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M icrobiology, V irology and I mmunology Department. Classification and modern methods of diagnostics of infectious diseases. METHODS OF EXAMINATION IN MICROBIOLOGY. BACTERIOSCOPIC BACTERIOLOGICAL SEROLOGICAL BIOLOGICAL ALLERGIC EXPRESS-DIAGNOSIS. BACTERISCOPIC METHOD. - PowerPoint PPT Presentation
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Microbiology, Virology and Immunology Department
Classification and modern methods of diagnostics of
infectious diseases
BACTERIOSCOPIC
BACTERIOLOGICAL
SEROLOGICAL
BIOLOGICAL
ALLERGIC
EXPRESS-DIAGNOSIS
METHODS OF EXAMINATION IN MICROBIOLOGY
BACTERISCOPIC METHOD
Staphylococcus aureus
Escherichia coli
M. tuberculosis
M. avium
Borrelia
BACTERISCOPIC METHOD
Neisseria gonorrhoeae
Yersinia pestis
Corynebacterium diphtheriae
Bacillus anthracis
BACTERISCOPIC METHOD
Clostridium tetani
Clostridium botulinum
BACTERISCOPIC METHOD
Isolation of pure culture
BACTERIOLOGICAL METHOD
Examples of varous colony morphologies. The appearance of colonies on a plate is species specific and can be very helpful in identifying.
Pure culture of conditionally-pathogenic microbes may be causative agents of disease according to the such signs:
microbes are present in tested material from pathologic focus in the amount of 104-105 colony-forming units (CFU) in 1 ml or 1 g;
repeated isolation from the same material the same culture;
increasing in the patient’s serum antibodies to the autostrains or microbial culture, which can be causative agent.
to take material before antibacterial therapy beginning or after some time after antibiotic inoculation which is necessary for its excretion from the organism (as a rule 8-10 hours);
to take material from infectious focus or examine proper discharges;
hold on to the strict aseptic for the purpose to prevent contamination of the specimen by microflora of environment;
Necessary rules before collection of tested material:
material is taken into the sterile boxes; clinical specimen with anaerobic bacteria must be protected from atmosphere oxygen action;
the collection of an adequate specimen is useless if the time between collection and culturing allows the disease-producing organism to die (in another cases it’s necessary to use the refrigerator or special transport media);
Necessary rules before collection of tested material:
isolation of viruses, Rickettsia, Chlamidia is made in specialize laboratories;
to clinical specimen a proper document is added, which has data , which has data for correct microbiological examination.
Necessary rules before collection of tested material:
Most specimens received in a clinical microbiology lab are plated onto Blood Agar
BACTERIOLOGICAL METHOD
Gold’s streak
BACTERIOLOGICAL METHOD
Staphylococcus Streptococcus
BACTERIOLOGICAL METHOD
BACTERIOLOGICAL METHOD
Mannitol Salt Medium
Most non-pathogenic staphylococci will not ferment mannitol
Klebsiella pneumoniae
BACTERIOLOGICAL METHOD isolation of pure culture
Biochemical Reactions on API Strip
Proteus mirabilis
Lactose Positive ColoniesMacConkey Agar
Indole Reactions
Positive
Negative
Agglutination test
BACTERIOLOGICAL METHOD Serological method
Agglutination test
BACTERIOLOGICAL METHOD Serological method
IHT
BACTERIOLOGICAL METHOD Serological method
Ring precipitation test
BACTERIOLOGICAL METHOD Serological method
Doublel Immune Diffusion (Ouchterlony’) Test
BACTERIOLOGICAL METHOD Serological method
Radial Immune Diffusion (Mancini’s) Test
BACTERIOLOGICAL METHOD Serological method
CFT
BACTERIOLOGICAL METHOD Serological method
ELISA
BACTERIOLOGICAL METHOD Serological method
Bordetella pertussis
SEROLOGICAL METHODS
Treponema pallidumTreponema pallidum
IFA
Express-diagnosis
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Molecular DiagnosticsImmunological Diagnostics Methods
Immunofluorescence
Fluorochromes-Fluorescein (490→517nm)-Rhodamine (515→546nm)-Phycoerythrin
mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope.
Protein A has the ability to bind to IgG
BIOLOGICAL METHOD
botulismtetanus
TBC in guinea pigTBC in rabbit
BIOLOGICAL METHOD
Mantoux’s test
ALLERGIC METHOD
Applications of Molecular Based Testing in Clinical Microbiology
Rapid or high-throughput identification of microorganisms
Detection and analysis of resistance genesGenotypingClassificationDiscovery of new microorganisms
Specimen CollectionPreserve viability/nucleic acid integrity of
target microorganismsAvoid contaminationAppropriate time and site of collection
(blood, urine, other)Use proper equipment (coagulant, wood,
or plastic swab shafts)Commercial collection kits are availableThe Clinical and Laboratory Standards
Institute (CLSI) has guidelines for proper specimen handling
Sample PreparationConsider the specimen type (stool, plasma,
CSF)More rigorous lysis procedures are
required to penetrate cell walls Consider the number of organisms in the
sampleInactivate inhibitors (acidic polysaccharides
in sputum or polymerase inhibitors in CSF)Inactivate RNases
36
Molecular DiagnosticsImmunological Diagnostics Methods
ELISA• Addition of a specific antibody (primary
antibody) which will bind to the test molecule if it is present.
• Washing to remove unbound molecules.
• Addition of secondary antibody which will bind to the primary antibody.
• The secondary antibody usually has attached to it an enzyme e.g. alkaline phosphatase.
• Wash to remove unbound antibody.
• Addition of a colourless substrate which will react with the secondary antibody to give a colour reaction which indicates a positive result.
-> can be used for quasi High-throughput!!!
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Molecular DiagnosticsImmunological Diagnostics Methods
ELISA -Variants
Detection based on enzyme catalyzed reactions:
1.alkaline ⓟ2.horseradish peroxidase3. β-galactosidase
Detection based on fluorescent labeled secondary antibody
38
Molecular DiagnosticsImmunological Diagnostics Methods
Western blotSDS-Page: separates the components according to their molecular weight.
Blot: the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current.
Immunoreaction: probed with Ab & then radiolabeled or enzyme-linked 2nd Ab.
Detection: a position is visualized by means of an ELISA reaction.
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Molecular DiagnosticsDNA Diagnostic Systems
PCR based methods
-> The presence of the appropriate amplified size fragment confirms the presence of the target.-> Specific primers are now available for the detection of many pathogens including bacteria (E. coli, M. tuberculosis), viruses (HIV) and fungi.
Example: Using PCR to Detect for HIV
• RT-PCR (reverse transcriptase PCR).• HIV has a ssRNA genome.
• Specific primers are used to amplify a 156 bp portion of the HIV gag gene.
• Using standards the amount of PCR product can be used to determine the viral load.
• PCR can also be used as a prognostic tool to determine viral load.
• This method can also be used to determine the effectiveness antiviral therapy.
Other examples:-> Using PCR to Detect DMD deletions (60% of mutations are deletions)
Polymerase Chain ReactionCapable of amplifying tiny quantities of nucleic acid.Cells separated and lysed.Double stranded DNA separated into single strands.Primers, small segments of DNA no more than 20-30 nucleotides
long added.Primers are complementary to segments of opposite strands of
that flank the target sequence.Only the segments of target DNA between the primers will be
replicated.Each cycle of PCR consists of three cycles:
denaturation of target DNA to separate 2 strands.annealing step in which the reaction mix is cooled to allow primers to
anneal to target sequenceExtension reaction in which primers initiate DNA synthesis using a
DNA polymerase.These three steps constitute a thermal cycle
Each PCR cycle results in a doubling of target sequences and typically allowed to run through 30 cycles, one cylce takes approximately 60-90 seconds.
(55°C-65°C).
(95°C)
(72°C)
The final product can be checked on an agarsoe gel to make sure it has correct size and can be sequenced.
The final product is available in high conc. and can be used for cloning (gene that has a product), diagnosis (gene of a virus) or fingerprinting (forensic investigation in crime scenes).
Denature
Continue 30 cycles
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PCR Polymerase Chain Reaction
PCR tubesThermocycler
Agarose gel to detect the productsSequencing
Gel Electrophoresis apparatus Loading the gel
After reaction is complete, the DNA is loaded into an agarose gel, in order to visualize the bands.
The DNA is mixed with Glycerol and specific reagents when loading into the gel.
An electric current is applied so the DNA moves towards the + ve electrode according to it s size
Small fragments move faster and reach the end of the gel, larger fragments move slower and are at the beginning the gel.
Gel is stained to view the DNA bands.
Agarose gel stained with Ethedium bromide and visualized under UV
Larger bands up
Smaller bands downD
irection of migration
In forensic medicine: For instance, trace amounts of DNA, in fluids such as blood
or semen or in tissue such as hair, can be amplified by PCR and analyzed to see whether the DNA is identical to that of a person suspected of committing a crime.
In clinical diagnosis: The technique enabled clinicians to detect infection by the
AIDS virus when other methods have failed, in addition to other diseases.
Diagnosis genetic diseases, such as sickle cell anemia in fetus still in its mother’s uterus, by amplifying the genetic information provided by just a few fetal cells, which can be obtained without harming the fetus.
Applications of PCR
Target Microorganisms for Molecular-Based TestingThose that are difficult or time-consuming
to isolatee.g., Mycobacteria
Hazardous organismse.g., Histoplasma, Coccidiodes
Those without reliable testing methodse.g., HIV, HCV
High-volume testse.g., S. pyogenes, N. gonorrhoeae, C.
trachomatis
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