PRESENTER: Tittu Joseph

MODERATOR: Dr. Beena Anthony

Definitions

Aetiology

Laboratory Diagnosis

Summary

Diarrhoea :Passage of 3 or more loose or liquid stools per day, or more frequently than is normal for the individual.

Gastroenteritis : Inflammation of mucous membrane of stomach & intestine resulting in frequent loose motions with or without mucous & with or without blood, pain abdomen and with or without fever.

Dysentery : Passage of blood and mucous with motion, often associated with tenesmus.

A) Bacteria

Invasive Bacterial

Pathogens:

Salmonella sp

Shigella sp

Enteroinvasive Esch. coli

(EIEC)

Enterohaemorrhagic Esch.

coli (EHEC)

Vibrio parahaemolytius

Campylobacter jejuni

Yersinia enterocolitica

Non-invasive Bacterial

Pathogens:

Enterotoxigenic Esch. coli

(ETEC)

Enteropathogenic Esch.

coli (EPEC)

Vibrio cholerae

Shigella dysentriae type 1

Staphylococcus aureus

Clostridium perfringens

Clostridium difficile

B)Viruses: Rotavirus

Norwalk virus

Adenovirus

Astrovirus

Calicivirus

C) Protozoa: Entamoeba histolytica

Giardia lamblia

Cryptosporidium parvum

D)Immuno-

compromised

patients:

Salmonella sp

Clostridium difficile

Mycobacterium avium

complex (MAC)

Cryptosporidium

Microsporidium

Giardia

Entamoeba histolytica

Isospora belli

Enterovirus

Cytomegalovirus

SPECIMENS:

Watery stool

COLLECTION AND TRANSPORT:

Preferably prior to the start of antibiotics.

Should be immediately transported.

Transport media - Venkatraman-Ramakrishnan (VR)

fluid, Cary-Blair medium, alkaline peptone water

/Monsur’s medium.

DIRECT MICROSCOPY:

For rapid diagnosis.

Dark field or phase contrast microscope.

Darting motility.

CULTURE:

Selective media - Alkaline Bile Salt

Agar(BSA), Thiosulphate Citrate Bile

Sucrose agar(TCBS) or Monsur’s Gelatin

Taurocholate Tryticase Tellurite

agar(GTTA).

Non-selective media - Blood agar and

MacConkey’s agar.

Incubated at 37°C for overnight.

COLONY MORPHOLOGY AND STAINING:

Pale colonies on MacConkey’s agar, TCBS

shows yellow colonies & BSA translucent

colonies are present.

Gram staining: Gram negative comma

shaped bacilli.

BIOCHEMICAL REACTION:

Ferments glucose, mannitol, sucrose,

maltose, mannose with acid production.

Catalase and oxidase positive.

SPECIMENS:

Fresh stool is collected.

Ideal specimen is a direct swab of an ucler taken under

sigmoidoscopic examination.

TRASPORT:

Should be transported immediately.

Sach’s buffered glycerol saline, pH 7.0-7.4

Alkaline transport media used for vibrios are inhibitory

to Shigella.

DIRECT MICROSCOPY:

Saline and iodine preparation shows large number of

pus cells, RBCs and macrophages.

CULTURE:

Selective media - MacConkey’s agar,Deoxycholatecitrate agar(DCA) or Xylose Lysine Deoxycholateagar(XLD).

Selenite F broth is used as enrichment media which inhibits growth of normal flora like Esch. coli and helps in the rapid growth of enteric pathogens.

Incubated for 24hrs at 37°C.

COLONY MORPHOLOGY AND STAINING:

Non-lactose fermenting colonies on MacConkey’s agar.

Gram negative bacilli and are non-motile.

BIOCHEMICAL REACTIONS:

Glucose fermentation without gas production, catalasepositive, oxidase negative.

SLIDE AGGLUTINATION TEST:

Slide agglutination with polyvalent antisera and monovalent sera.

FEATURE: AMOEBIC

DYSENTRY:

BACILLARY

DYSENTRY:

Number 6-8 motions/day >10 motions/day

Amount Copious Small

Odour Offensive Odourless

Colour Dark red Bright red

Reaction Acidic Alkaline

Nature Blood & mucous

mixed with

faeces.

Blood & mucous

but no faeces.

Consistency Not adherent to

container.

Adherent to

container.

FEATURE: AMOEBIC

DYSENTRY:

BACILLARY

DYSENTRY:

RBCs In clumps Discrete or in

rouleaux formation

Pus cells Scanty Numerous

Eosinophils Present Scarce

Macrophages Very few Numerous; many of

them contain RBCs;

hence mistaken for

E.histolytica

Ghost cells Absent Numerous

Pyknotic bodies Present Absent

Charcot-Leyden

crystals

Present Absent

Parasite Trophozoites of

E.histolytica

present

Absent

Bacteria Many motile

bacteria

Absent

SPECIMEN:

Faeces

DIRECT MICROSCOPY:

Gram staining: Gram negative, curved bacilli.

Dark ground or phase contrast microscopy: Darting or tumbling motility of the spiral rods.

CULTURE:

Transport media: Cary-Blair media.

Selective media: Butzler’s selective medium, Skirrow’sCampylobacter selective medium and Campy BAP selective media.

Campy BAP: Lysed blood agar, vamcomycin, polymyxinB, trimethoprim, cephalothin and amphotericin B.

Incubated at 42°C for 48hrs.

BIOCHEMICAL REACTIONS: Oxidase and catalasepositive .

SPECIMEN:

Faeces.

CULTURE:

Selective medium: Cycloserine-cefoxitin-fructose

agar(CCFA).

Colonies appear yellow due to fructose fermentation.

DEMONSTRATION OF TOXIN:

Toxin can be demonstrated in the faeces of patients by

its characteristic effects on Hep-2 and human diploid

cell cultures, or by ELISA.

Toxin is specifically neutralized by atiserum of

Clostridium sordelli.

SPECIMEN:

Faeces

CULTURE:

Blood agar and MacConkey’s agar.

Incubated at 37°C for 24hrs.

Colonies show β-haemolysis on Blood agar.

Pink lactose fermenting colonies seen on MacConkey’s

agar.

Identification of ETEC depends upon the demonstration

of Heat Labile toxin and Heat Stable toxin using

methods like ELISA, Radioimmunoassay(RIA).

Verocytotoxin producing Esch. coli(VTEC) can be

detected by its cytotoxic effect on Vero and HeLa cells.

Sereny test: Done by inoculating suspension of bacteria

into guinea pig's eye. Severe

mucopurulent conjunctivitis and

severe keratitis indicates a positive test.

Done for identification of EIEC.

BIOCHEMICAL REACTIONS:

Glucose ,lactose, mannitol and indole positive with

acid and gas production.

Sucrose, urease and citrate negative.

Rotavirus And Norwalk Virus:

Demonstration of viruses:

During the acute stage of the disease, virus particles

may be present in the faeces and can be demonstrated

by:

ELISA

Latex Agglutination Test

Counterimmunoelectrophoresis(CIEP)

Electron microscopy

Immunoelectron microscopy

Antibody Detection:

IgM and IgG antibodies can be detected in the blood by

ELISA and compliment fixation test.

Polymerase Chain Reaction(PCR)

STOOL EXAMINATION: (Microscopic examination)

Normal saline preparation: Actively motile trophozoites.

Iodine preparation: Cysts or dead trophozoites.

[Excretion of cysts in the stool is often intermittent, therefore, at least three consecutive specimens should be examined]

Charcot-Leyden crystals may appear in saline preparation. These are diamond-shaped crystals, clear and refractile.

Concentration method such as formal ether may be used for concentration of amoebic cysts in the stool when the number of amoebae are scanty.

STOOL ANTIGEN DETECTION:

ELISA is used to detect antigens of E.histolytica in faeces.

BLOOD EXAMINATION: Leucocytosis.

Serological tests:

In early cases, its always negative.

Indirect haemagglutination assay(IHA), indirect

fluorescent antibody(IFA) and ELISA.

DNA probes

Polymerase Chain Reaction

Stool Examination: (Microscopic examination)

Cysts in formed stools and trophozoites of the parasite

in diarrhoeal stools by wet preparation(Normal saline

and iodine)

Multiple stool samples may not show any parasites as

they are attached firmly to the mucosa by means of

suckling disc.

Concentration techniques like zinc sulphate floatation

or formalin ethyl acetate are used if the cysts are

sparse.

Antigen Detection:

Fluorescent method using monoclonal antibodies and

ELISA test are done for detection of Giardia antigen in

faeces.

Not in routine use.

Antibody Detection:

Anti-Giardia antibodies may be detected in the patient

serum by using ELISA and indirect fluorescent antibody

tests.

May indicate present or past infection and hence not

useful in diagnosis.

Stool Examination:

Three consecutive stool specimens should be examined.

Direct wet mount – Highly refractile, spherical oocysts.

Staining methods – Modified Ziehl-Neelsen staining.

Oocysts appear bright red(acid-fast).

Immunofluorescent Antibody (IFA) test :

Specific and most sensitive method for identification of

C.parvum in the faeces.

Concentration technique – Sheather’s sugar

concentration technique.

ANTIGEN DETECTION IN FAECES:

ELISA- To detect Cryptosporidial antigen in the faeces.

Immunochromatographic test- simple and rapid.

Direct Microscopy: Gram Staining, Simple Staining.

Blood agar

MacConkey’s Agar

Selective Media: TCBS agar- V.cholerae

DCA – Shigella

CCFA – C.difficile

Parasites: Saline preparation

Iodine preparation – Cysts

Modified acid-fast staining – Cryptosporidia

- Microsporidia

Antigen Detection: Viruses – Rotavirus, Norwalk virus.

Toxin Detection: C.difficile