Stool specimen lecture NO 9 Dalia Kamal Eldien Mohammed

introduction  Proper gastrointestinal function is needed to eliminate toxic substances, pathogenic microbes, and undigested food particles from the body to prevent health problems. A stool analysis is a series of tests done on a stool (feces) sample to help diagnose certain conditions affecting the digestive tract . These conditions can include infection (such as from parasites, viruses, or bacteria), poor nutrient absorption, or cancer (checking for hidden occult blood).

Stool analysis include: fecal occult blood test can be used to diagnose many conditions that cause bleeding in the gastrointestinal system including colorectal cancer or stomach cancer Chemical tests Fecal PH: test may be used to determine lactose intolerance or the presence of an infection. Fecal fat test: that checks for the male absorption of fat. Fecal elastase: levels are becoming the mainstay of pancreatitis diagnosis. Microbiology tests Parasite testing

Microbiology tests& Parasite testing large numbers of abnormal bacteria, viruses, fungi, or parasites can grow in the intestines and cause infections and diseases. Although more than 50 different kinds of bacteria normally live in the intestines Acute infective diarrhoea and gastroenteritis (diarrhoea with vomiting) are major causes of ill health and premature, Loss of water and electrolytes from the body can lead to severe dehydration which if untreated can be rapidly fatal in young children, especially those that are malnourished, hypoglycaemic

Cause of diarrhoea Invasive organisms such as shigellae, campylobacters, some salmonellae, and E. histolytica are associated with dysentery (passing of blood and mucus in stools). Organisms such as Rotaviruses, V. cholerae, and enterotoxigenic E. coli, cause watery (secretory) diarrhoea. Diarrhoea is also associated with HIV disease, malaria, severe malnutrition, pneumonia, hepatitis, cirrhosis of the liver, inflammation of the pancreas, tuberculosis of the intestine, colitis, previous surgery of the bowel, and malignant diseases of the intestinal tract. Food poisoning also is another cause of diarrhoea

Possible pathogens Gram positive Gram negative Clostridium perfringens Shigella species types A and C Salmonella serovars Clostridium difficile Campylobacter Bacillus cereus (toxin) Yersinia enterocolitica Staphylococcus aureus Escherichia coli (ETEC, EIEC, EPEC, VTEC) Vibrio cholerae 01, 0139 Other Vibrio species Aeromonas species

VIRUSES: Mainly Rotaviruses and occasionally Adenoviruses, Astrovirus, Calcivirus and Corona virus. PARASITES Entamoeba histolytica, Giardia lamblia, intestinal coccidia (Isospora, Cryptosporidium, Cyclospora) and other protozoan enteric pathogens

Laboratory diagnosis Specimen: Faeces for microbiological examination should be collected during the acute stage of diarrhoea. Give the patient a clean, dry, disinfectant-free suitable wide-necked container Ask the patient to avoid contaminating the faeces with urine(Urinate before collecting). Rectal swabs: Only when it is not possible to obtain faeces

Macroscopical exam Color of the specimen Consistency: Whether it is formed, semi formed, unformed or fluid Presence of blood, mucus or pus Odor Presence of worms seen in a freshly passed stool eg adult stages of Ascaris lumbricoides and Enterobius vermicularis Normal faeces: Appear brown and formed or semi formed. Infant faeces are yellow-green and semi formed.

Microscopical exam 1-Saline and eosin preparations:- to detect E. histolytica and other parasites Place a drop of fresh physiological saline on one end of a slide and a drop of eosin stain on the other. Using a piece of stick or wire loop, mix a small amount of fresh specimen (especially mucus and blood) with each drop Cover each preparation with a cover glass. Examine the preparations using the X10 and X40 objectives :

Wet preparation

report the presence of :  Large numbers of pus cells  RBCs  Amoebas, flagellates- motile E. histolytica trophozoites containing red cells, motile G. lamblia trophozoites, motile Strongyloides larvae.  Eggs, larvae & cysts. Using of Eosin 1%: this provide a pink background and that will help to clear the unstained objects.

Entamoeba histolytica cyst& trophozoite

Giardia lamblia cyst& trophozoite

Enterobius vermicularis egg (Pin worm)

Ascaris lumbricoides egg& worm

Hook worm egg

Taenia saginata scolex& gravite

Fasciola hepatica

Schistosoma eggs

Continue stool exam 2-Basic fuchsin smear to detect campylobacters Prepare when the specimen is unformed and, or, contains mucus, pus, or blood and is from a child under 2 years. Make a thin smear of the specimen on a slide. When dry, gently heat-fix. Stain by covering the smear with 10 g/l basic fuchsin for 10–20 seconds. Wash well with water and allow to air dry, use X100 Look for abundant small, delicate, spiral curved bacteria (often likened to gull wings), S- shapes

campylobacters

3-Motility test and Gram stained smear when cholera is suspected Examine an alkaline peptone water culture (sample from the surface of the culture) for vibrios showing a rapid and darting motility. The preparation is best examined using dark-field microscopy A Direct Gram stain is not useful beyond its determination of the presence of leukocytes, as it will not differentiate suspected pathogens from normal microbial flora.

Stool cultures Routine stool cultures should be examined for the presence for Salmonella, Shigella, and Campylobacter species at minimum Often cultures for Vibrio, Yersinia and E. coli O157:H7 are performed upon doctor request. Enterohemorrhagic E. coli (EHEC) has been isolated from patients who have hemorrhagic colitis and hemolytic-uremic syndrome (HUS).

Inoculate the media making the first streak then use a sterile loop to streak for isolation. Non inhibitory media should always be inoculated first. Routine stool culture MacConkey agar Hektoen enteric (HE) agar (or XLD agar or SS agar) Skirrow& Butzler's (Selective agar for Campylobacter ) Gram negative(GN) broth or Selenite F – enrichment media enhances the growth of Salmonella and Shigella while suppressing the growth of normal bowel flora, they are subcultured to HE at 24 hours.

Yersinia culture a. Cefsulodin-Irgasan-Novobiocin (CIN) agar or Yersinia Selective agar b. Phosphate buffered saline (PBS) broth – suppresses the growth of normal bowel flora allowing easier detection of Yersinia species, but it also slows the growth of the Yersinia. Vibrio culture a. TCBS agar b. Alkaline peptone broth

Incubate media 1. Temperature: a. Campylobacter: 42ºC b. PBS broth: 4ºC c. CIN plate: room temperature d. All other plates: 37ºC 2. Atmosphere: a. Campylobacter: Microaerophilic (increased CO2) b. BAP: either aerobically or CO2 c. All other plates: aerobically 3. Time: 18-24 hours for all, 48 hours and 72 hours for Campylobacter

Colonial morphology After 24 hours incubation, examine the HE and MAC plates for non-lactose fermenting colonies and H2S producing colonies. These colony types are suspicious for Salmonella species and Shigella species NLF but no H2S, but may also be normal enteric flora – so biochemical screens must be performed to rule in or out the presence of these pathogens. a. For colonies that are suspicious for Salmonella or Shigella perform KIA(kliglar iron agar) and LIA (lysine iron agar) b. If screen is positive perform biochemical ID panels such as API 20 E. c. Colonies that appear to be Salmonella species or Shigella species are confirmed by performing serological agglutination tests.

Salmonella sp. after 24 hours growth on XLD

Salmonella sp. On Hektoen enteric

 shigella on XLD

A susceptibility test is performed only on any confirmed colonies of Salmonella and Shigella species when requested. Treatment with antibiotics is not recommended for Salmonella species because it may induce a carrier state in the patient. In most case treatment of the clinical symptoms such as dehydration is sufficient. If no suspicious colonies are found or if all biochemical screens are negative, the report is sent out as: No Salmonella or Shigella isolated

Serological Testing for Salmonella and Shigella A. All isolates that biochemically resemble either Salmonella species or Shigella species must be confirmed by serological methods following site specific procedures. B. For suspected Shigella species, test the following somatic (“O”) antigens: Antigen A = Shigella dysenteriae Antigen B = Shigella flexneri Antigen C = Shigella boydii Antigen D = Shigella sonnei C. For suspected Salmonella species, test the following somatic (“O”) antigens:

Campylobacter Colonies are tested for oxidase test. Any oxidase positive colonies are Gram stained to look for the typical curved rods of Campylobacter species. Sodium Hippurate test will confirm the species C. jejuni. Susceptibility tests are not run on Campylobacter species because their resistance patterns are well established.

Yersinia After 24 hours incubation, examine the CIN plate for dark red colonies with a “bull’s eye” center surrounded by transparent border colonies that indicate mannitol fermentation and is suspicious for Yersinia. Confirm identification of suspicious organisms with various biochemical ID panels. Vibrio After 24 hours incubation, examine the TCBS agar for yellow colonies that indicate sucrose fermentation. Screen suspicious colonies biochemically.

yersinia enterocolitica on CIN agar

Vibrio on TCBS

Escherichia coli O157:H7 Setup stool culture with a MacConkey agar with 1% D-sorbitol (instead of lactose). After 24 hours of incubation, examine Mac-Sorbitol plate for colorless colonies (E. coli O157:H7 is sorbitol negative and most other normal flora strains of E. coli are sorbitol positive) Confirm identification of suspicious organisms with various biochemical ID panels and O157:H7 antisera.

Textbook of Diagnostic Microbiology, Mahon & Manuselis, 3rd edition, Chapter 34, pages 957- 978.