Microbiology, Virology and Immunology Department Classification and modern methods of diagnostics of infectious diseases

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


Neisseria gonorrhoeae

Yersinia pestis

Corynebacterium diphtheriae

Bacillus anthracis


Clostridium tetani

Clostridium botulinum


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);


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.


Most specimens received in a clinical microbiology lab are plated onto Blood Agar


Gold’s streak


Staphylococcus Streptococcus



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


IHT


Ring precipitation test


Doublel Immune Diffusion (Ouchterlony’) Test


Radial Immune Diffusion (Mancini’s) Test


CFT


ELISA


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

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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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ELISA -Variants

Detection based on enzyme catalyzed reactions:

1.alkaline ⓟ2.horseradish peroxidase3. β-galactosidase

Detection based on fluorescent labeled secondary antibody

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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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Microbiology, Virology and Immunology Department Classification and modern methods of diagnostics of infectious diseases