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Laboratory Diagnosis
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Implicating organisms include:
Bacteria
Parasites
Viruses
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Toxin producing:- Preformed toxin
- Toxin formed in vivo
Invasive
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Specimens Faeces, Rectal swab Others
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The use of a preservative,e.g buffered glycerol salineis recommended if there isdelay in submittingspecimen to the Laboratory
Stool iscollected in awide mouthcontainer
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Culture media forthe isolation ofSalmonella andShigella :MacConkeys AgarSalmonella and
Shigella Agar (SS)GN Broth
MacConkeys AgarDifferential willdifferentiatelactose and nonlactose fermenters
SalmonellaShigella (SS) AgarSelective Agarfor Salmonellaand Shigella
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A small amount of thestool is removed with aswab and plated to theculture media.Culture media areincubated
The swab is then placed in a GNbroth e.g. Selenite broth andincubated
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GN or Selenite brothincubated on day 1Is sub-cultured toMacConkeys and SSagar
This procedureincreases the chance ofisolating the pathogen
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MacConkey s agar showing Normal flora SS agar showing Normal flora
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Lactose fermentingcolonies (pink)
Non-Lactosefermenting coloni(pale )
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NoteSalmonella andShigella are nonlactose fermenters
Non- lactosefermentingcolonies areremoved andinoculated to :KliglersagarUrea
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A puregrowth ofnon lactosefermentingcolonies
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NoteThe media is so designed toshow a slant and a butt
Slant-for the detection ofLactosefermentation
Butt-for the detection ofglucose fermentationThe medium contains twosugars, Glucose andLactose along withcompounds to detect theproduction of H2S
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Organisms that producethe enzyme urease willform alkaline endproducts which willchange the colour of thismedia to pink
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Dextrose fermented, little or no gas, no H2S, urea negative:
Possible Shigella, Shigelloides
Dextrose fermented, gas H2S positive, urea negative:
Possible Salmonella, Arizona,
Citrobacter, Edwardsiella
Dextrose fermented, gas/no gas, little or no H2S, urea negative:
Possible Salmonella (e.g.Salmonella
typhii)
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Kligler and urea
Kligler Alkaline SlantH2S obscuring
Acid butt,Gas
Urea Negative
NoteSalmonella typhi will give similarreaction except there is a smallamount of H2S and no gas in the butt
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Kligler and urea
Kligler Alkaline Slant
Acid buttUrea Negative
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Further tests depend on resultsof kligler and ureae.g. 1. Slide agglutination- using O and H antisera for suspicious
Salmonella sp.- using O, H and Vi for suspiciousSalmonella typhi
- using species specific for Shigella spp.2. Further biochemical tests to confirm
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Kligler alkaline - slant-acid - butt with H2S and gas
Urea ve Lactose veCitrate +ve Sucrose veMotility +ve Dulcitol +veIndole ve Malonate -veLysine +ve Dextrose +ve
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Specimens:stool
Implicated food
Laboratory Investigation:- Gram stain from stool-gram positive cocci
- faecal leukocytes
- Culture : blood agar
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Colonies ofStaphylococcusaureus on blood agar
Gram stain of Staphylococcus aureus
Note!Serological tests using
specific antisera are used to
identify toxigenic strains
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MICROSCOPYMorphologically they may becurved or spiral shaped.GRAM REACTION:Organism is a gram negativecurved bacillus
On wet preparation, the organismhas a characteristic cork screwlike darting motility but are rigidrather than flexous
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ISOLATIONSpecimen is inoculated untoCampylobacter blood agar (CBA)IncubationCBA is placed in a microaerophilicatmosphere (90% Nitrogen, 5%Oxygen,5% carbon dioxide) and incubatedat 42oC for 48hrs.
Microaerophilic Usually generatedin a gas jar using amicroaerophilicgas sachet.
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CULTURAL CHARACTERISTICS:Colonies appear as water droplets ormelted paraffin and slightly gray.Theymay also appear tan or pink.After 24hours incubation colonies may appear tobe more solidified paraffin.Organism is :oxidase positiveCatalase positiveDoes not ferment glucoseReduce nitrates to nitritesIs susceptible to 30mcg Naladixic acidGrows in 1% glycine
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Gram positive sporulating bacilli
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Specimen of choice :stool rice water stool)
Definitive diagnosis :Demonstration of Vibriocholerain the patients stool.Culture Media Thiosulphate Citrate Bile- Salt(TCBS) agar.
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Definition:Gram negative rodsmotileaerobic and facultativeanaerobescatalase - negativeOxidase - positive
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A selective agarused for theisolation of Vibriocolera
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Colonialappearance ofyellow colonieson TCBS medium
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Cultural Characteristics :Oxidase testBiochemical testsSpecific antisera in slide agglutination testsFluorescence antibody (FA) tests
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V. cholera biotype V. cholera biotypeCholera (classical) El TorVP Reaction Negative PositiveSensitivity toPolymixin B Sensitive Resistant(50g disc)Sensitivity toMukerjee Sensitive ResistantIV phage
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V. cholera V.parahaemolyticusColonial appearance yellow colonies deep blue or greenon TCBS mediumGrowth in Peptone water no Growth Growthwith 7% NaCl conc.Acid from sucrose Positive Negative
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paired sera A fourfold rise or fall in vibriocidal titre isconsidered diagnostic.
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Escher ichia col i :
Colonial morphology: flat dry LF
Gram reaction: Gram negative
bacilli
Oxidase: negative
Kliglers: Acid/Acid GasUrea: negative
Citrate: negative
Motility: Motile
Indole: Positive
Lysine: Variable
flat dry lactose
fermenter (LF)
Note!
Serological tests using
specific antisera are used to
identify the various
pathogenic strains
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Specimens:Mainly stool/rectal swab
Blood
Laboratory investigations:Electron Microscopy (EM)
Isolation of Virus:
Tissue culture
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Include:RotavirusNorwalkCalicivirusesEnteric adenoviruses40 @ 41AstrovirusesHepatitis A
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Tissue culture cell line
Normal cell line Cell line showing CPE
Note!
Roundingof cells
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Enzyme-linked immunosorbent Assay (ELISA) TEST
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Giardia lamblia
In immunocompromised patients:
Cryptosporidium sp.
Isospora sp.
Micrsporidium sp
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Specimens: Stool
Blood
Small bowel fluid samples (e.g. entero string test)
Small bowel biopsy material
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Watery samples aremore likely tocontaintrophozoites of
iardia lamblia
Note theconsistencyof the stoolsample
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Laboratory investigations:1. Wet mount ( saline and/or iodine)
preparation (Giardia lamblia
trophozoites)
2. Modified ZN (Cryptosporidium sp. Isospora sp.
micrsporidium sp.
3. ELISA
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NoteOocysts are acid fast
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Oocyst of Isospora belli
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1.Salmonella typhi2. Salmonella paratyphi A
3.Salmonella parathphi B
4.Salmonella paratyphi C
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Blood cultures Urine samples
Stool samples
Bone marrow culture
Clotted blood samples for serology
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Submitted in all suspectedcases
1st-10thday of illness
Taken before treatment
The clot is cultures andserum used to perform
Widal serology testing
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Urine is concentrated and deposit used toinoculate appropriate media
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Stool will be positive in 2ndto 3rdweek ofinfection
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Bile aspirated by means of duodenal tube Valuable in the later stages of the illness and
in carrier detection
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CULTURE MEDIA USED
MacConkey
Salmonella and Shigella Agar (selective)
GN Broth( enrichment broth)
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NoteNon lactosefermenting colonies
RememberBiochemical reactions as
outlined in previous slides
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