LAB DIAGNOSIS DIARRHOEAL DISEASES

CAUSES- DIARRHOEA
BACTERIA Enteropathogenic E. coli (EPEC, ETEC, EIEC), Staphylococcus aureus, V. cholerae, V. parahemolyticus, salmonellae, shigella, Clostridium perfringens, Clostridium botulinum, B. cereus, campylobacter, VIRUSES rotavirus, Norwalk virus ,adenovirus PARASITES E. histolytica, G. lamblia, strongyloides, Balantidium coli OTHERS IBD, Malabsorption syndromes

TYPES
ACUTE DIARRHOEA:(3 or More loose stools/day for <4 wks) Infectious -Watery: E. coli (EPEC, ETEC), Staphylococcus aureus, V. cholerae, Clostridium perfringens, rotavirus, adenovirus -Dysentry: E. coli (EIEC, EHEC), campylobacter, Clostridium difficile, E. histolytica Non-Infectious: IBD, food intolerance

CHRONIC DIARRHOEA:(3 or More loose stools/day for >4 wks) Watery -Osmotic: Carbohydrate malabsorption, Osmotic laxative -Secretory: Bacterial toxins, Laxative abuse, Hormonal disorders Inflammatory: Invasive bacterial and parasitic inf, IBD, Pseudomembranous enterocolitis Fatty diarrhoea: Malabsorption

STOOL-Preferred sample
COLLECTION Container should be clean, of sufficient size,with a 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

Rectal swab specimen is used : (1) when it is desirable to collect the feces immediately 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 the stool to the laboratory as the collecting tube contains a transport medium, which can act as a preservative.

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 taurocholatetellurite peptone water pH 9.2 Both are good as Tpt & enrichment media For salmonella selenite broth For Rotavirus examination, a small amount of stool or rectal swab is put into 1 ml phosphate buffered saline solution and frozen at 20 C

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 used for microscopy for several months

 The examination of faeces for parasitological diagnosis is done to detect: Adult worms Segments of tapeworms

Ova and cysts

Larvae Trophozoites Cellular exudates such as WBCs, RBCs, macrophages

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 stool Trophozoites 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

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

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 or larvae, 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 one drop 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 drop of iodine. Put the cover slip separately on both and examine under the microscope.

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Concentration techniques
If the number of parasites in the stool specimens is low,examination of a direct wet mount may not detect them, hence the stool should be concentrated Eggs, cysts and larvae are recovered after concentration procedures whereas trophozoites get destroyed 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 ethyl acetate).

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Formal ether sedimentation technique Advantages:

Faecal odour is removed. The sensitivity of detecting the ova or cysts increases by 8-10 folds. The examination is easier than examining a direct wet smear. The size and shape of the parasitic structures is maintained. It is inexpensive, easy to perform and can be done at any 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 the microscope 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 in this fixative for at least half an hour Schauddins fluid consists of saturated solution of mercuric chloride, ethanol and glacial acetic acid 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 of methylene blue on a glass slide Use 40 X magnification large no. of polymorphonuclear leukocytes indicates diffuse colonic inflammation caused by an invasive enteric pathogen

Fecal leucocytes presence indicate an invasive bacterial causes, like shigella, yersinia, campylobacter, enteroinvasive E. co1i (EIEC), salmonella, amoebic colitis, idiopathic inflammatory bowel disease or pseudomembranous colitis. Absence of fecal leucocytes indicates noninvasive bacterial causes, like cholera,enterotoxigenic E. coli (ETEC) or viral gastroenteritis. Giardiasis and parasitic infection generally do not produce fecal leucocytes. Fats or oils should point toward one of the diseases that cause chronic malabsorption as in chronic pancreatitis, sprue or other small bowel disease. RBC always suggests hemorrhage.

E. Hystolytica Quadrinucleate cyst 15-40 u Motility on wet mount Ingested RBCs ELISA & PCR used to be differente from nonpathogenic Entamoeba dispar

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 Enterotest can 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

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 <50mOsm/Kg- Secretory diarrhoea Fecal pH: <5.6 in carbohydrate malabsorption