Micro exam 3

Amoeba cyst form

Formed stool, rigid, non-motile, infectious form

Amoeba trophozoite form

Liquid stool, active feeding, active disease

Amoeba motility mechanism

Pseudopods

Amoeba life cycle

Ingestion of mature cyst, troph migrate to intestine or other body sites and multiply. cysts and trophs passed in feces

E histolytica epidemeology

Warm climates, STI, transmission via mechanical vector

E histolytica clinical manifestations

Amoebic dysentery (invasive intestinal amebiasis), extraintestinal amebiasis, often spreads to liver

E histolytica cyst

10-20um, Round, cigar shaped chromatoidal bars. 1, 2, or 4 nuclei. Small central karyosome. Even PC

E histolytica troph

15-25 um, One nucleus with even PC and central karyosome, granular cytoplasm, may have ingested RBCs

Charcot leyden crystals

Breakdown product of eosinophils, may appear as chromatoidal bars but they are outside the org

Entamoeba hartmanii cyst

<10 um, 1, 2 or 4 nuclei. “Small race” E. histolytica (even PC, central karyosome, chromatoidal bars)

Entamoeba hartmanii troph

3-12 um, 1 nucleus. No ingested RBC

Entamoeba coli cyst

10-35 um, 1-8 nuclei, sharp chromatoidal bars, eccentric karyosome, irregular PC, granular cytoplasm

Entamoeba coli troph

12-30um, short blunt pseudopods, dirty cytoplasm/ingested bacteria, 1 nucleus with eccentric karyosome and irregular PC

Endolimax nana cyst

15-12 um, 1-4 nuclei, irregular clumped karyosome, no PC, oval

Endolimax nana troph

5-12 um, blunt hyaline pseudopods, large irregular karyosome with no PC

Iodamoeba butschlii cyst

6-15 um, well defined glycogen mass, 1 nucleus with huge karyosome and no PC

Iodamoeba butschlii troph

Rarely seen, 6-20 um, nucleus similar to E nana

Blastocystis hominis

Lab should report quantitatively. Strict anaerobe, has 4 forms, diagnosis based on finding vacuolar form in stool

Blastocystis hominis vacuolar

5-15 um, up to 4 nuclei, large central vacuole takes up 90% of cell volume, push nuclei to side

Free living amoeba

Found in many water sources. Acanthamoeba, Balamuthia and Naegleria. We are not a part of their life cycle

GAE

Granulomatous amoebic encephalitis. Acanthamoeba and Balamuthia

PAM

Primary amoebic meningoencephalitis. Naegleria

Naegleria fowleri

Free living amoeba, Causes PAM, only troph found in CSF, lives in warm water. Don’t usually ID

Acanthamoeba

Free living amoeba, causes GAE, contaminated contact lens use. Cyst is doubled walled, troph has central prominent karyosome

Balamuthia mandrillaris

Free living amoeba, causes GAE, found in soil and dust

What flagellates lack a cyst stage?

Dientamoeba fragilis, Trichomonas vaginalis, and Pentatrichomonas hominis

Flagellate structure

Troph flagella arise from basal body, cytostome (mouth), undulating membrane. Cyst has axostyle. Both have parabasal bodies

Giardia epidemiology

Intestinal flagellate, nicknamed beaver fever (bodies of water), highly contagious (infectious dose <10 cysts)

Giardia dx

DFA (direct fluorescent antibody), EIA (enzyme immunoassay). It is a reportable disease

Giardia cyst

9-12 um, oval, 2-4 nuclei, central axostyle, 4 median bodies, internal flagella. Cytoplasm often shrinks from cyst wall

Giardia troph

10-20 um, 2 axostyles and 2 median bodies, ventral sucking disc for attachment, 4 pairs of flagella (8 total), 2 nuclei with large central karyosomes, no PC.

Dientamoeba

Troph form only, looks like amoeba but is a flagellate

Dientamoeba transmission/CM

Ingestion of helminth egg, infection stays in intestine

What characteristic motility feature of giardia?

Falling leaf

Dientamoeba troph

6-15um, Binucleate, no external flagella. Large karyosome, no PC. May have vacuoles with ingested bacteria

Chilomastix cyst

6-10 um, Lemon shaped with nipple like projection, large single nucleus with distinct central karyosome. Prominent cytostomal fibers (shephards crook)

Chilomastix troph

6-20 um, Pear shaped, curved and twisted posteriorly, large single nucleus with distinct central karyosome, cytostomal fibrils

Pentatrichomonas hominis troph

6-14 um, teardrop shaped, quick jerky movement undulating membrane and axostyle through body. No cyst form

Enteromonas hominis cyst

Ellipsoidal, 4-8um. 1, 2 or 4 nuclei (binucleate most common), large central karyosome, no PC

Intestinal flagellates

Giardia and dientamoeba

Atrial (Urethral and vagina) flagellates

Trichomonas vaginalis

Non pathogenic flagellates

Chilomastix, Rerotamonas, pentatrichomonas, enteromonas

Enteromonas hominis troph

Oval, 4-10 um, 3 anterior flagella 1 posterior, one side of body is flattened, jerky motility

Retortamonas intestinalis cyst

4×6 um, pear shaped (resembles chilomastix), single large nucleus, 2 fibrils (bird beak arrangement)

retortamonas intestinalis troph

4-9 um, Pear/oval shaped, jerky movement, promininent cytostomal groove extending half of body, exterior flagella, one nucleus

Trichomonas overview

STI, Worldwide, multiplies in genitourinary tract, male is resevoir

Trichomoniasis

Caused by trichomonas, symptoms are itching and discharge for women, men often don’t experience symptoms

Trichomonas diagnosis

Molecular testing, wet prep, urinalysis, gram stain

Trichomonas vaginalis troph

7-23 um, jerky non directional movement, 4 anterior flagella, short undulating membrane, long axostyle, large nucleus and small kinetoplast. No cyst

Balantidium coli overview

Intestinal ciliate, only one pathogenic to humans. Largest protozoan parasite. Pig is natural host, human is accidental host. Most common in Philippines

Balantidium coli CM

Severe infections termed Balantidiasis, ulceration in intestines

Balantidium coli cyst

45-65 um, round to elliptical shape. Cilia inside double cyst wall. Nuclei, macro (kidney shaped) and micronucleus

Balantidium coli troph

30-100 um, cilia present along periphery, anterior end is tapered with cytostome. Contractile vacuoles in cytoplasm. 2 nuclei with macro (kidney shaped) and micronucleus

Blood and tissue flagellates

Leishmania and Trypanosoma. Transmitted by insect vector, which is the intermediate host and required for their life cycle

What flagellate forms are found in humans?

Amsatigote and trypomastigote

Amastigote

Found in tissue, seen with Leishmania and Trypanosoma cruzi. Retracted flagellum, kinetoplast and nucleus. 2-3 um, so can only see nucleus on scope

Flagellate development steps

Amastigote, promastigote, epimastigote, trypomastigote

Promastigote

In sand fly, not in humans. anterior kinetoplast where anterior flagella attaches. central nucleus

Epimastigote

In tsetse fly and reduviid bug (not in humans). Single anterior flagellum, undulating membrane down half of body that is attached anterior to nucleus. Kinetoplast is anterior to the posterior nucleus

Trypomastigote

In human blood and CSF, Trypanosoma. Free anterior flagellum, undulating membrane on entire bdoy. Kinetoplast is posterior of nucleus

Leishmaniae vector

Sand fly

Cutaneous Leishmaniasis

skin sores, develops within a few weeks or months. Usually painless but can be painful. L. tropica complex, L mexicana complex, and L braziliensis complex

Visceral leishamniasis

Affects internal organs (spleen, liver BM). Can be life threatening. Develops within months to years

Leishmania tropica complex

Old world cutaneous (E hemisphere). Re papule at site, lesions are benign and self healing. secondary bacterial infections common.

Leishmania mexicana complex

New World cutaneous (W hemisphere). 60% occur on ear lobes. Has other mammal resevoirs

Leishmania braziliensis complex

Mucocutaneous. Aggresive chronic cutaneous ulcers with mucous membrane spread. Can be deadly and/or recur years later causing espundia (destruction of nose and ear cartilage)

Leishmania donovani complex

Visceral, AKA Kala-azar. Organisms disseminate and spread to the viscera. Found intracellularly as LD bodies. Death in 2-3 years if untreated

Leishmania life cycle

Promastigote injected into human during sandly meal, promastigote becomes amastigote which replicates and infects other cells

Leishmania diagnosis

Giemsa and H&E stains. Amastigotes of Leishmania and Trypanosoma cruzi are indistinguishable.

African trypanosomiasis

Sleeping sickness. Tsetse fly is insect vector and intermediate host. Includes Trypanosoma brucei rhodesiense (E Africa) and Trypanosoma brucei gambiense (W Africa)

Trypanosoma brucei gambiense

90%, W Africa, mild and more chronic form

Trypanosoma brucei rhodesiense

10%, East Africa. Less prevalent, acute with rapid course and death

Trypanosoma brucei CM

Inflammation at bite site, lymphadenopathy in neck = Winterbottoms sign. CNS involvement causing coma (Sleeping sickness)

African Trypanosomiasis life cycle

Tsetse fly takes blood meal, injects trypomastigote which multiply.

African trypanosomiasis diagnosis

Winterbottoms sign, trypomastigote in blood.

American trypanosomiasis

Chagas disease, caused by Trypanosoma cruzei. Intracellular amastigote develops in tissues, trypomastigote circulates in blood.

American trypanosomiasis transmission

Feces of reuviid bug

American trypanosomiasis CM

Acute: Primary lesion at bite site called chagoma

Chronic: can be decades long with few orgs in the blood.

Symptoms: Edema of eyelids (Romanas sign)

Trypanosoma cruzi life cycle

Reduviid bug takes blood meal, trypomastigote in feces enters wound site. Amastigotes multiply in tissue and some transform into trypomastigotes and circulate in the blood

Trypanosoma cruzi diagnosis

Romanas sign (Unilateral edema of eyelids). Cruzi —> Chagas disease —> Curls (trypomastigote)

Immunoassay of stool sample

Ag-Ab complexes used to detect specific parasites. Rapid detection, specific to an org, but can’t determine number of org

Saline mount of feces

Used to see motility

Iodine mount for feces

Used to see nuclear structures

Trichrome stain

Used to see morphology

Film array (IA)

PCR multiplex w 22 targets. Bacteria, viruses and parasites

ELISA

Ab to capture the Ag (E histolytica). Enzyme linked to secondary Ab

DFA

Ab linked toa fluorocrhome binds cell Ag (cell wall). Giardia, cryptosporidium

Coccidia

Complex life cycles that include sexual and asexual stages. May include intermediate host and definitive host. Fecal oral route, includes oocysts, sporocysts, sporozoites.

Coccidia intermediate host stage

Asexual

Coccidia definitive host stage

Sexual

Cryptosporidium

Most important coccidia, watery diarrhea, increased risk for those with weak immune systems

Cryptosporidium CM

Immunocompetent: incubation (2-10 days), self limiting. General GI symptoms

Immunocompromised: Chronic diarrhea —> dehydration

Cryptosporidium epidemiology

Lives in intestine, infected person sheds parasites in stool. Found all over the world

Cryptosporidium oocyst

Infective and diagnostic

Cryptosporidium diagnosis

Acid-fast staining they appear pink and 4-5 um, contains 4 sporozoites. DFA, EIA, PCR

Cyclospora

Coccidia, humans are only known host. Endemic in Nepal, Pakistan, and India

Cyclospora CM

Cyclospora infects small intestine causing self-limiting diarrhea. Sometimes asymptomatic and recurring

Cyclospora life cycle

Oocyst in stool is immature (unsporulated) and not infective. Oocyst sporulates in the environment and gets ingested again

Cyclospora diagnosis

Acid fast they appear light pink to deep red/purple and 8-10um, contain immature sporoblast (bigger than crypto). Cyclospora oocysts are auto fluorescent

Cystoisospora

Coccidia found worldwide. both sexual and asexual repro occur in GI tract