Microscopic exam. Fat colorless, neutral, fatty acids, crystals and soaps. Undigested food, meat fibers, starch none Eggs and segment of parasite

Transcription

1 Stool examination Stool analysis determines the various properties of the stool for diagnostic purposes. Frequently ordered tests on faeces includes tests for leukocytes, blood, fat, parasites, and pathogens. Bacteria, viruses, intestinal parasites and other malfunctions can be revealed from stool samples. Stool cultures are necessary in epidemiology and public health studies.

2 Microscopic exam Fat colorless, neutral, fatty acids, crystals and soaps. Undigested food, meat fibers, starch none Eggs and segment of parasite none Yeasts none Leukocytes none

3 Chemical examination Water Ph Occult blood Minerals Reducing substances up to 75% neutral to alkaline None variable <0.25 g/dl

4 Patient preparation & sample collection After patient has been prepared & instructed for stool sampling do: Collect faeces in dry, clean, urine free container with cover. Collect entire stool specimen and transfer it in container using tongue depressor, spatula or wooden stick. A sample of 2.5cm or 64.7mg is sufficient. Warm stool is best for detection of ova &parasites. Do not refrigerate specimen for (o & p).

5 Techniques used in diagnosis Wet preparation technique. Concentration technique : a/ Flotation technique b/sedimentation technique Staining procedures: a/ Trichrome b/ Iron haematoxlin PCR Antigen detection Cultivation or culture

6 Types of specimens Ordinary defecated sample Stool bag sample Rectal swab sample Adhesive tape sample

7 Diagnostic value of stool sample Detection of ova and parasites Detection of worms and segments Culture for bacteria cholera and salmonella Culture for viruses Rota and Polio Diagnosis of malabsorption Detection of occult blood

8 Macroscopic analysis Color : Yellow Green Black Red Clay Others Consistency: Soft Watery Mucoid bloody Ph : Alkaline or acidic Odor : varies Observe and report : Mucus Pus Blood Segment or Worm.

9 Microscopic examination On a clean slide on a drop of a suitable solution e.g. saline, iodine, methylene blue eosine, formal saline or Zink sulphate emulsify a suitable amount of stool and make a thin homogenous film covered with cover glass examine on a microscope under 10x power then 40x power and then report.

10 Macroscopic analysis Color : Yellow Green Black Red Clay Others Consistency: Soft Watery Mucoid bloody Ph : Alkaline or acidic Odor : varies Observe and report : Mucus Pus Blood Segment or Worm.

11 precautions Always wear protective gloves Reject contaminated samples Reject dry & delayed samples >1hour Use only covered stool containers Examine samples within one hour Do not discard sample till result verification

12 Stool Analysis What is the stool or feces? 1. Waste residue of indigestible material (cellulose during the previous 4 days) 2. Bile pigments and salts 3. Intestinal secretions, including mucus 4. Leukocytes that migrate from the bloodstream 5. Epithelial cells that have been shade 6. Bacteria and Inorphosphatesganic material(10-20%) chiefly calcium and. Undigested and unabsorbed food.

13 Random Collection 1. Universal precaution 2. Collect stool in a dry,clean container 3. uncontaminated with urine or other body secretions, such as menstrual blood 4. Collect the stool with a clean tongue blade or similar object. 5. Deliver immediately after collection

14 Ova and parasites collection 1. Warm stools are best for detecting ova or parasites. Do not refrigerate specimen for ova or parasites. 2. If the stool should be collect in 10 % formalin or PVA fixative, storage temperature is not critical. 3. Because of the cyclic life cycle of parasites, three separate random stool specimens are recommended.

15 Enteric pathogen collection 1. Some coliform bacilli produce antibiotic substances that destroy enteric pathogen.refrigerate specimen immediately. 2. A diarrheal stool will usually give accurate results. 3. A freshly passed stool is the specimen of choice. 4. Stool specimen should be collected before antibiotic therapy, or as early in the course of the disease. 5. If blood or mucous is present, it should be included in the specimen

16 Interfering factors 1. Patients receiving tetracycline, anti-diarrheal drugs, barium, bismuth, oil, iron, or magnesium may not yield accurate results. 2. Bismuth found in toilet tissue interferes with the results. 3. Do not collect stool from the toilet bowl. A clean, dry bedpan is the best. 4. Lifestyle, personal habits, environments may interfere with proper sample procurement.

17 Normal values in stool exam Amount Color Odor Consistency Size and shape Gross blood Mucus Pus Parasites g/d Brown varies with ph plastic, soft, bulky and small Formed None None None None

18 Normal values in stool analysis Microscopic examination Normal values Fat (Colorless, neutral fat (18%)and fatty acid crystals and soaps) Undigested food amount None to small Meat fibers, Starch, Trypsin None Eggs and segments of parasites None Yeasts None Leukocytes None

19 Normal values in stool analysis Chemical examination Normal values Water Up to 75 % ph Occult blood Negative Urobilinogen g/24hr Porphyrins Coporphyrins: g/24hr Uroporphyrins:10-40 mg/24hr Nitrogen <2.5 g/24hr

20 Normal values in stool analysis Chemical examination Normal values Bile children Negative in adults :positive in Trypsin amounts greater amounts in units/g( positive in small in adults; present in normal children. Osmolarity osmol- used mosm with serum arity to calculate osmotic gap Sodium meq / 24hr

21 Normal values in stool analysis Chemical examination Chloride Potassium Lipids ( fatty acid) Normal values meq / 24 hr meq /24 hr 0-6 g / 24 hr

22 Clinical Implications 1. Fecal consistency may be altered in various disease states a. Diarrhea mixed with mucous and red blood cells is associated with 1. Typhus 2. Typhoid 3. Cholera 4. Amoebiasis 5. Large bowel cancer

23 Clinical Implications b. Diarrhea mixed with mucus and white blood cells is associated with 1. Ulcerative colitis 2. Regional enteritis 3. Shigellosis 4. Salmonellosis 5. Intestinal tuberculosis

24 Clinical Implications C. Pasty stool is associated with a high fat content in the stool: 1. A significant increase of fat is usually detected on gross examination 2. With common bile duct obstruction, the fat gives the stool a putty- like appearance. 3. In cystic fibrosis, the increase of neutral fat gives a greasy, butter stool appearance.

25 Stool Odor Normal value Varies with ph of stool and diet. Indole and sketole are the substances that produce normal odor formed by intestinal bacteria putrefaction and fermentation. Clinical implication. 1. A foul odor is caused by degradation of undigested protein. 2. A foul odor is produced by excessive carbohydrate ingestion. 3. A sickly sweet odor is produced by volatile fatty acids and undigested lactose

26 Stool color Normal value : Brown Clinical implication: 1. Yellow to yellow-green : severe diarrhea 2. Green : severe diarrhea bile Black: resulting from bleeding into the upper gastrointestinal tract (>100 ml blood) 3. Tan or Clay colored : blockage of the common bile duct. 4. Pale greasy acholic (no bile secretion) stool found in pancreatic insufficiency.

27 Stool ph Normal value : Neutral to acid or alkaline Clinical implication 1. Increased ph ( alkaline) a. protein break down b. Villous adenoma c. Colitis d.antibiotic use 2. Decreased ph ( acid) a. Carbohydrate malabsorption b. Fat malabsorption c. Disaccharidase deficiency

28 Stool color(con) 4. Maroon-to-red-to-pink : possible result of bleeding from the lower gastrointestinal tract (e.g. Tumors, hemorrhoids, fissures, inflammatory process) 5. Blood streak on the outer surface of usually indicates hemorrhoids or anal abnormalities. 6. Blood in stool can arise from abnormalities higher in the colon. In some case the transit time is rapid blood from stomach or duodenum can appear as bright or dark red or maroon in stool.

29 Blood in Stool Normal value : Negative Clinical Implication : 1. Dark red to tarry black indicates a loss of 0.50 to 0.75 ml of blood from the upper GI tract. 2. Positive for occult blood may be caused by a. Carcinoma of colon b. Ulcerative colitis c. Adenoma d. Diaphramatic hernia e. Gastric carcinoma f. Diverticulitis g. Ulcers

30 Mucous in Stool Normal value : Negative for mucous Clinical Implication: 1. Translucent gelatinous mucous clinging to the surface of formed stool occurs in a. Spastic constipation b. Mucous colitis c. Emotionally disturbed patients d. Excessive straining at stool 2. Bloody mucous clinging to the surface suggests a. Neoplasm b. Inflammation of the rectal canal

31 Mucous in Stool (con) 3. Mucous with pus and blood is associated with a. Ulcerative colitis b. Bacilliary dysentery c. Ulcerating cancer of colon diverticulitis e. Intestinal tuberculosis d. Acute

32 Fat in Stool Normal value : fat in stool will account for up to 20 % of total solids. Lipids are measured as fatty acids (0-6.0 g/24hr) Clinical Implication : 1. Increased fat or fatty acids is associated with the malabsorption syndromes a. Non tropical sprue b. Crohn s disease c. Whipple s disease d. Cystic fibrosis e. Enteritis and pancreatic diseases f. Surgical removal of a section of the intestine

33 Urobilinogen in Stool Normal value : Ehrlich units / 24 hr Ehrlich units/100 g Clinical Implication: 1. Increased values are associated with Hemolytic anemias 2. Decreased values are associated with a. Complete biliary obstruction b. Severe liver disease, infectious hepatitis c. Oral antibiotic therapy that alters intestinal bacteria flora d. Infants are negative up to 6 months of age

34 Bile in Stool Normal value Adults negative Children may be positive Clinical Implication: 1. Bile may be present in diarrheal stools. 2. Increased bile levels occur in Hemolytic anemia

35 Trypsin in Stool Normal value : Positive in small amounts in 95 % of normal persons. Clinical Implication : Decreased amounts occur in a. Pancreatic deficiency b. Malabsorption syndromes c. Screen for cystic fibrosis

36 Leukocytes in Stool Normal value : Negative Clinical Implication 1. Large amounts of leukocytes a. Chronic ulcerative colitis b. Chronic bacillary dysentery c. Localized abscess d. Fistulas of sigmoid rectum or anus 2. Mononuclear leukocytes appear in Typhoid

37 Leukocytes in Stool (con) 3. Polymorphonuclear leukocytes appear in a. Shigellosis b. Salmonellosis c. Yersinia d. Invasive Escherichia coli diarrhea e. Ulcerative colitis 4. Absence of leukocytes is associated with a. Cholera c. Viral diarrhea b. Non specific diarrhea d. Amebic colitis e. Noninvasive E.coli diarrhea f. Toxigenic bacteria Staphylococci spp., Clostridium Cholera g. Parasites-Giardia

38 Porphyrins in Stool Normal value : Coproporphyrin g / 24hr Urophorphyrin g / 24 hr. These values vary from Lab to Lab. Clinical Implication: 1. Increased fecal coproporphyrin is associated with a. Coproporphyria (hereditary) b. Porphyria variegata c. Protoporphyria d. Hemolytic anemia 2. Increased fecal protoporphyrin is associated with a. Porphyria veriegata c. Acquired liver disease b. Protoporphyria

39 Stool Electrolytes Normal values : Sodium meq / 24 hr Chloride meq / 24 hr Potassium meq /24 hr Clinical Implication : 1. Idiopathic proctocolitis Sodium and Chloride Normal Potassium 2. Cholera Sodium and Chloride

40 Why study Parasitology Many of these parasites are causative agents of major public health problems of the world. Recent estimates of prevalence of parasites in the world are: Ascaris Hookworms Whipworms Filarial worms Malaria Schistosomes Amebiasis Taenia tapeworms Clonorchis Chagas Disease 1.5 billion 1.3 billion 1 billion 657 million 500 million 210 million 50 million 50 million 20 million 15 million These parasites cause varying morbidities and even mortalities 40

41 41

42 DIAGNOSIS DIRECT INDIRECT MOLECULAR Urine Stool Sputum Biopsy Blood Aspirates IHAT LAT IFAT ELISA CFT DEIDT PCR DNA probes 42

43 STOOL EXAMINATION MACROSCOPIC Consistency Colour Composition OTHERS MICROSCOPIC Culture Cellophane tape Permanent Diect saline smear Baeremann tech. Temprory Iodine smear Ova quantitaion (Stoll & Kato) Concentration techniques Floatation Sedimentation Saline Formol ether Sat saline Zinc sulphate Sheather s sugar 43

44 WET MOUNT PREPARATION It is a fast, simple, procedure and provides a quick answer when positive It provides an estimate of the parasitic burden It can be used as a safe guard It may be more cost effective to delete the direct smear and begin the stool examination with the concentration procedure. Results should be confirmed by permanent stained smears

45 Lugol iodine acetic acid solution causes trophozoite forms to become nonmotile. the Using a fine Pasteur pipette, allow a drop of methylene blue solution to run under the coverslip over the saline preparation (Fig. 7). This will stain the nuclei of any cells present and distinguish the lobed nuclei of polymorphs from the large single nuclei of mucosal cells. If a drop of eosin solution is added, the whole field becomes stained except for the protozoa (particularly amoebae), which remain colourless and are thus easily recognized. 45

46 STOOL EXAMINATION MACROSCOPIC EXAMINATION COLOUR Pale=Steatorrhea (G.l) CONSISTENCY -Liquid (Troph) -Formed (Cyst) -Semi formed (Cyst) COMPOSITION?? Blood?? Mucus (dysentry) Adult PARASITES *Ascaris worm *E. vermicularis *T. saginata 46

47 STOOL EXAMINATION Temporary Saline smear Iodine smear saline Iodine 1% Huge number of: Huge number of: Eggs Cyst morphological details Protozoal troph. Motility (Amoeb, flagellates) 47

48 Staining the saline preparation with methylene blue

49 Microscopic examination of fecal material WET MOUNT STAINED SMEAR Concentration methods generally appear unsuitable for B.hominis, because they cause disruption of the vacuolar, multivacuolar and granular forms of the organism.

50 Concentration procedures Flotation techniques Zinc sulfate Sedimentation techniques Formalin ethyl acetate The preparation is clean. Cysts are suitable for inoculation into culture media It is the method of choice in most laboratories It is ideal for large volume laboratories It can be performed on fresh material and on specimens fixed in most of the available preservatives The sedimentation technique used at (CDC) It can only be performed on fresh material or on specimen preserved in preservatives containing formalin Contain an excess of fecal debris that could mask the presence of parasitic cysts.

51 STOOL EXAMINATION Scanty infection Concentration techniques Sedimentation Heavy eggs (Ascaris Floatation Non Operculated eggs egg) Trematodes ( S. m.) Operculated eggs Cestode (Trematodes) Larvae (Strong sterc.) Nematode(Hookworms,Trichoston g) Cysts

52 STOOL EXAMINATION Saline sedimentation Mesh wire gauze Saline Emulsify Conical flask 10 g stool Sediment 52

53 STOOL EXAMINATION Formol Ether Sed. Conc. Ether Ether 10% Formalin debris formalin 1 g stool Sediment Thorough mixing Conical flask centrif. tube Ether adsorbs fecal debris & floats. Formalin fixes & preserves the specimen. 53

54 Floatation concentration STOOL EXAMINATION Clean light eggs & cysts Sat saline Sheather s sugar Zn sulphate Tin container Cestode eggs (non op) Nematode eggs????? Hookworms??????? Trichostong Egg of S.m. Eggs of small tapeworms Cysts 20 min Seive Crypto, Iso. oocysts Centrif. 2 min 54

55 STOOL EXAMINATION Kato technique Mesh screen Hole Remove the template Template Cellophane soaked by glycerin (clears faeces( Egg count/ g stool Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni

56 STOOL EXAMINATION Stoll s technique Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni 24 hr stool 60 CC 4 g Stool 56 CC Shake well 0.15 CC NaOH Egg count/ slide Eggs/1g= Eggs/slideX Erlynmeyer flask Egg/day=Eggs/1g 56 X stool

57 STOOL EXAMINATION Baermann s technique Stool/soil seive CC Warm water Glass funnel 30 min centrifuge clamp Detec. Of Nematode L. /stool, soil

58 Cultivation of intestinal protozoa Xenic in which the parasite is grown in the presence of an undefined flora. D. fragilis,b. coli, E. histolytica and B. hominis Monoxenic in which the parasite is grown in the presence of a single additional species E. histolytica and B. coli Axenic in which the parasite is grown in the absence of any other metabolizing cells. G. intestinalis, E.histolytica and B.hominis Cultures are primarily research tools rather than diagnostic tools.

59 STOOL EXAMINATION Cultures for Nematode larvae Filter paper culture Filter paper Slide Sealed petri dish Water Scanty infection Larvae of: St. stercoralis (A,L) Hookworms 59 Trichostrong

60 INDIRECT IMMUNOLOGICAL METHODS Scanty infection. Tissue parasite no portal of exit (Hydatid dis.) Migratory stage (Fasciola) Chronic infection fibrosis (Bilharziasis) 60

61 STOOL EXAMINATION Permanent Stained smears Iron haematoxylin stain Trichrome stain Modified Ziehl Neelsen stain (Crptosporidum.) 61

62 INDIRECT IMMUNOLOGICAL METHODS Antigen detection ore specific ore accurate. ctive infection arly uantitative Antibody detection Ab remain in serum for months even after cure 62

63 Antigen detection offer advantages in labor, time, simple to perform, and batching efficiency that may lead to reduce costs offer both increased sensitivity and specificity compared to conventional staining methods These reagents are particularly helpful in an outbreak or when screening patients with minimal symptoms Can detect only one or two pathogens at one time It is costly

64 Low sensitivity of microscopic examination Many E.histolytica infections were confused with E.dispar The only way to distinguish E.dispar from E.histolytica microscopically is erythrophagocytosis. False-positive results due to misidentification of macrophages and nonpathogenic species of Entamoeba Antigen based ELISA Some of the assays differentiate E.histolytica from E. dispar They have excellent sensitivity and specificity They are readily usable by even non experienced laboratory personnel Used as large scale screening tools in epidemiological studies

65 INDIRECT IMMUNOLOGICAL METHODS IHAT Ag Sensitized heep s RBC (O ve) LAT Ag + + Latex particlepatient s serum (?? AB) Patient s serum (?? AB) Agglutination 65 Agglutination

66 Immunofluorescent-antibody test (IFAT) using monoclonal probes specific for Enterocytozoon bieneusi or Encephalitozoon intestinalis. The sensitivity and specificity of IFAT were 100% Moreover, Secies identification by IFAT was more rapid and less expensive than that by PCR. IFAT is a suitable test for detection of microsporidia in developing countries.

67 INDIRECT IMMUNOLOGICAL METHODS INDIRECT FLUORESCENT ANTIBODY TEST fluorescein Anti human AB Patient s serum (?? AB) parasite 67

68 INDIRECT IMMUNOLOGICAL METHODS ELISA OPD Peroxidase E OPD Anti human AB atient s serum (?? AB) Ag AB Flat bottom plastic micrititre plate 68

69 INDIRECT IMMUNOLOGICAL METHODS CFT Sheep s Anti sheeprbc AB AB comple ment Patient s serum (?? AB) Ag Tube / microplate 69

70 INDIRECT IMMUNOLOGICAL METHODS Double Electro Immuno Diffusion Line of ppt Electric current A b Ag Buffere d gel 70

71 INDIRECT IMMUNOLOGICAL METHODS Immunodiagnostic Strip Test (Dip Stick Test) Pt bld (?Ag) Coloured dye Monoclonal Ab trocellulose strip Malaria, Filaria, African tryp. Ag

72 PCR PCR was more sensitive, specific, and easier to interpret Nonhuman pathogens can be distinguished from human pathogens It takes longer time It is technically complex and is costly. Thus it may be not well suited for use in developing countries.

73 MOLECULAR BIOLOGICAL TECHNIQUES Polymerase Chain Reaction (PCR) Single stranded DNA Replication 73 Detection T cruzi, T gondii

74 10 X Objective 74

75 40 X Objective 75

76 2 VIAL COLLECTION KITS 5% OR 10% FORMALIN PVA ONCENTRATION PERMENANT STAINED SMEAR Trichrome or Iron haematoxylin GENERAL MORPHOLOGY PRECISE MORPHOLOGY

77 Non-parasitic structures found in faeces: Care must be taken not to report as parasites those structures that can be normally found in faeces such as: muscle fibres, vegetable fibres, starch cells (stain blueblack with iodine), pollen grains, fatty acid crystals, soaps, spores, yeasts, and hairs. Large numbers of fat globules may be seen in faeces when there is malabsorption. Charcot Leyden crystals (breakdown products of eosinophils) can sometimes be seen in faeces (also in sputum) in parasitic infections. They appear as slender crystals with pointed ends, about 30 40m in length 77

78 Structures found in faeces that required differentiation from parasites. Structures found in faeces that 78required differentiation from parasites.

79 Image illustrating Red Blood Cells in slide preparation. Image illustrating Yeast Cells in slide preparation79 Note similarity to parasitic oocysts. Image illustrating Fat Globules in slide preparation Image illustrating Vegetable cell in slide preparation.

80 Image illustrating Vegetable Spiral in slide preparation. Image illustrating Vegetable cell in slide preparation. 80 Image illustrating a Vegetable Spiral in slide preparation. Such spirals may appear similar to proglottids.

81 Image illustrating pollen in slide preparation that could be mistaken for a Taenia egg. The shell is thinner, of non-uniform thickness, and no hooks are visible. Image illustrating pollen resembling a Hymenolepis nana egg. Hooks and polar filaments are not visible. Image illustrating pollen in slide preparation using a color filter Image illustrating geranium pollen cells in slide preparation. 81

82 Image illustrating peach hair in slide preparation. Note the similarity to Strongyloides stercoralis. Image illustrating vegetable hairs in slide preparation. 82