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Diagnosis of Infectious Diseases

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Title: Diagnosis of Infectious Diseases


1
Diagnosis of Infectious Diseases
2
Laboratory Investigation of Microbial infections
  • Examining specimens to detect isolate and
    identify pathogens
  • 1- Microscopy
  • 2- Culture techniques
  • 3- Biochemical reactions

  • 4- Serological identification

  • 5- Molecular biology techniques

  • 6- Bacteriophage typing

3
1- Microscopy
  • Microorganisms can be examined microscopically
    for
  • a- Bacterial motility
  • Hanging drop method
  • A drop of bacterial suspension is placed
    between a cover slip and glass slid
  • b- Morphology and staining reactions of bacteria
  • Simple stain methylene blue stain
  • Gram stain differentiation between Gmve and
    Gmve bacteria
  • . Primary stain (Crystal
    violet)
  • . Mordant (Grams Iodine mixture)
  • . Decolorization (ethyl
    alcohol)
  • . Secondary stain (
    Saffranin)
  • Ziehl-Neelsen stain staining acid fast
    bacilli
  • . Apply strong carbol
    fuchsin with heat
  • . Decolorization (H2SO4 20
    and ethyl alcohol
  • . Counter stain (methylen
    blue)

4
2- Culture Techniques
  • Culture media are used for
  • - Isolation and identification of pathogenic
    organisms
  • - Antimicrobial sensitivity tests
  • Types of culture media
  • a- Liquid media
  • - Nutrient broth meat extract and
    peptone
  • - Peptone water for preparation sugar
    media
  • - Growth of bacteria detected by
    turbidity
  • b- Solid media
  • - Colonial appearance
  • - Hemolytic activity
  • - Pigment production

5
Types of solid media
  • 1- simple media
  • Nutrient agar
  • 2- Enriched media media of high nutritive
    value
  • . Blood agar
  • . Chocolate agar
  • . Lofflers serum
  • 3- Selective media allow needed bacteria to
    grow
  • . LowensteinJensen medium
  • . MacConkeys agar
  • . Mannitol Salt Agar
  • 4- Indicator media to different. between lact.
    and non lact. ferment
  • . MacConkeys medium
  • . Eosine Methlyne blue Agar
  • 5- Anaerobic media for anaerobic cultivation
  • . Deep agar, Robertsons Cooked Meat
    Medium

6
Colonial appearance on culture media
  • Colony morphology
  • . Shape . Size . Edge of
    colony . Color
  • Growth pattern in broth
  • . Uniform turbidity
  • . Sediment or surface pellicle
  • Pigment production
  • . Endopigment production (Staph.
    aureus)
  • . Exopigment production (Ps.
    aeruginosa)
  • Haemolysis on blood agar
  • . Complete haemolysis (Strept.
    Pyogenes)
  • . Partial haemolysis
    (Strept. Viridans)
  • Growth on MacConkeys medium
  • . Rose pink colonies (Lactose
    fermenters)
  • . Pale yellow colonies (Non
    lactose fermenters)

7
3- Biochemical Reaction
  • Use of substrates and sugars to identify
    pathogens
  • a- Sugar fermentation
  • Organisms ferment sugar with production of
    acid only
  • Organisms ferment sugar with production of
    acid and gas
  • Organisms do not ferment sugar
  • b- Production of indole
  • Depends on production of indole from amino
    acid tryptophan
  • Indole is detected by addition of Kovacs
    reagent
  • Appearance of red ring on the surface
  • e- H2S production
  • Depends on production H2S from protein or
    polypeptides
  • Detection by using a strip of filter paper
    containing lead acetate

8
3- Biochemical Reaction (cont.)
  • c- Methyl red reaction (MR)
  • Fermentation of glucose with production
    of huge amount of acid
  • Lowering pH is detected by methyl red
    indicator
  • d- Voges proskaurs reaction (VP)
  • Production of acetyl methyl carbinol from
    glucose fermentation
  • Acetyl methyl carbinol is detected by
    addition KOH
  • Color of medium turns pink (positive)
  • e- Action on milk
  • Fermentation of lactose with acid
    production
  • Red color if litmus indicator is added

9
3- Biochemical Reaction (cont.)
  • f- Oxidase test
  • Some bacteria produce Oxidase enzyme
  • Detection by adding few drops of colorless
    oxidase reagent
  • Colonies turn deep purple in color
    (positive)
  • g- Catalase test
  • Some bacteria produce catalase enzyme
  • Addition of H2O2 lead to production of gas
    bubbles (O2 production)
  • h- Coagulase test
  • Some bacteria produce coagulase enzyme
  • Coagulase enzyme converts fibrinogen to
    fibrin (plasma clot)
  • Detected by slide or test tube method
  • i- Urease test
  • Some bacteria produce urease enzyme
  • Urease enzyme hydrolyze urea with
    production of NH3
  • Alklinity of media and change color of
    indicator from yellow to pink

10
4- Animal pathogenicity
  • Animal pathogenicity test
  • Animals commonly used are guinea pigs,
    rabbits, mice
  • Importance of pathogenicity test
  • - Differentiate pathogenic and non pathogenic
  • - Isolation organism in pure form
  • - To test ability of toxin production
  • - Evaluation of vaccines and antibiotics

11
Serological identification
  • A- Direct serological tests
  • - Identification of unknown organism
  • - Detection of microbial antigens by using
    specific
  • known antibodies
  • - Serogrouping and serotyping of isolated
    organism
  • B- Indirect serological tests
  • - Detection of specific and non specific
    antibodies
  • (IgM IgG) by using antigens or
    organisms

12
Molecular Biology Techniques
  • A- Genetic probes (DNA or RNA probes)
  • Detection of a segment of DNA sequence
    (gene) in unknown
  • organism using a labeled probe
  • Probe consists of specific short sequence
    of labeled single-
  • stranded DNA or RNA that form
    strong covalently
  • bonded hybrid with specific
    complementary strand of
  • nucleic acid of organism in
    question
  • B- Polymerase chain reaction (PCR)
  • Amplification of a short sequence of
    target DNA or RNA Then
  • It is detected by a labeled probe
  • C- Plasmid profile analysis
  • Isolation of plasmids from bacteria and
    determination of their
  • size and number compared with standard
    strains by agarose
  • gel electrophoresis

13
  • Bacteriophage

14
Bacteriophage
  • Bacteriophages are viruses
  • that parasitize bacterial cell
  • Replication of Bacteriophage
  • A- Lytic or vegetative cycle
  • End by lysis of bacterial cell and release of
    copies of the phage
  • 1) Adsorption
  • Adsorption occurs between attachment sites
    on the phage (tail fibres) and specific receptor
    sites on bacteria
  • It is specific strep (sensitivity of
    bacteria to different phages)
  • 2) Penetration
  • The tail sheath will contract and inject
    DNA into bacterial cell

15
A- Lytic or Vegetative Cycle
  • 3) Eclipse phase
  • Viral DNA directs the host cell metabolism to
    synthesize new enzymes and proteins for phage
    synthesis
  • 4) Intracellular synthesis
  • Host cell machinery is directed by genetic
    information provided by phage nucleic acid to
    synthesize phage coats and nucleic a.
  • 5) Assembly
  • Protein subunits of the phage head and tail
    aggregate
  • Each capsid acquires nucleic acid molecule to
    become a mature phage particle
  • 6) Release
  • Accumulation of huge number of phage
  • The cell bursts and phage particles are
    released

16
II- Temperate Phage cycle Lysogenic cycle
  • Adsorption and penetration take place as in
    lytic cycle
  • Virus DNA integrate with host chromosome
    (Prophage)
  • and replicate as part of host chromosome
  • The bacterial cell is called alysogenic
    bacterium
  • Lysogenic bacterium has certain characters
  • a- Immune to infection by another phage
  • b- Acquire new properties e.g. production of
    exotoxin
  • Diphtheria bacilli, Cl. Botulinum, Strpt.
    Pyogen
  • erthrogenic toxin

17
Outcome of Temperate cycle
  • 1) The cell continue carrying prophage
    indefinitely, passing it to daughter cells
  • 2) The prophage detach from the bacterial
    chromosome and start a lytic cycle
  • 3) As prohage is detached it may carry genetic
    material of bacterial chromosome
  • As it infects another bacterium , it will
    transmit to it new characters

18
Practical applications using phages
  • Phages are important as a research tools
  • Phages are used as vectors in DNA recombinant
    technology
  • Phage typing of bacteria is important in
    tracing source of infection for epidemiologic
    purposes

19
Antimicrobial Susceptibility testing
  • Introduction
  • Identification of a bacterial isolate from a
    patient provides guidance in the choice of an
    appropriate antibiotic for treatment
  • Many bacterial species are not uniformly
    susceptible to a particular anti-bacterial
    compound
  • This is particularly evident among the
    Enterobacteriaceae, Staphylococcus spp., and
    Pseudomonas spp.
  • The wide variation in susceptibility and high
    frequencies of drug resistance among strains in
    many bacterial species necessitates the
    determination of levels of resistance or
    susceptibility as a basis for the selection of
    the proper antibiotic for chemotherapy

20
  • Antimicrobial Susceptibility testing can be down
    by three ways
  • Minimum Inhibitory Concentration (MIC)
  • Disk Diffusion Method
  • Minimum Bactericidal Concentration (MBC)

21
1. Minimum Inhibitory Concentration (MIC)
  • Principle
  • The tube dilution test is the standard method for
    determining levels of resistance to an
    antibiotic.
  • Serial dilutions of the antibiotic are made in a
    liquid medium which is inoculated with a
    standardized number of organisms and incubated
    for a prescribed time.
  • The lowest concentration of antibiotic preventing
    appearance of turbidity is considered to be the
    minimal inhibitory concentration (MIC).

22
  • Different concentrations of Gentamycin in
    Nutrient broth
  • Conc. in mcg/ml
  • 0.1 0.2 0.4 0.8 1.6 3.1

Gentamicin, generally considered a bacteriocidal
antibiotic, for this bacterium, has an MIC of 0.8
mcg/ml
23
  • Different concentrations of Tetracycline in
    Nutrient broth
  • Conc. in mcg/ml
  • 0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5

Tetracycline, generally considered a
bacteriostatic antibiotic, for this bacterium,
has an MIC of 1.6 mcg/ml
24
2. Disk-diffusion Method (Kirby-Bauer Method)
  • The disk-diffusion method (Kirby-Bauer) is more
    suitable for routine testing in a clinical
    laboratory where a large number of isolates are
    tested for susceptibility to numerous
    antibiotics.
  • An agar plate is uniformly inoculated with the
    test organism
  • A paper disk impregnated with a fixed
    concentration of an antibiotic is placed on the
    agar surface.
  • Growth of the organism and diffusion of the
    antibiotic commence simultaneously resulting in a
    circular zone of inhibition in which the amount
    of antibiotic exceeds inhibitory concentrations.
  • The diameter of the inhibition zone is a function
    of the amount of drug in the disk and
    susceptibility of the microorganism.

25
  • This test must be rigorously standardized since
    zone size is also dependent on
  • inoculum size,
  • medium composition,
  • temperature of incubation,
  • excess moisture and
  • thickness of the agar.
  • Zone diameter can be correlated with
    susceptibility as measured by the dilution
    method.
  • Further correlations using zone diameter allow
    the designation of an organism as "susceptible",
    "intermediate", or "resistant" to concentrations
    of an antibiotic which can be attained in the
    blood or other body fluids of patients requiring
    chemotherapy.

26
  • Using a dispenser, antibiotic-impregnated disks
    are placed onto the agar surface.
  • As the bacteria on the lawn grow, they are
    inhibited to varying degrees by the antibiotic
    diffusing from the disk.

27
Staphylococcus aureus (MRSA)
  • Note the yellowish pigmentation of the bacterial
    lawn, and the lack of inhibition by the Oxacillin
    disk

28
Streptococcus pneumoniae (Pneumococcus)
  • The brownish tint of the blood agar plate outside
    the zones of bacterial inhibition is caused by
    alpha-haemolysis.

29
Pseudomonas aeruginosa
  • The greenish tint of the lawn and plate in
    general is caused by the diffusible pigment made
    by the Pseudomonas aeruginosa itself.
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