Diarrhoea Lab Diagnosis

7/31/2019 Diarrhoea Lab Diagnosis

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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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Diarrhoea Lab Diagnosis