Intestinal and Luminal protozoa

Intestinal and Luminal protozoa Bushehr University of Medical Sciences Department: Microbiology and Parasitology Module: Medical Parasitology Instructor: Mohammad Rayani, PhD 1

Flagellates: Giardia lamblia Dientamoeba fragilis Chilomastix mesnili Trichomonas hominis Enteromonas hominis Retortamonas intestinalis Ameba: Entamoeba histolytica Entamoeba dispar Entamoeba coli Entamoeba hartmanni Endolimax nana Iodamoeba bütschlii Apicomplexa: Cryptosporidium hominis Cryptosporidium parvum Cyclospora cayetanensis Isospora belli Other: Blastocystis hominis Balantidium coli INTESTINAL PROTOZOA unicellular eukaryotic organisms 2

Intestinal protozoans Divided into classes based on motility Majority live in colon Few in small intestine - Cryptosporidium parvum, Isopora belli, Giardia lamblia Transmission direct, fecal/oral route Difficult ID small size similar morphological characteristics Dr. M. Rayani (Bushehr University of Medical Sciences) 3

Intestinal protozoans cont. Permanent stained smear - MOST important for ID Developmental stages Trophozoite motile, feeding stage reproduces by binary fission Cyst - resistant stage, infective for humans 4

INTESTINAL AND LUMINAL PROTOZOA AMOEBA Entamoeba histolytica dispar hartmani coli gingevalis Endolimax nana Iodamoeba butschlii 5

Characteristics of Intestinal amebae Move by cytoplasmic protrusions (pseudopods); most slow, random motility Identification based on: size number of nuclei nuclear structure presence of specific internal structures Treatment - only Entamoeba histolytica Dr. M. Rayani (Bushehr University of Medical Sciences) 6

Amebiasis Cause: Entamoeba histolytica A parasitic ameba; class Sarcodina Transmitted via contaminated food & water Cysts are shed in the feces When ingested: Excystation occurs in the intestine Trophozoites grow & reproduce in the intestinal tract 7

Amebiasis Symptoms Abdominal pain Little diarrhea but often blood in the stool ( amebic dysentery ) Possibly of intestinal ulceration; perforation; infection of internal organs such as liver & lungs 8

Entamoeba histolytica - Pathogen Trophozoite 15-20 um progressive motility small, central karyosome fine, evenly distributed chromatin red blood cells diagnostic Cyst mature: 4 nuclei cigar-shaped chromatoid bodies 9

Entamoeba histolytica cont. Only pathogenic ameba World-wide, 40,000-100,000 deaths/year Transmitted - fecal contamination of food/ water; direct with dirty hands & objects Disease: invasive intestinal/extraintestinal infection causing liver, lung or brain abscesses. Bloody stools, increased liver enzymes Treatment: metronidazale 10

Entamoeba coli (non-pathogen) Trophozoite average size 25um (15-30um) large ± eccentric karyosome coarse, uneven/dark solid chromatin ring cytoplasm: purple with vacuoles Cyst mature: 8 nuclei (5+ diagnostic) chromotoidal bars (if present) - splintered 11

Endolimax nana (non-pathogen) Trophozoite large karyosome no peripheral chromatin vacuolated cytoplasm average size 10um (5-12 um) Cyst oval/spherical, 5-10 um up to 4 large karyosomes 12

Iodamoeba butschlii (non-pathogen) Less commonly seen than E. coli & E. nana Nucleus single karyosome no peripheral chromatin Trophozoite: 12-15 um, vacuolated cytoplasm Cysts :large glycogen vacuole 13

INTESTINAL AND LUMINAL PROTOZOA The Urogenital & Digestive Tract Flagellates Giardia lamblia Trichomonas vaginalis Tenax hominis Chilomastix mesnili Retortamonas intestinalis Enteromonas hominis Dientamoeba fragilis 14

Giardiasis Cause: Giardia lamblia A flagellate of class Mastigophora The trophozoite has four pairs of flagella & two nuclei; giving it a face-like appearance Grows in the intestinal tract Cysts are shed in the feces Dr. M. Rayani (Bushehr University of Medical Sciences) 15

Giardiasis Transmission and Symptoms Transmitted via contaminated water & food Foul-smelling profuse diarrhea Sometimes chronic Often misdiagnosed Diagnosis via microscopic examination Enterotest capsule 16

Trichomoniasis Cause: Trichomonas vaginalis Flagellate of the class Mastigophora No cyst stage Transmission & Symptoms Transmitted via sexual contact Genital itching Painful urination with a white, mucoid discharge Occasional reduction of sperm count or erosion of the cervix Dr. M. Rayani (Bushehr University of Medical Sciences) 17

Trichomonas vaginalis (urogenital pathogen) one of most common STDs women: itching, discharge, dysuria men: generally asymptomatic keep specimens moist for motility Wet mount: 5-18 um, look for jerky motility Culture - most sensitive, takes up to 7 days 18

Chilomastix mesnili (non-pathogen) World-wide distribution Found in chimpanzees and monkeys Trophozoite pear shaped 10-20 mu by 3-10 mu elongated cytostome Cyst - lemon shaped with anterior protrusion 19

Dientamoeba fragilis (pathogen) Transmission - no cyst form (oral/fecal route unlikely) Incidence 9x higher if E. vermicularis infection Usually asymptomatic but can be carrier 50-80% binucleate, 4-8 karyosomes, no chromatin, 5-12 um Food vacuoles in cytoplasm 20

Balantidiasis Cause: Balantidium coli Ciliated, in Class Ciliophora Trophozoites grow in the intestinal tract Cysts are shed in the feces and may remain embedded in intestinal walls, causing chronic infections Transmission & Symptoms Contaminated water & food Ulceration in intestines Profuse diarrhea Dr. M. Rayani (Bushehr University of Medical Sciences) 21

Balantidium coli (pathogen) only infectious ciliate (covers surface) largest protozoan generally asymptomatic or self-limiting diarrhea, vomiting Trophozoite - oval, vacuoles in cytoplasm Cyst - 45-75 um; 2 kidney-shaped nuclei 22

Balantidium coli Worldwide Symptoms: mostly asymptomatic or similar to amebasis Reservoirs: Human and animals (pigs, chimpanzees, ) Transmission: waterborne, foodborne Prevention: personal hygiene, water treatment, food safety program 23

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Coccidia characterized by thick-walled oocysts excreted in feces In Humans Cryptosporidium Isospora Cyclospora Sarcocystis Toxoplasma 25

Cryptosporidium 4-5 mm oocysts 4 sporozoites no sporocysts Cyclospora 8-10 mm oocyts 2 sporocysts 2 sporozoites each Isospora belli 30 x 12 mm oocyts 2 sporocysts 4 sporozoites each 26

Cryptosporidium 4-5 mm oocysts 4 sporozoites no sporocysts Cyclospora 8-10 mm oocyts 2 sporocysts 2 sporozoites each Isospora belli 30 x 12 mm oocyts 2 sporocysts 4 sporozoites each 27

Cryptosporidium 4-5 mm oocysts 4 sporozoites no sporocysts Cyclospora 8-10 mm oocyts 2 sporocysts 2 sporozoites each Isospora belli 30 x 12 mm oocyts 2 sporocysts 4 sporozoites each 28

Cryptosporidium parvum Coccidian protozoa Located worldwide Primarily infects small intestine Transmission Fecal-oral Person-to-person Animal-to-person Waterborne Foodborne self-limiting diarrhea in immunocompetent persons watery diarrhea associated with AIDS (life threatening) Dr. M. Rayani (Bushehr University of Medical Sciences) 29

Cryptosporidiosis Cause: Cryptosporidium species In class Sporozoa A common protozoan parasite in humans Transmission and Symptoms Contaminated water (waterborne disease outbreak) Mild diarrhea in non-immunosuppressed persons Severe diarrhea in immunosuppressed persons Is a minute coccidian parasite with worldwide distribution. 20 species of the parasite have been described from a variety of vertebrates including mammals, birds, reptiles and fish. 30

Cryptosporidiosis Pathogenesis oocysts infective when passed +/- auto-infection if cysts rupture in gut Clinical infection - immunocompetent: rapid but selflimiting diarrhea and cramps AIDS patient life-threatening diarrhea (3-17 L/day) electrolyte imbalance possible malabsorption due to injury to villi Modified Ziehl-Nielsen acid-fast stain oocyst: red, 4-6 um, yeast: green 31

Cryptosporidium Life Cycle Infectious form = oocyst Sporozoites invade intestinal epithelial cells Merogony produce merozoites Gametogony produce micro- and macrogametes Sporogony produce sporozoites completed on host cell thin (autoinfection) or thick walled oocysts 32

Molecular Epidemiology 2 major genotypes identified: genotype 1 (C. hominis) only human sources non-infective for mice or calves anthroponotic transmission genotype 2 (C. parvum) human and bovine sources infective for mice and calves zoonotic transmission 33

Cryptosporidiosis: Epidemiology Cosmopolitan ( A serious problem in the warmer ports). The children are more commonly infected than are adults and that nonbreast-fed infants have more cryptosporidiosis than breast-fed infants. Immune deficiency increase risk of disease Infection is by fecal-oral contamination A number of animals can serve as reservoirs of infection Important for travelers (One of the causative travelers diarrhea & in boarding house). Transmited to human by infected water & food & any contaminated sources 34

Cryptosporidiosis: Symptoms & pathgenesis - In immunecompetent persons Self-limited diarrhea that usually lasts about 2 weeks & less commonly is accompanied by Abdominal discomfort, Anorexia, Fever, Nausea, Weight loss - Immunodeficient patients typically have severe diarrhea some times 17 liter water loss/days accompanied by these symptoms. - In patients with AIDS may couse life-threatening disease and has been found in sputum in lang biopsy material and in the biliary tract and has been associated with malabsorption. 35

Cryptosporidiosis: Diagnosis Finding oocyst in fecal material Acid fast modified stains Oocyst: - 4-5 - Round - Without Sporocyst - With 4 Sporozoite are passed in the feces Concentration procedures to improve sensitivity: Sucrose solution or Sheather, s technique Formalin-ether (Formalin ethyl asetate ) 36

Cryptosporidiosis: Prevention Prevention: personal hygiene, surface disinfection, water treatment, food safety program Especial in AIDS disease: Boil drinking water for 1 minue Filter drinking water with devices that remove particles 1 m and larger Use bottled drinking water, especially water obtained from underground sources, i.e., springs or welds which are less likely to be contaminated by Cryptosporidium oocysts Treatment Diease is self-limited infection in immunocompetent patients In AIDS patients: Oral paromomycin 1gr 3times/days, 2 weeks appears to suitable 37

Isosporiasis Clinical infection same as C. parvum Life cycle similar to C. parvum except not infective for 24-48 hours Disease - AIDS patients Diarrhea and weight loss for immunodeficient & suppressed persistent diarrhea that leads to dehydration and electrolyte imbalance, which can be fatal most infections are asymptomatic & self-limiting Dr. M. Rayani (Bushehr University of Medical Sciences) 38

Isosporiasis wide geographical distribution (higher prevalence in warmer climates) monoxenous, probably not zoonosis invades intestinal epithelial cells often asymptomatic symptoms range from mild gastro-intestinal distress to severe dysentery often self-limiting, but can become chronic (wasting, anorexia) symptoms more severe in AIDS patients 39

Isosporiasis: Pathology / clinical symptoms Most infections are asymptomatic and self-limited Symptomatic disease in immune competent individuals includes diarrhea and malabsorption AIDS patients have severe persistent diarrhea that leads to dehydration and electrolyte imbalance, which can be fatal Mucosal epithelial cells in which I. belli develop rupture when the organsims are released creating the symptoms described above 40

Isosporiasis: Diagnosis Wet preps Modified acid-fast stain Permanent smears: best if preserved in SAF 10% formalin Mature oocyst: Oval, 20-30 um with 2 sporocysts 41

Cyclospora Infection with cyclospora spp. is widely distributed probably cosmopolitan. Countries initially identified as having endemic cyclosporiasis: Haiti, Guatemala, Peru and Nepal Infection most common in HIV/AIDS patients. Also important cyclosporosis in travelers. Associated with imported food items, specially raspberries and green leafy vegetables such as basil and mesclun lettuce. Dr. M. Rayani (Bushehr University of Medical Sciences) 42

Pathology Cyclospora infects enterocytes of the small bowel where various stages, sexual and asexual stages have been observed. Villous blunting, mild crypt hyperplasi. The main symptom is watery diarrhea, loss of appetite, weight loss, abdominal bloating and cramping, nausea, fatigue and low grade fever. Incubation period averages one week and illness lasts 6 weeks. Clinical Features symptoms similar to Cryptosporidium and Isospora watery diarrhea/frequent stools 1-2 week duration typical relapses over 1-2 months In the immunocompromised patient, severe diarrhea can last up to 4 months or longe. Extra-intestinal infection appears to be more common in AIDS patients 43

Cyclospora: Epidemiology Food-borne pathogen Not sure if water could also have a role in transmission Infected humans are the only known sources of oocysts Not other reservoir host identified No methods available to grow them in the lab Marked seasonality of cyclospora: temp, humidity, may facilitate sporulation associated with food-borne outbreaks oocysts detected on market vegetables in Peru presumed source: contaminated water or human waste as fertilizer 44

Cyclospora: Diagnosis and treatment Detection of oocysts in stool sample by microscopy Recovery of oocysts in intestinal fluid or small bowel biopsy specimens Demonstration of oocyst sporulation PCR amplification of Cyclospora DNA Can be successfully treated with trimethoprim (TMP)- sulfamethoxasole: TMP 160 mg/ SMX 800 mg bid 7-10d 45

Sarcocystis rare human infection heteroxenous parasite predator-prey life cycle humans support both stages taxonomic confusion generally named after host species Sarcocystis bovihominis Sarcocystis suihominis ingest undercooked meat transient mild to severe diarrhea excrete sporulated sporocysts 13x10 mm 4 sporozoites Dr. M. Rayani (Bushehr University of Medical Sciences) 46

Intestinal and Luminal protozoa Microsporidia spp. New Phylum 143 genera, >1200 species 14 identified human pathogens Produce very resistant spores Usuaually small (1-4 m) A unique organalle (polar tubule) Coiled inside the spores Inject infective spore contents into the host cells Dr. M. Rayani (Bushehr University of Medical Sciences) 47

Microsporidia 99% in AIDS patients (dramatic increases) Identification spores 1.5-3 um confused with yeast spores modified trichrome stain Electron microscopy - biopsy (definitive) 48

The infective form of microsporidia is the resistant spore and it can survive for a long time in the environment. 1 The spore extrudes its polar tubule and infects the host cell. 2 The spore injects the infective sporoplasm into the eukaryotic host cell through the polar tubule. 3 Inside the cell, the sporoplasm undergoes extensive multiplication either by merogony (binary fission) or schizogony (multiple fission). 4 This development can occur either in direct contact with the host cell cytoplasm (e.g., E. bieneusi) or inside a vacuole termed parasitophorous vacuole (e.g., E. intestinalis). Either free in the cytoplasm or inside a parasitophorous vacuole, microsporidia develop by sporogony to mature spores.. 5 During sporogony, a thick wall is formed around the spore, which provides resistance to adverse environmental conditions. When the spores increase in number and completely fill the host cell cytoplasm, the cell membrane is disrupted and releases the spores to the surroundings. 6 These free mature spores can infect new cells thus continuing the cycle. 49

Epidemiology (Microsporidia spp.) Worldwide Both developed and developing countries Symptoms: Immunocompetent people: asymptomatic or self-limiting diarrhea Immunocompromised people: Chronic diarrhea Disseminated diseases (keraconjunctivitis, bronchitis, pnuemonia, hepatitis, ) Reservoirs: human and animals (rabbits, mice, dogs, pigs, cats, cattle, wild birds (parrots), insects?) Transmission: uncertain Airborne transmission? Waterborne transmission?? Transplacental transmission? (Encephalitozoon spp.) 50

Blastocystis hominis Possible Pathogen Controversial: Some labs evaluate when symptoms with no other enteric pathogens more than 5/HPF 5-15 um cytoplasm - dark green nuclei - dark purple/black Dr. M. Rayani (Bushehr University of Medical Sciences) 51

Blastocystis hominis (Possible Pathogen) one of the most common parasites found in the human intestinal tract (resides in the colon and cecum). a nonpathogenic protozoan parasite may be associated with clinical illnesses such as diarrhea, abdominal pain, abdominal ballooning, anorexia, vomiting, and urticaria Controversial: Some labs evaluate when symptoms with no other enteric pathogens 5-15 um, cytoplasm - dark green, nuclei - dark purple/black 52

Blastocystis hominis (Possible Pathogen) B. hominis reproduces asexually, most likely by binary fission. B. hominis does not have a cell wall and therefore there is great variation in its size and shape, ranging from 5 to 40 μm. Multiple forms of B. hominis have been described in culture including amoeboid, vacuolar, granular. The amoeboid form is more likely associated with disease. Acquisition of B. hominis is thought to occur as a result of frequent animal human, human human and human animal transmission. 53