Medical parasitology lab Cryptosporidium parvum Infect human and

Medical parasitology lab

*Cryptosporidium parvum Ø Infect human and most mammals. Ø The infective stage is oocyst containing sporozoites measuring 4 -6µ in diameter. Ø The diagnostics stage is oocyst containing 4 sporozoites. Ø Diagnosis: Ø Detecting oocyst in stool. Ø Acid-fast stain

*Isospora belli Ø Isosporiasis is a human intestinal disease caused by the parasite Isospora belli. Ø The coccidian parasite Isospora belli infects the epithelial cells of the small intestine, and is the least common of the three intestinal coccidia that infect humans. Ø Infection causes acute, non-bloody diarrhea with crampy abdominal pain, which can last for weeks and result in malabsorption and weight loss. In immunodepressed patients, and in infants and children, the diarrhea can be severe. Eosinophilia may be present. Ø Diagnosis: Ø Acid- fast stain

Cryptosporidium parvum_ wet mount *

* Oocysts in clinical specimens may be difficult to identify with out special staining. * Modified acid-fast (partial acid-fast) stains are recommended for identifying these organisms. * This test detects coccidian parasites (Cryptosporidium, Cyclospora, or Isospora belli) in stool. It is used to evaluate chronic diarrhea. Cryptosporidium parvum oocyst Isospora belli oocyst

* Principle of acid fast stain * The oocysts absorb the red from the carbol-fuchsin stain and may appear in a range of colors from pink to dark purple with bright red being typically seen. * The background material typically stains blue or light red. * Specimen Collection Concentrated sediment of fresh or formalin preserved stool may be used. Other types of clinical specimens such as bile , duodenal fluid, pulmonary fluid (induced sputum, bronchial washings, biopsy specimens may also be used to stain for organisms.

procedure 1 2 3 1 -carbol-fuchsin for 3 -5 mint 2 -rinse by tap water 3 - add acid alcohol as decolorizer for 1 mints 4 - rinse by tapewater 5 - add methylene blue as counter stain for 1 mint Rinse by tapewater then examine under oil immersion. 4 5

* Iron Hematoxylin Stain *The iron hematoxylin stain reveals excellent morphology of the intestinal protozoa. *Iron hematoxylin was the stain used for most of the original morphological descriptions of intestinal protozoa found in humans. *Under oil immersion power (1, 000), one can examine the diagnostic features used to identify the protozoan parasite. Appearance of select protozoan structures and background material on the iron hematoxylin stain Protozoa cytoplasm Blue to purple Protozoa nuclear material Dark blue to dark purple Debris and background material Light blue sometimes with pink tint

Method 1 *

Place approxiamtely 50 ml of fixative solution, solution 1, solution 2 in separate jars. Place approximately 50 ml of deionized water in separate coplin jar. * Place the stool smear in fixative solution for 30 seconds. Remove slide from the coplin jar and let air dry.

Place the stablized smear in solution 1 for 15 seconds. Remove slide from deionzed water. Rinse briefly with deioized water. Let air dry. Place slide in solution 2 for 15 seconds. Rinse briefly with deionized water. Let air dry. Remove slide from deionized water. And let air dry.

Method 2 *

Prepare slide for staining (for SAF smears. Place slide in 70% ethanol for 5 min. Place slides in 50% ethanol for 2 min.

Wash slide in running tap water (constant stream of water into the container) for 10 min. Place slide in iron hematoxylin working solution for 4 to 5 min. Wash slide in running tap water (constant stream of water into the container) for 10 min. Differentiate slides (one by one) in destaining solution for 30 seconds. Place slides under running tap water (best if tepid) for 10 min.

Place slide in 95% ethanol for 5 min. * Place slide in two changes of 100% ethanol for 5 min each. * Place slide in two changes of xylene for 5 min each. Add Per mount to the stained area of the slide and cover with a coverslip. Examine the smear microscopically with the 100 x objective.

*Nematodes = roundworm • General characters 1. 2. Non segmented Sex are separate Male is smaller than female and its posterior end is curved ventrally. Female are either • Larviparous or viviparous: giving birth to larvae • Oviparous: laying egg • Oviviviparous: laying eggs which contain larvae and which hatch out immediatly

Tissue Nematodes * Trichinella spiralis Intestinal Nematodes * Entrobius vermicularis * Trichuris trichiura * Ancylostoma duodenale * Strongyloides stercoralis * Ascaris lumbricoides

Trichinella spiralis Ø Adult inhabit the small intestine of the rats, pigs and human. Both males and females lie freely in the lumen of the intestine of pigs, rats and human. Ø Fertilized female only penetrate the mucosa where the larviposit, they do not lay eggs.

* Trichinella spiralis encysted in muscle larvae

Trichinella spiralis Disease trichinosis, trichiniasis or trichinelliasis infective stage Larvae is live encysted in the flesh of the host, diagnostic stage. Larvae Route of infection dead end host. Diagnosis: eating undercooked pork containing infective encysted larvae. Human 1. 2. 3. Finding larvae in the blood during migration or in muscle after encystation. Immunological tests. X- ray to detect the calcified larvae in muscle.

*Entrobius vermicularis Eggs Adults

• Young and mature worms are present in small intestine (at terminal ileum till fertilization).

Egg Morphology : 20 -50 u, transparent with double walled shell. • Oval, It may show one side convex and the other flat. • Shell: double layered, thick, colorless. • Embryo stage of development varies may be unembryonated, • embryonated, mature. Microscopically examined the slide under low power. Reduced light is recommeneded as the eggs will appeear colorless, making them difficult to detect under high light intensity.

*These cells are readily differentiated from parasitic forms because they lack internal structures. Entrobius vermicularis

* The female is 0. 88 -1. 3 cm long, it has a long tapering tail resembling 1/3 its length, its straight. Gravid females are present at lower rectum where they lay ova at perianal region around anus (oviparous). The tail of female pointed resembles pinhead The male is shorter than female (2 -5 mm) the tail is curved strongly to ventral side, and has a single spicule.

Entrobius vermicularis Alternative names human pin worm and seat worm Disease oxyurdiasis. Infective stage Embryonated Eggs Definitive host human Diagnosis: When found in feces, the eggs are typically unembryonated as seen in this wet preparation. The typical embryonated (infective) eggs are usually seen in the cellulose tape preparation the most reliable means for detecting the parasite . 1. Egg in stool is rarely detected but adults present after purgative. 2. Egg can be detected at perianal area as follow: 1. N. I. H method (National Institute of Health), its based on swabbing the perianal area with a cellophane paper. 2. The adhesive cellulose tape (scotch tape), better result. 3. The Vaseline cloth wiping, the collected ova examined .

* Adhesive tape method for the detection of pinworm eggs 1. Adhesive tape 2. Use gloves !(pinworm eggs are infectious!) 3. Prepare the tape then Take sample on peri-anal skin in the morning 4. Stick tape on microscope slide 5. Place a drop of Xylene on the edge of the tape to remove airbubbles

Trichuris trichiura

• • • Adult inhabit the large intestine in the caecum of man. The adult male smaller than female, male 3. 4 -4. 5 cm, female 4 -5 cm. Its commonly called whip worm because of the shape of this worm (anterior thin and posterior thick).

v Trichuris trichiura eggs have distinct shape. (oviparous) v Shell: smooth, yellow brown color due to bite contact. v Hyaline plug at each pole. v Diagnosis: v Stool examination to detect eggs.