PCR a guide

PCR a guide 

The results of a PCR reaction are usually visualized (made visible) using Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it separates DNA fragments according to size. A standard, or DNA ladder, is typically included so that the size of the fragments in the PCR sample can be determined.

DNA fragments of the same length form a "band" on the gel, which can be seen by eye if the gel is stained with a DNA-binding dye. For example, a PCR reaction producing a 400400400 base pair (bp) fragment would look like this on a gel:

Using PCR, a DNA sequence can be amplified millions or billions of times, producing enough DNA copies to be analyzed using other techniques. For instance, the DNA may be visualized by gel electrophoresis, sent for or digested with restriction enzymes and into a plasmid.

PCR is used in many research labs, and it also has practical applications in forensics, genetic testing, and diagnostics. For instance, PCR is used to amplify genes associated with genetic disorders from the DNA of patients (or from fetal DNA, in the case of prenatal testing). PCR can also be used to test for a bacterium or DNA virus in a patient's body: if the pathogen is present, it may be possible to amplify regions of its DNA from a blood or tissue sample.

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Antibiotic – a substance produced by living organisms with activity against bacteria.

Antibiotic – a substance produced by living organisms with activity against bacteria.

Intrinsic resistance refers to resistance which is an inherent and unchanging property of a particular organism. • Acquired resistance refers to the appearance of resistance in organisms which were initially susceptible to a particular antibiotic.

Low level resistance: when an organism becomes resistant to the normal clinical dose of the antibiotic but may be treated by the use of higher doses. • High level resistance: when an organism becomes resistant to the highest clinically achievable dose of the antibiotic, i.e. it is no longer treatable with that antibiotic.

Resistance mechanisms fall into four main categories: 1. Modification of the target site 2. Metabolic bypass (reduced physiological importance of the target site) 3. Decreased drug accumulation in the bacterial cell 4. Antibiotic inactivation

1. Modification of target site • Occurs primarily through mutation. • Example: Streptomycin resistance in E.coli 1) Streptomycin inhibits protein synthesis by binding to the bacterial ribosome. 2) High level resistant E. coli have been found with a mutation at the Lys42 or Lys87 positions of the S12 ribosomal protein. 3) Mutations in rRNA genes have also been found to cause streptomycin resistance.

2. Metabolic bypass Reduces the importance of the antibiotic target site by providing an alternative pathway. Examples include: Methicillin resistance in staphylococci - due to the possession of an alternate penicillin binding protein (PBP2). Vancomycin resistance in enterococci - due to the possession of the vanA gene cluster which synthesises a modified pentapeptide building block for cell wall synthesis.

3. Decreased drug accumulation Can be achieved in one of two ways: 1. Prevent uptake of the drug by the cell 2. Active efflux of the drug from the cell Limited uptake • Responsible for a lot of intrinsic resistance, e.g. penicillins do not easily cross the outer membrane of Gram negative cells; Mycobacteria are impermeable to many drugs. • Alteration in outer membrane proteins can lead to low level resistance to several classes of drugs: • 1) Loss of D2 porin in Pseudomonas aeruginosa leads to imipenem resistance • 2) Altered porin expression in Salmonella has been associated with combined low level resistance to βlactams, aminoglycosides, chloramphenicol, tetracyclines, trimethoprim and quinolones.

Active efflux • The best studied example is tetracycline resistance – found in a wide range of both Gram positive and Gram negative bacteria. • It depends on the presence of an inner membrane protein (tet). This forms a multimer spanning the membrane and binds the drug in the presence of Mg2+, leading to its export from the cell in an energy dependent manner.

4. Antibiotic inactivation • This is the most commonly encountered mechanism of drug resistance. • Resistance can be achieved in one of two types of reaction: I. Hydrolysis of the antibiotic molecule e.g. β-lactamases II. Addition of groups to the antibiotic molecule e.g. chloramphenicol acetyl-transferase The genes for these enzymes are often associated with transposons and plasmids.

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Mycology (continued)

Mycology continued

Mycology is the branch of biology concerned with the study of fungi, including their genetic and biochemical properties, their taxonomy and their use to humans as a source for tinder, medicine, food, and entheogens, as well as their dangers, such as toxicity or infection.

Concerned with diagnosis, management, and prevention of fungal diseases or mycoses • Mycoses are among the most difficult diseases to diagnose and treat – Signs of mycoses are often missed or misinterpreted – Fungi are often resistant to some antifungal agents
Diagnosis of invasive fungal disease (IFD) is challenging because current diagnostic methods lack sensitivity and specificity, or take too long to yield a result to be clinically useful. Such limitations have consequences; delayed diagnosis leads to delayed treatment. Speed to diagnosis is a key risk factor in patient outcomes (Barnes 2008). Diagnosis of fungal infection is further complicated by problematic developments in the field of medical mycology. First and foremost is the loss of senior mycology experts in the field who were trained in classical mycology, which has created crises-level problems in clinical mycology (Steinbach et al. 2003). This problem has been compounded over the last 30 years as the spectrum of fungi-causing infections has exploded owing to AIDS and the use of highly immunosuppressive agents for treatment of a variety of diseases. These patients are susceptible to infections from fungi rarely seen, or never reported as a human pathogen, which can cause identification problems for even the most experienced mycologists. Whereas mycologists in the past needed to be able to identify ∼50 commonly encountered fungi, and ∼300 total fungi that were pathogenic for humans, the number of potential fungal pathogens is likely many times what is described in textbooks, and will continue to grow as the severely immunosuppressed patient population continues to grow 

Diagnosis of fungal infection has relied primarily on methods such as direct microscopic examination of clinical samples, histopathology, and culture. Such approaches are dependent on personnel with relatively high levels of specific mycology training. The growth in the number of fungi that clinical mycologists must identify has forced investigators to develop and apply new methods for fungal identification that go beyond classical phenotypic methods. As a consequence, there is an increased emphasis on the use of molecular methods and antigen detection as surrogates for culture in diagnosis of fungal diseases.

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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!

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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)

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