environment in terms of climate change.

It is important that that employees share their job-related with each other, so that they will be able to perform their job better and eventually lead to higher organization performance. The more knowledgeable individuals are, the more they will be able to innovate. For a company to stay in the game, the employees need to make the most of the knowledge they have- sharing and absorbing it readily.

2.How do you think Thames Water can fulfil its environmental responsibility by sharing scientific knowledge?

·         its written on corporate responsibility and sustainability report. ''responsible and sustainable use of natural resources''.

·         Sending a monthly scientific magazine to all workforce of latest scientific advancements, holding seminars to the public and visiting primary and secondary schools to spread awareness to the general public.

·         creating posters and/or advertising.

Environmentalism is an ideology that evokes the necessity and responsibility of humans to respect, protect and preserve the natural world.* Promoting environmental awareness is a crucial part of being an environmental steward. A way to promote this is by proposing environmentally sustainable and beneficial projects and starting community gardens. As well as constantly educating people. By this way people can understand the economic and biological importance of eliminating the harmful products created by man.

3. What previous experience do you have in teaching or tutoring?

·         very rewarding to tutor people.

·         I have tutored 9 year olds at a elite private school for three years.

·         I am able to explain theories and complex information in a simple and easy to understand and friendly way similarly I way will help my colleagues in understanding.

4. Please give an example when you used your communication skills in teaching or 

tutoring?

·         I helped teach someone how to code and create website as well as buy a website and domain name.

·         I used to be in a computer club.

·         looking for someone with it experience and I know how to use Microsoft office.

Effective communication is the key to human interaction. Whenever I teach, I communicate in a respectful manner. I use a tone that is honest and tactful. When taking on a listening role, I make eye contact and focus on the speaker.

5. How do you see your career progress within the area of teaching?

·         I personally love to teach very rewarding.

·         I like the career prospects and development such as promotional opportunities of career advancement to become of senior instructor &  part of the senior training strategic team.

·         looking to stay with Thames Water for the a long period of time in my life.

·         I can handle responsibility.

Developing my skills as a teacher will not only help you deliver engaging and informative lessons, but it will also help me progress in my teaching career. Continuing professional development (CPD) is a great way of showing employers that I am committed to the company and that I will keep up to date with new approaches of teaching. Personal development way is the best way to learn new skills. And may one day I will have the expertise to have a leadership role.

6. What do you think you will personally bring to the role?

·         I have learned business studies so can help cater to achieve the company goals as well as my scientific understanding.

·         I am determined individual so will try my upmost best to spread the awareness due my duties such as assisting in workshops.

·         I enjoy teaching and open to learn from others.

7. Why are you interested in working  for Thames Water?

·         I like to drink water.

·         I like the career prospects and development such as promotional opportunities of career advancement to become of senior instructor & senior training strategic team.

·          I enjoy the mix of the business and science fields which I have qualifications for both and learned them at A-level.

·         Thames water is the largest water services company in the UK and it will be a privilege to serve the people of the UK providing them with a vital necessity of water.

·        The Thames Water reputation is certainly a key factor, I would be proud to work for a company with such a long history of leadership in the industry.

·        * A friends that works there has told me that the culture supports learning and development and really rewards hard work.

·        * Also my proven track record in teaching and my superb communication skills make me and excellent match for the job requirements.

·        * Also, the role excites me because I love the idea of sharing facts and interacting with people.

**REMEMBER TO STAY POSITIVE LIKE A PROTON!!**