PARASITOLOGY - PROTOZOA

What’s Amoebiasis

All clinical conditions produced by Entamoeba histolytica

What differentiates Entamoeba histolytica from other non-virulent Entamoeba species?

Entamoeba histolytica is hematophagous

E. histolytica in the population

• 90% Asymptomatic carriage or subclinical presentation (No dysentery, however if subclinical infection, they can suddenly develop extraintestinal amoebiasis

• 10% Invasive

Describe Entamoeba histolytica’s Trophozoite and cyst forms

Trophozoite: Amoeboid shape, motile by Pseudopodia, and hematophagous

Cyst: Spherical, non-motile, and infective.
→ It has 1-4 nuclei
→ Chromatoid bodies: Semi-crystalline arrays of ribosomes - reserve for excystation)
→ Central karyosome: Inactive chromatin, each associated with its corresponding nucleus
→ Glycogen masses

Top 3 deadliest protozoa worldwide

1. Plasmodium spp (malaria)

2. Leishmania spp

3. Entamoeba histolytica

What is requireme

What makes the diagnosis of amebiasis difficult

Overlap with IBD

Acute amoebic dysentery clinical presentation

• Frequent mucoid stools

• Rectal blood release without diarrhea (Especially children)

• Dysentery and Constipation alteration

• Abdominal pain with tenderness

• Tenesmus

• Possibly: Fulminant + Necrotizing colitis

What are some features that help differentiate Amoebic vs Bacillary dysentery

In Amoebic:

• Less motions/day

• Lots of feces vs few

• Darker color

• Blood + Mucus + feces (Little feces in bacillary)

• Acid pH (Alkaline bacillary)

• Stools do not adhere to container (Do in bacillary)

• Clumped RBCs in amoebic

• Pyknotic bodies PRESENT

• Charcot Leydon crystals PRESENT (Residues of EOPs)

• ABSENT Ghost cells

Laboratory diagnosis of Intestinal Amoebiasis

• Stools + Saline: Motile trophozoites with ingested RBCs

• Stools + Iodine: Should stain Yellow: Nucleus + central karyosome + brown glycogen mass

• Biopsy + H&E

• Stools + Trichrome stain

How can we determine E. histolytica asymptomatic carriage

If cysts can be found in feces in least 3 stool specimens

Non-histological lab diagnosis of E. histolytica and their main purpose

• Sigmoidoscopy + biopsy

• Serology and PCR: To differentiate between E. histolytica and E. dispar (In saymptomatic carriage screening cuz they look identical but the latter is completely non-pathogeneic)

E. histolytica tissue bipsy apearance

Flask-shaped ulcer appearance

On screening, ulcerations look like buttons

Amoeboid dysentry sequalae

Ulcerative post-dysenteric colitis:

• Edema of the mucosa + Other irregularities

• Spasms

• Loss of haustration → Smooth colon appearance of X-Ray (Important sign of ulcerative colitis in general)

Amoebiasis intestinal complications

• Perforation (Rare)

• Peritonitis (40% Mortality)

• Amoeboma

• Blood invasion and extraintestinal infections

Describe Amoeboma

Due to chronic infection by E. histolytica

→ Tumor-like inflammatory msses in the colon (neoplasms) that can cause intussusception and obstruction

Hepatic Amebiasis Pathogenesis

1. Trophozoites reach liver via the hepatic portal vein and multiply locally

2. They cause inflammation and endothelial damage which can cause vessel obstruction

3. Endothelial damage can lead to thrombosis

4. Liver cell necrosis due to the pathogen itself and worsened by endothelial damage and iscchemia

Hepatic amebiasis diagnosis

• Imagining

• Blood test: WBC/ESR/CRP/SGPT/Alkaline phosphatase

• Liver Aspirate microscopy

  • Anchovy sauce-like pus: Mix of sloughed liver tissue and blood, and contains NO TRPHOZOITES

Which regions of a liver abcess have the most trophozoites?

Peripheral zones

Complications of hepatic amoebiasis

Rupture of abscess into:
→ Right pleural cavity
→ Right lung
→ Peritoneal cavity
→ Below diaphragm
(Can be fatal)

Metastasis to lungs, skin, spleen, brain

Luminal Amebisis treatment

• Paromomycin (Humatin)

• Diodoquin

• Furamide

Cyst passers

Invasive amebiasis treatment

• Metronidazole (Flagyl) or Tinidazole + A lumen-acting drug
  *Note: Metronidazole is actually activated by one of E. histolytica’s fermentative enzymes

• Emetine or DehydroEmetine for severe disease (Fulminant & Necrotizing colitis)

Amebiasis prevention

Hygene, safe water drinking, avoid raw vegetables and fruits

Note: If kept moist, cysts can survive even if refregirated for up to a month
Note: Cysts are fairly resistant to drinking water chlorination

Giardia lambia habitat in body

Duodenum and upper jejunum

Giardia lambia Trophozoite morphology

Tennis-racket-shape, dorsally convex, and ventrally concave with sucking disc.

2 Oval nuclei with karyosome

2 axostyle traversing the body longitudinally

2 coma-shaped parabasal that lie perpendicular to the axostyle

8 flagella (4 pairs), that grant motility

Giardia cyst morphology

Oval, the axostyles are diagonal, parabasal bodies still pependicular, 4 nuclei, flagellar reminants in the cysts (Retrated flagella)

G. lambia pathogenesis

1. The cysts are ingested

2. The cysts cross the stomach easily because they are acid-resistant

3. Once in the small intestine, the alkaline-sensitive cysts excyst and release alkaline-tolerant trophozoites

4. The G. lambia trophozoites bind to the mucosa with their sucking discs

5. Overtime, this causes abnormal villous architecture

6. Mucoid diarrhea and Stearrhoea due to fat absorption impairment

7. Encystation in Large intestine

Name and explain the symptoms caused by Giardiasis

Duodenitis: Caused by mechanical irritation by the pathogen

Mild or Severe Diarrhea: The pathogen’s attachment and exotoxin production triggers cytokine production and inflammation which increases gut permeability, mobility, and electrolyte release (Increased water content)

Mucoid Diarrhea: Inflammation and cytokine release triggers hypersecretion of goblet cells

Villous Atrophy and Blunting of Brush border: Related to immunodeficency and a decreased IgA release
# Fat malabsorption → Steatorrhea
# Less surface area to release saccharidases → More undigested carbs → Microbiota selection for fermentative bacteria → Gas production and flatulence
# B9 and ADEK Vitamin deficencies

What kinds of immunodefiencies increase susceptibility to giardia

Hypogammaglobulinemia

Low sIgA

Low gastric acidity or achlorohydria

Giardiasis symptoms in immunodefiency

• Persistent diarrhea

• Steatorrhea

• Malabsorption and hypoproteinemia

• B9, B12, A, D, E, K deficencies

• Cholangitis (Bile duct inflammation and Cholcystis (Gallbladder inflammation )

Laboratory diagnosis of Giardiasis

• Symptomatic: Diarrhea + Mucus + Cysts in stools

• Asymptomatic: Cysts in stools (Might need to sccreen for 4 days in a row)
  *Note: Iodine stain

• Positive string test

• Serology

• PCR

Giardiasis treatment

Metronidazole or Tinidazole and recently Albendazole

Clinical significance of Coccidians

Causes diarrhea almost exclusively in AIDS patients

Which organisms causes Coccidiasis

Coccidians from the phylum Apicomplexa:

• Cryptosporidium pavum (Zoonosis)

• Cryptosporidium hominis (Human-Human)

• Cyclospora cayetanesis

• Isospora belli

Cryptosporidium pavum pathogenesis

1. Ingestion of Oosysts (Infective form)

2. Oocyst releases sporozoite

3. Sporozoite attaches to enterocytes and becomes trophozoite

4. Trophozoite matures into Type I Meront

5. Type I Meront hatches and releases type I merozoites

6. Type I merozoite either reforms a trophozoite to release more type I merozoite (Asexual), OR, transforms into Type II Meront (Sexual)

7. Type II Meronts release Type II Merozoites

8. Type II Merozoites either become Microgamont (Male), or Macrogamont (Female)

9. The male Microgamont hatches, releasing microgametes that will fertilize the female Macrogamont → Zygote formation

10. The zygote either becomes a thin-walled oocyst that will progress the infection, OR, a thick-walled oocyst which will exit the host

Cryptosporidium outbreak causes

• Close-contact with animals

• Oocysts are robust and have low ID, and small

• Oocysts resistant to chlorine

• Nosocomial infection

Cryptosporidiosis lab diagnosis

Modified Kinyoun’s Acid-Fast stain (For all coccidians)

Iodine stain

Fluerescent antibodies, PCR

Trichomonas vaginalis habitat

Female lower genital tract / male urethra and prostate

Trichomonas infectious form

Tichomonas exist as trophozoites ECLUSIVELY

Trichomoniasis symptoms in males vs females

Males: Asymptomatic usually

Females: Purulent greenish discharge, dysuria & dyspareunia, itching, burining sensation

Trichomoniasis diagnosis

Demonstration of actively-motile trophozoites in wet films

Direct fluorescent antibodies (DFA)

Punctate cervical hemorrhages (Strawbeey cervix)

Trichomoniasis treatment

Metronidazole

Explain the 3 parts of Plasmodium’s life cycle in details

Plasmodium uses mosquitos as their definitive hosts (Support sexual reproduction), and humans as their intermediate hosts (Support asexual reproduction and maturation)

Exo-erythrocytic Cycle:

1. Infected mosquito takes a blood meal and injects its plasmodium-infused saliva

2. The form injected into humans is the sporozoite form, it travels hematogenously to the liver, where it infects hepatocytes (Using Kupffer cells as an entry point)

3. The protozoan replicates inside the infected cells, eventually the parasite load will push the hepatocyte nucleus against the membrane, this cell form is known as a Schizont (Tissue Schizont)

   1. Alternatively, the sporozoite could just stay dormant and would thus be known as a Hypnozoite

4. The schizont eventually ruptures, releasing Plasmodium Merozoites with RBC tropism

Erythrocytic Cycle:

1. Plasmodium merozoites infect RBCs in the circulation

2. Inside RBCs merozoites mature into Immature Trophozoites, which look like a wedding ring inside an RBC - Most commonly found stage in the blood

3. From this stage, we get divergent paths

A: Continuation of the Asexual Erythrocytic cycle through trophozoite maturation and schizont (Blood-stage schizont) formation then schizont rupture to give merozoites again which directly infect RBCs
B: Switch to gametocyte formation of male and female gametocytes in preparation for the Sporogonic Cycle

Sporogonic cycle:

1. When a mosquito takes a blood meal from an infected human, it uptakes the gametocytes (Other forms of the parasite die), and becomes infected itself

2. The drop in temperature inside the mosquito’s gut triggers the maturation of male and female gametocytes into micro and macro-gametes respectively

3. The microgamete fertilizes the macrogamete to give an Ookinete

4. The ookinete encysts and becomes an Oocyst

5. Rupture of the oocyst in the salivary glands of the mosquito releases the sporozoites, which can start the Exo-Erythrogenic cycle again

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Sporogonic cycle

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Exo-erythrocytic and Erythrocytic Cycle

What’s an important cause of Malaria Relapse

Persistence of Plasmodium parasites in the liver during the exoerythrocytic cycle

Name the different Plasmodium species and their different affinities of RBCs

• P. falciparum → Infects RBCs of all ages, even reticulocytes in the BM

• P. vivax and ovale → Prefer to invade younger RBCs

• P. malariae → Affinity for older RBCs

How do malaria relapses occur?

During the initial sporozoite stage, some just stay dormant inside hepatocytes, and become known as hypnozoite. At some point, they can regain virulence.

*Note: Hypnozoites only in Po, Pv & Pm (None in Pf → no relapses in Pf)

*Note: Re-infection is possible due to a high degree of antigenic variation resulting from gene shuffeling (Applies to all Plasmodia but most clinically relavent for Pf)

How can people get non-vector acquired Malaria

It can be acquired:

• Through contaminated blood transfusions

• Congenitally - transplacentally, but only if there’s a placental defect cuz normal placenta is a good enough barrier

• Needle sharing among drug addicts

*Note: In this case the infectious form is Trophozoites → Trophozoite-induced malaria

High-risk individuals for malaria

Pregnant women, babies, young children, elderly.

Plasmodium incubation period

Acute malaria symptoms

Malaria causes febrile paroxysms that are associated with the repture of erythrocytic schizonts in the circulation.

3 stages:

• Cold stage: Intense cold, but patient is febrile (15-60 minutes)

• Hot stage: Intense heat, febrile, headache, body & joint pain, vomitting & diarrhea (2-6 hours)

• Perspiration stage: Profuse sweating, fall in temperature, exhaustion & weakness followed by sleep (2-4 hours not including the sleep period)

Other symptoms: Jaundice and Anemia (due to hemolysis, pigments from the parasite contribute to jaundice color), splenomegaly

Distinguish the fever patterns of Plasmodium based on the different species

It depends on what type of RBCs they infect

• Pf: Any age, and even reticulocytes → Subtertian/Irregular episodes (Most severe)

• Pv/o: Younger RBCs → Episode every other day - Every 48 hours

• Pm: Older RBCs → Episode every 72 hours

Severe malaria complications

Cerebral Malaria

Complication of Pf infection.

→ Proteins of trophozoite-containing RBCs have receptors (PfEMP) on endothelial cells that result in their adhesion and aggregation of infected and non-infected RBCs (Rosetting) → Obstruction of capillaries

High TNF:

• Fever

• Confusion, seizures, and coma

• Metabolic acidosis and RDS6

Black Water Fever

Pf complication

Due to presence of Hb in urine following a hemolytic attack → Dark red/ brown-black

In addition to fever, rigor, biious vomiting, icterus, circulatory collapse, acute renal failure.

Laboratory diagnosis of Malaria

Identification of Malarial parasites in peripheral blood films using Thick or Thin smears (Thick smears 30-40 times more sensitive)

*Note: RBCs may appear elongated

Giemsa stain (10% solution for 10 min)

Serology: HRP2 (His-rish protein) Ag / pLDH (parasite LDH)
→ Both produced by the parasite during their growth & differentiation in RBCs

Which forms are most reliable for the diagnosis of plasmodium

Immature trophozoites: Rings inside RBCs

Other forms are rarely seen

Malaria prophylaxis

1. Causual Prophylaxis: Kill parasites in hepatocytes: Primaquine (++++) / Malarone (++)

2. Suppressive Prophylaxis: Kill asexual parasites in RBCs: Chloroquine/ malarone/ mefloquine/ Doxycyline (all ++++)

3. Gametocytoicidal Prophylaxis: Kill gameocytes: Primaquine (++++)

Antimalarial treatment

Babesiosis causative agents

Aka periplasmosis, caused by apicomplexan parasites of the Babesia genus

Babesia morphology

Babesia life cycle

Babesia uses ticks as their definitive hosts and many mammals as their intermediate hosts.

Intemediate host: Tick blood meal causes sporozoite injections → RBC infection to obtain trophozoites (ring inside RBC) → Trophozoite undergoes merogony to give Merozoites
→ Merozoites either reinfect RBCs asexually or differentiate into gametes ready to be uptaken by ticks again
*Note: both babesia sporozoites and merozoites have RBC tropism

Definitive host: Microgamete fertilizes macrogamete → Ookinete enters salivary glands → Sporogony → Sporozoite release upon blood meal

Babiasis incubation period

1-4 Weeks

Babesiosis clinical manifestation

• Fever

• Chills and sweating

• Myalgias

• Fatigue

• Hepatosplenomegaly

• Hemolytic anemia.

When is Babesiosis more severe

Pathogen specie: B. divergens infection more severe than B. microti

Host: Immunosuppressed, splenecctomized, and elderly individuals

Laboratory diagnosis of Babesiosis

Blood smear stained with giemsa → Look for ring form or maltese cross inside RBCs

Serology

PCR

How can you morphologically differentiate between Plasmodium and Babesia-infected RBCs?

Babesia is pleomorphic

Babesia-infected RBCs may be vacuolated (In plasmodium, hepatocytes get vacuolated, but not RBCs)

Babesia does not produce any pigment

Babesiosis Treatment

Azithromycin, Clindamycin, Altovaquone, ELQs (Endochin-like quinones)

Toxoplasma gondii phylum

Phylum: Apicomplexa

Toxoplasma host types

Definitive: Felines

Intermediate: All warm-blooded animals

Toxoplasma localization in felines

It is an obligate IC parasite, and can be found in a cat’s intestinal epithelium

Describe toxoplasma tachyzoites

It is the infectious form of the parasite, and also the form that can travel trans-placentally

• Crescent-shaped

• Pointed anterior end and rounded exterior end

• May be found single:

  • Free

  • IC trophozoites

  • Proliferative forms

• Or may be found in masses called pseudocysts

• Invasion: They invade cells from their apical side using AMA1 receptors to attach to RON2 host cell receptors to get endocytosed (Clathrin-mediated)

Describe toxoplasma tissue cysts

Cyst-like forms - an adaptation against immune responses

Describe toxoplasma’s life cycle

Cat - Sexual cycle:

1. Cat ingests tissue cysts in mice

2. Upon digestion by cat, the cyst wall will dissolve, releasing bradyzoites into the intestines

3. Bradyzoites invade cat enterocytes and start dividing by schizogony

4. Many divisions take place and, and eventually, the schizonts rupture to release Merozoites

5. Merozoites will infect more cells and keep dividing or form micro and macro gametes that may mate inside or outside the enterocytes

6. Fertilization will lead to the formation oocysts

7. Immature oocysts are excreted via feces

8. Maturation takes place in the soil by sporulation

9. 1 Mature Oocyst will contain 2 sporocysts, and each sporocyst will contain 8 sporozoites - This is the infectious mature oocyst

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Humans - Asexual:

1. Ingest Oocyst (cat feces-contaminated food)- > Sporozoite infectious form / Ingest cyst (Raw meat) → Bradyzoite infectious form

2. Both Sporozoites and Bradyzoites will infect enterocytes and give rise to Tachyzoites (Multiply quickly)

3. Tachyzoites will move from the intestines to other tissues - Acute phase of toxoplasmosis

4. Tachyzoites infect Macrophages, and replicate by endodyogeny

5. After a few divisions - > Rosette shape + Acidocalcisome accumulation

6. Eventually tachyzoites will be carried to the CNS hematogenously and will invade neurons and transform to bradyzoites intracellularly

7. Bradyzoites will secrete a cyst wall and form tissue cysts an immune evasion mechanism

8. Tissue cysts, can stay dormant for as long as the host is alive, and are the hallmark of toxoplasmosis’s chronic phase

Post-natally-acquired toxoplasmosis clinical presentation

Asymptomatic, subclinical, or mononucleosis-like (but unlike mono, heterophile antibody test will be negative)

Note: this is the acute form of the disease

Toxoplasmosis presentation in IC hosts

Systemic form

Almost always resulting from reactivation of latent infections in AIDS

Symptoms include Myocarditis, pneumonitis, chorioretinitis, and eventually brain abscesses and fatal encephalitis

*Note: Ring-enhancing lesions show up on MRI

Congenital Toxoplasmosis

Occurs in 1 in 1000 pregnancies

May cause damage or death of the fetus

Symptom Triad: Chorioretinitis, hydrocephalus, intracranial calcifications

Neonatal toxoplasmosis

The risk of vertical transmission to the fetus increases with the age of pregnancy, however the severity of the lesions decreases

Toxoplasmosis diagnosis

Toxoplasmosis serology interpretation

Laboratory diagnosis of toxoplasmosis

Take tissue biopsy for tachyzoite - Stain with Giemsa

PCR of amniotic fluid for possible intrauterine disease

Toxoplasmosis treatment & Prophylaxis

Recommended for pregnant women, IC patients, people with ocular diseases

Sufadiazine + Pyrimethamine (Potential teratogen, only use after 3rd trimester)
Spiramycin if pregnant woman is infected during 1st trimester

TMP-SMX: For AIDS with <100 cells/mm3 CD4+

Toxoplasmosis prevention

Causative agents of Amoebic Meningoenccephalitis

Naegleria fowleri & Acanthamoeba

Amoebic Meningoencephalitis Life cycles and treatments

Naegleria fowleri: Flagellated Trophozoites enter the olfactory neuroepithelium and cause amebic meningoencephalitis in healthy individuals
*Note: In healthy individuals

Acanthamoeba / Balamuthia mandrillaris: Cysts or trophozoites enter through LRT or through ulcerated/ broken skin → Granulomatous amebic Encephalitis
*Note: In IC patients

Brain-eating amoeba treatment

Amphotericin B or Rifampin