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LAB DIAGNOSIS DIARRHOEAL
DISEASES
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CAUSES- DIARRHOEA
BACTERIA
Enteropathogenic E. coli (EPEC, ETEC, EIEC), Staphylococcus aureus,V. cholerae, V. parahemolyticus, salmonellae, shigella, Clostridiumperfringens, Clostridium botulinum, B. cereus, campylobacter,
VIRUSES
rotavirus, Norwalk virus ,adenovirus
PARASITES
E. histolytica, G. lamblia, strongyloides, Balantidium coli
OTHERS
IBD, Malabsorption syndromes
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TYPES
ACUTE DIARRHOEA:(3 or More loose stools/dayfor
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CHRONIC DIARRHOEA:(3 or More loose stools/dayfor >4 wks)
Watery
-Osmotic: Carbohydrate malabsorption, Osmoticlaxative
-Secretory: Bacterial toxins, Laxative abuse,Hormonal disorders
Inflammatory: Invasive bacterial and parasitic inf,
IBD, Pseudomembranous enterocolitis
Fatty diarrhoea: Malabsorption
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STOOL-Preferred sample
COLLECTION
Container should be clean, of sufficient size,witha tight-fitting lid.
Stool must be fresh No antiseptics should have been used
Stool must not be mixed with urine
Oil, oily emulsion ,antibiotics , antacids not to be
given to patient 7 days before examination 20-40 gms of formed stools or 5-6 tablespoons of
watery stools collected
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Rectal swab specimen is used :
(1) when it is desirable to collect the fecesimmediately in the absence of a bowel
movement (2) when transport of the stool to the laboratory
would pose problems
(3) when there may be delay in transporting thestool to the laboratory as the collecting tubecontains a transport medium, which can act as apreservative.
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Transport A simple transport medium is the glycerol saline mixture
For V. cholerae, one can use-Venkatraman-Ramakrishnan (VR) medium- 20 g sea salt &5g peptone in 1 L DW pH-8.8
-Cary Blair medium-Buffered solu of NaCl, sod
thioglycollate, disod PO4 and CaCl2- pH-8.4-Alkaline Peptone water pH 8.6 & Monsurs taurocholate-tellurite peptone water pH 9.2Both are good as Tpt &enrichment media
For salmonellaselenite broth For Rotavirus examination, a small amount of stool or
rectal swab is put into 1 ml phosphate buffered salinesolution and frozen at 20 C
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Distant Transport and preservation
10 % formol saline can be used. This is
prepared by adding 100 ml formaldehyde to
900 ml of 0.85% saline
PVA (Poyvinyl alcohol) fixative consists of
saturated mercuric chloride, glycerol, and
glacial acetic acid.Mix 1 ml of stool specimen
in 5 ml of PVA., this preparation can be usedfor microscopy for several months
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The examination of faeces for parasitological diagnosisis done to detect:
Adult worms
Segments of tapeworms
Ova and cysts
Larvae
Trophozoites
Cellular exudates such as WBCs, RBCs, macrophages
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Macroscopic examination
Various points to be noted are:
Consistency: The consistency of the stool could be formed,
soft, loose or watery.
The cysts are found maximum in the formed stoolTrophozoites are most abundant in watery stool
Presence of blood and mucus.
Presence of round worms, thread worms or tapeworm
segments. Colour and smell of the stool
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Naked eye examination
Amoebic dysentry
Colour- dark red
Blood and mucus mixed
with feces
Offensive odour
Not adherent to the
container
Bacillary dysentry
Bright red
Blood and mucus, no
faeces
Odourless
Adherent to the bottom
of the container
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Microscopic examination (temporary
wet mounts)
It is the simplest and easiest technique. A wet mount
can be prepared directly from faecal material or from
the concentrated specimen
Saline wet mount: It is used to detect worm eggs orlarvae, protozoan trophozoites and cysts. In addition
it can reveal the presence of RBCs and WBCs.
Iodine wet mount: It is used to stain glycogen and
nuclei of the cysts.
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Microscopic examination (temporary
wet mounts) contd
Place a drop of saline on left half of the slide and onedrop of iodine on the right half.
With an wire loop pickup a small portion of the
specimen (equivalent to the size of a match head)and mix with saline drop.
Similarly pickup similar amount and mix with a dropof iodine.
Put the cover slip separately on both and examineunder the microscope.
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Concentration techniques
If the number of parasites in the stool specimens islow,examination of a direct wet mount may notdetect them, hence the stool should be concentrated
Eggs, cysts and larvae are recovered afterconcentration procedures whereas trophozoites getdestroyed during the procedure
This makes direct wet mount examination obligatory
as the initial phase of microscopic examination.
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Concentration techniques
Grouped under 2 categories:
Sedimentation procedures: In which the eggs
and cysts settle down at the bottom.
Flotation procedures: In which the eggs and
cysts float at the surface due to specific gravity
gradient.
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Concentration techniques contd
The basic disadvantage of sedimentation technique is
that examination of the sediment is often difficult
due to the presence of excessive faecal debris that
may mask the presence of the parasites. The basic disadvantage of flotation technique is that
not all eggs and cysts float in the flotation
procedures.
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Concentration techniques contd
Two commonly used concentration
techniques:
Formalin-ether
Saturated salt solution technique
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Formal ether sedimentation
technique Take faeces in 10 ml of
water and mix
thoroughly.centrifuge. Resuspend the sediment
in7 ml of
10%formaldehyde.Add 3
ml of ether (or ethylacetate).
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Formal ether sedimentation technique
Advantages:
Faecal odour is removed.
The sensitivity of detecting the ova or cystsincreases by 8-10 folds.
The examination is easier than examining a directwet smear.
The size and shape of the parasitic structures ismaintained.
It is inexpensive, easy to perform and can be done atany level of health infrastructure.
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Formal ether sedimentation technique
disadvantages
Faecal debris may mask the parasitic
structure.
Trophozoite forms are not detected in this
method.
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Saturated salt flotation technique
Add salt solution so that
the container is nearly
full, glass slide laid on the
top of the container
Allowed to stand for 20
minutes after which the
glass slide is quickly lifted,
and examined under themicroscope after putting a
coverslip
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Preparation of faecal smears for
staining
Spread the sample evenly along the slide
Fix immediately in Schaudinns fluid; leave inthis fixative for at least half an hour
Schauddins fluid consists of saturated solutionof mercuric chloride, ethanol and glacial aceticacid
Iron haemotoxylin stain
Trichrome stain
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Direct microscopic examination
Fecal leucocytes
One drop of stool (preferably including blood
and mucus) is mixed with 2 drops ofmethylene blue on a glass slide
Use 40 X magnificationlarge no. of
polymorphonuclear leukocytes indicatesdiffuse colonic inflammation caused by aninvasive enteric pathogen
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Fecal leucocytes presence indicate an invasive bacterial causes,
like shigella, yersinia, campylobacter,enteroinvasive E. co1i (EIEC), salmonella, amoebiccolitis, idiopathic inflammatory bowel disease orpseudomembranous colitis.
Absence of fecal leucocytes indicates non-
invasive bacterial causes, likecholera,enterotoxigenic E. coli (ETEC) or viralgastroenteritis. Giardiasis and parasitic infectiongenerally do not produce fecal leucocytes.
Fats or oils should point toward one of the diseasesthat cause chronic malabsorption as in chronicpancreatitis, sprue or other small bowel disease.
RBC always suggests hemorrhage.
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E. Hystolytica
Quadrinucleate cyst
15-40 u Motility on wet mount
Ingested RBCs
ELISA & PCR used to be differente fromnonpathogenic Entamoeba dispar
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E histolytica quadrinucleate
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Ascarias lumbricoides
Unfertilized egg
90x55 micrometer ,
brownish. Elongated ovoidal in
shape.
Egg shell is thinner
than the fertilized
Ascarias egg.
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Ascaris lumbricoides
Fertilized egg
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Hookworm egg
Faecal smear , Wet
mount.
A four-cell stage egg ,40x60 micrometer.
A thin egg shell.
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Giardia lamblia Giemsa
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Giardia lambia
Foul smelling diarrhea
Malabsorption like syndrome with
steatorrhea, weight loss, anorexia
Shedding of cysts is irregular in stools
If multiple specimens fail to reveal the
organism, a duodenal aspirate or Enterotestcan be used
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Cryptosporidia acid fast
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CULTURE
Fecal suspension of 1:10 dil in 2-3ml of phosphate buffer
saline or 0.1% peptone water is inoculated in Shigella -MacConkey agar; desoxycholate citrate agar
(DCA), xylose lysine desoxycholate medium (XLD);
Salmonella - MacConkey agar; brilliant green agar;
bismuth sulfite agar; salmonella shigella agar (SSA);
E. coli - MacConkey agar
Y. enterocolitica - MacConkey agar; SSA;
V. cholerae, Non-01 V. cholerae, V. parahemolyticus -TCBS agar; tellurite taurocholate agar;
Campylobacter - Campy-BAP; Skirrow's; Butzler's
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CHEMICAL EXAMINATION
Occult blood: Hookworms, Amoebiasis, UC
Excess Fat excretion: (Oil red O, Sudan III,
Sudan IV) >60 fat droplets/ HPF- Steatorrhoea
Fecal Osmotic Gap: 290-2(Na+K)
>150mOsm/Kg Osmotic diarrhoea
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