Diagnosis of Infectious Diseases

8/3/2019 Diagnosis of Infectious Diseases

1/29

Diagnosis of InfectiousDiseases


2/29

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


3/29

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 andglass slid

b- Morphology and staining reactions of bacteria:Simple stain: methylene blue stain

Gram stain: differentiation between Gm+ve 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)


4/29

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


5/29

Types of solid media1- 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


6/29

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)


7/29

3- Biochemical Reaction

Use of substrates and sugars to identify pathogens:

a- Sugar fermentation:Organisms ferment sugar with production of acid onlyOrganisms ferment sugar with production of acid and gasOrganisms do not ferment sugar

b- Production of indole:Depends on production of indole from amino acid tryptophanIndole is detected by addition of Kovacs reagentAppearance of red ring on the surface

e- H2S production:Depends on production H2S from protein or polypeptidesDetection by using a strip of filter paper containing lead acetate


8/29

3- Biochemical Reaction (cont.)

c- Methyl red reaction (MR):Fermentation of glucose with production of huge amount of acidLowering pH is detected by methyl red indicator

d- Voges proskaurs reaction (VP):

Production of acetyl methyl carbinol from glucose fermentationAcetyl methyl carbinol is detected by addition KOHColor of medium turns pink (positive)

e- Action on milk:

Fermentation of lactose with acid productionRed color if litmus indicator is added


9/29

3- Biochemical Reaction (cont.)f- Oxidase test:

Some bacteria produce Oxidase enzyme

Detection by adding few drops of colorless oxidase reagentColonies turn deep purple in color (positive)

g- Catalase test:

Some bacteria produce catalase enzyme

Addition of H2

O2

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 mediaand change color of indicator from yellow to pink


10/29

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


11/29

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


12/29

Molecular Biology TechniquesA- Genetic probes (DNA or RNA probes):

Detection of a segment of DNA sequence (gene) in unknownorganism 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 ofnucleic 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


13/29

Bacteriophage


14/29

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 (tailfibres) 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


15/29

A- Lytic or Vegetative Cycle

3) Eclipse phase:

Viral DNA directs the host cell metabolism to synthesize newenzymes and proteins for phage synthesis

4) Intracellular synthesis:

Host cell machinery is directed by genetic information providedby 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 maturephage particle

6) Release:

Accumulation of huge number of phage

The cell bursts and phage particles are released


16/29

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


17/29

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 chromosomeAs it infects another bacterium , it will transmit toit new characters


18/29

Practical applications using phages

* Phages are important as a research tools

* Phages are used as vectors in DNArecombinant technology

* Phage typing of bacteria is important intracingsource of infection forepidemiologic purposes


19/29

Antimicrobial Susceptibility testing

Introduction: Identification of a bacterial isolate from a patient

provides guidance in the choice of an appropriateantibiotic for treatment

Many bacterial species are not uniformlysusceptible to a particular anti-bacterial

compound This is particularly evident among the

Enterobacteriaceae, Staphylococcus spp., andPseudomonas spp.

The wide variation in susceptibility and highfrequencies of drug resistance among strains inmany bacterial species necessitates thedetermination of levels of resistance orsusceptibility as a basis for the selection of theproper antibiotic for chemotherapy


20/29

Antimicrobial Susceptibility testing can bedown by three ways:

1.Minimum Inhibitory Concentration (MIC)

2.Disk Diffusion Method

3.Minimum Bactericidal Concentration (MBC)


21/29

1.Minimum Inhibitory Concentration (MIC) :

Principle:

The tube dilution test is the standard method fordetermining levels of resistance to an antibiotic.

Serial dilutions of the antibiotic are made in a liquidmedium which is inoculated with a standardizednumber of organisms and incubated for aprescribed time.

The lowest concentration of antibiotic preventingappearance of turbidity is considered to be the

minimal inhibitory concentration (MIC).


22/29

Different concentrations of Gentamycin inNutrient 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


23/29

Different concentrations of Tetracycline inNutrient broth:

Conc. in mcg/ml0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5

Tetracycline, generally considered a bacteriostatic antibiotic, for thisbacterium, has an MIC of 1.6 mcg/ml


24/29

2.Disk-diffusion Method (Kirby-Bauer Method):

The disk-diffusion method (Kirby-Bauer) is more

suitable for routine testing in a clinical laboratorywhere a large number of isolates are tested forsusceptibility to numerous antibiotics.

An agar plate is uniformly inoculated with the testorganism

A paper disk impregnated with a fixedconcentration of an antibiotic is placed on the agarsurface.

Growth of the organism and diffusion of the

antibiotic commence simultaneously resulting in acircular zone of inhibition in which the amount ofantibiotic exceeds inhibitory concentrations.

The diameter of the inhibition zone is a function ofthe amount of drug in the disk and susceptibility ofthe microorganism.


25/29

This test must be rigorously standardized since zonesize is also dependent on:

inoculum size, medium composition,

temperature of incubation,

excess moisture and

thickness of the agar.

Zone diameter can be correlated with susceptibilityas measured by the dilution method.

Further correlations using zone diameter allow the

designation of an organism as "susceptible","intermediate", or "resistant" to concentrations of anantibiotic which can be attained in the blood or otherbody fluids of patients requiring chemotherapy.


26/29

Using a dispenser, antibiotic-impregnated disks are placedonto the agar surface.

As the bacteria on the lawn grow, they are inhibited tovarying degrees by the antibiotic diffusing from the disk.


27/29

Staphylococcus aureus (MRSA)

Note the yellowish pigmentation of the bacteriallawn, and the lack of inhibition by the Oxacillin

disk


28/29

Streptococcus pneumoniae (Pneumococcus):

The brownish tint of the blood agar plate outside thezones of bacterial inhibition is caused by alpha-

haemolysis.


29/29

Pseudomonas aeruginosa:

The greenish tint of the lawn and plate in general iscaused by the diffusible pigment made by thePseudomonas aeruginosa itself.

Documents

Diagnosis of Infectious Diseases