This document discusses the definitions, etiology, and laboratory diagnosis of diarrheal diseases. It defines diarrhea, gastroenteritis, and dysentery. It lists common bacterial, viral, protozoan, and opportunistic pathogens that can cause these diseases. It then provides details on specimen collection and testing methods for identifying various pathogens, including culture-based and antigen/toxin detection techniques. These laboratory methods are described for bacteria like Salmonella, Shigella, Campylobacter, C. difficile, as well as parasites like Giardia, Cryptosporidium, and Entamoeba histolytica.

1. PRESENTER: Tittu Joseph
MODERATOR: Dr. Beena Anthony

2. Definitions
Aetiology
Laboratory Diagnosis
Summary

3. 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.

4. 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

5. 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

6.  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.

7.  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.

8.  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.

10. 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.

11.  CULTURE:
 Selective media - MacConkey’s agar,Deoxycholate
citrate agar(DCA) or Xylose Lysine Deoxycholate
agar(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, catalase
positive, oxidase negative.
 SLIDE AGGLUTINATION TEST:
 Slide agglutination with polyvalent antisera and
monovalent sera.

12. 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.

13. 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

14. Parasite Trophozoites of
E.histolytica
present
Absent
Bacteria Many motile
bacteria
Absent

15.  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’s
Campylobacter selective medium and Campy BAP
selective media.
 Campy BAP: Lysed blood agar, vamcomycin, polymyxin
B, trimethoprim, cephalothin and amphotericin B.
 Incubated at 42°C for 48hrs.
 BIOCHEMICAL REACTIONS: Oxidase and catalase
positive .

16.  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.

18.  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.

20.  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.

21.  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)

22.  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.

23.  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

25.  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.

26.  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.

27. 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.

29.  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