LAB 6 – Amoeba

• General Characteristics of Intestinal Protozoa

  • Small size

  • Morphological characteristics overlap among species

  • Must use permanent stained smear for definitive identification

  • Consistency of stool will suggest forms of protozoa present

  • Trophozoite (troph) – Motile, reproducing stage

  • Cyst – Nonmotile, nonfeeding stage; often is infective stage for human

• Entamoeba histolytica

  • Pathogenic

  • Worldwide

  • Variety of clinical conditions:

    • Asymptomatic (luminal amebiasis)

    • Invasive intestinal amebiasis

    • Invasive extraintestinal amebiasis

  • Morphology is similar to E. dispar

• Entamoeba histolytica/dispar troph

  • 10-60 µm (15-20µm most common)

  • Motile

  • Single nucleus

    • Chromatin: finely granular and evenly distributed around periphery

    • Karyosome: Small, centrally located

  • Cytoplasm:

    • Finely granular

  • May contain bacteria or RBCs

    • *E. dispar does not!

• Entamoeba histolytica/dispar cyst

  • 10-20 µm

  • Spherical

  • 4 nuclei in mature cyst

  • Nuclei:

    • Peripheral chromatin: fine, evenly distributed

    • Karyosome: Small, discrete, centrally located

  • Cytoplasm:

    • Chromatoidal bars may be present

• Entamoeba hartmanni troph

  • 5-12 µm

  • Motile

  • Nonpathogenic

  • Single nucleus:

    • Chromatin: small granules, evenly distributed

    • Karysome: Small, discrete, centric

  • Cytoplasm:

    • Finely granular

• Entamoeba hartmanni cyst

  • 5-10 µm

  • Spherical

  • 1-4 nuclei

  • Nuclei:

    • Peripheral chromatin: fine, evenly distributed

    • Karyosome: small, discrete, centric

  • Cytoplasm:

    • Chromatoidal bars with blunt ends

• Entamoeba coli troph

  • 15-50 µm (usually 20-25µm)

  • Nonpathogenic

  • Slow motility

  • Single nucleus:

    • Chromatin: heavy granules, irregular in size and distribution

    • Karyosome: large, may be diffuse, often eccentric

  • Cytoplasm:

    • Course

    • Often vacuolated

    • May contain bacteria or other debris

• Entamoeba coli cyst

  • 10-35 µm

  • Usually spherical

  • 8 nuclei

  • Nuclei:

    • Peripheral chromatin: course, granular

    • Karyosome: Large, eccentric or central

  • Cytoplasm:

    • Chromatoidal bars, usually have pointed ends

• Endolimax nana troph

  • 6-12 µm

  • Nonpathogenic

  • Sluggish motility

  • Single nucleus:

    • No peripheral chromatin

    • Karyosome: large, blot-like, centric

  • Cytoplasm:

    • Granular

    • May be vacuolated

• Endolimax nana cyst

  • 5-10 µm

  • Oval or spherical

  • Up to 4 nuclei

  • Nuclei:

    • No peripheral chromatin

    • Karyosome: large, blot-like, centric

  • Cytoplasm:

    • Small granules

    • Rarely have chromatoid bodies

• Iodamoeba butschlii troph

  • 8-10 µm

  • Nonpathogenic

  • Sluggish motility

  • Single nucleus:

    • No peripheral chromatin

    • Karyosome: large and central; sometimes surrounded by refractile granules

  • Cytoplasm:

    • Coarsely granular

    • May be vacuolated

    • May contain bacteria or other inclusions

• Iodamoeba butschlii cyst

  • 5-20 µm

  • Usually oval

  • 1 nucleus:

    • No peripheral chromatin

    • Karyosome: large, eccentric

  • Cytoplasm:

    • Some granules

    • Large glycogen vacuole

• Naegleria fowleri

  • Primary amoebic meningioencephalitis

  • Easily mistaken for WBCs

  • Examine for motility on wet mount

  • Culture

  • PCR

  • Antigen detection

• Acanthamoeba sp.

  • Granulomatous amebic encephalitis

  • Keratitis

  • Diagnose by presence of trophs or cysts

  • Stained slides of tissue or corneal scraping

  • Culture

  • PCR

LAB 7 – Flagellates

• Giardia lamblia/intestinalis trophs

  • Pathogenic

  • Found worldwide

  • 5-15 x 10-20 µm

  • Pear/teardrop shape

  • 2 nuclei

  • 4 pairs of flagella

  • Bilateral symmetry

  • Sucking disc – curved area used for attachment

  • Falling leaf motility

• Giardia lamblia/intestinalis cysts

  • 7-10 x 8-14 µm

  • Oval or round

  • Contains 4 nuclei, 2 axonemes, and 4 parabasal bodies

  • Often see halo effect where cyst has shrunk and pulled away from the cyst wall

• Dientamoeba fragilis

  • Pathogenic

  • Found worldwide

  • 5-18 µm

  • Variable shape

  • Only see trophozoites

  • 1 or 2 nuclei

  • Nuclear chromatin usually fragmented into granules

  • Vacuolated cytoplasm

  • Progressive motility

• Chilomastix mesnili trophs

  • Nonpathogenic

  • 4-8 x 10-20 µm

  • Pear-shaped

  • Single nucleus

  • May see oral groove next to nucleus

  • 4 flagella (difficult to see)

• Chilomastix mesnili cysts

  • 4-6 x 7-10 µm

  • Lemon or pear shaped

  • One nucleus

  • Large central karysome

  • Curved cytostomal fiber or “shepherd’s crook”

• Trichomonas vaginalis

  • Very common

  • Worldwide

  • 3-18 µm long

  • Variable in shape

  • Progressive motility

LAB 8 – Hemoflagellates

• Leishmania sp.

  • Vector-borne → sandfly

  • Causes leishmaniasis

  • Clinical conditions:

  • Cutaneous

  • Mucocutaneous

  • Visceral → Kala-azar

  • Promastigate – infective

  • Amastigote – diagnostic

• Laboratory Diagnosis

  • Leishmania amastigote

  • 3-5 µm

  • Oval

  • Large nucleus with small kinetoplast

  • Stains red/purple

  • Cytoplasm stains light blue

  • If specimen left at ambient temperature, amastigotes may transform into promastigotes

• Trypanosoma brucei

  • Vector borne → Tsete Fly

  • Two subspecies

  • Morphologically indistinguishable

  • Cause distinct disease patterns in humans

  • Trypanosoma brucei gambiense → West African Sleeping Sickness

  • Trypanosoma brucei rhodesiense → East African Sleeping Sickness

• Clinical Manifestations

  • 3 stages:

    • 1.Trypanosomal chancre develops at sight of bite

    • 2.Hemolytic stage

      • Fever, lymphadenopathy, pruritis

    • 3.Meningioencephalitic stage

      • Invasion of CNS

      • Headache, somnolence, abnormal behavior, loss of consciousness, coma

• Laboratory Diagnosis

  • Microscopic examination of chancre fluid, lymph node aspirate, blood, bone marrow, or CSF

  • Wet prep for motility

  • Giemsa-stained slides

  • Concentration technique → buffy coat

  • T. brucei trypomastigotes

  • 14-33 µm

  • Small posterior kinetoplast

  • Centrally located nucleus

  • Anterior flagellum

• Trypanosoma cruzi

  • Vector → triatome bug (kissing bug)

  • Worldwide

  • 10-12 million people infected annually

  • Causes → Chagas Disease

• Chaga’s Disease

  • Acute phase

    • Often asymptomatic or non specific symptoms

    • Nodular lesion where bitten by vector

    • Most cases resolve in a few months into a chronic form of the disease (dormant)

  • Chronic phase

    • Symptoms start many years later

    • Heart damage or GI involvement

    • Sometimes fatal

• Laboratory Diagnosis

  • Microscopic exam

  • Look for motility in fresh blood or buffy coat

  • Thin and thick blood smears (Giemsa stain)

  • Serological tests (EIA) for T. cruzi Ag

  • Molecular testing

  • T. cruzi trypomastigotes

  • 12-30 µm

  • “C” or “U” shaped

  • Large, posterior kinetoplast

  • Centrally located nucleus

  • Anterior flagellum

    LAB 9 — Hemosproridia

• Key Terms

  • Sporozoa — Protozoa with no obvious means of locomotion

  • Sporozoite — Infective stage transferred by vector

  • Schizont — Morphological form responsible for development and maturing of merozoites

  • Schizogany — Asexual multiplication that occurs in humans prior to invasion of RBCs

  • Merozoite — Asexual sporozoan trophozoites

  • Ring form — Ring-like structure that appears initially after invasion of RBCs by Plasmodium spp

  • Macrogametocyte — Female sex of Plasmodium spp

  • Microgametocyte— Male sex cell of Plasmodium spp

• Four Main Species of Plasmodium

  • Plasmodium falciparum

  • Plasmodium vivax

  • Plasmodium ovale

  • Plasmodium malariae

  • Most malarial infections caused by P. falciparum

• Epidemiology

  • Vector: female Anopheles mosquitos

  • ~250 million people infected annually

  • ~One million deaths annually

    • 90% deaths in Africa

  • Widespread in Africa, Asia, and Latin America

  • Usually associated with travel to endemic areas

• Malaria Life Cycle

  • Female Anopheles mosquito feeds on human

    • Infects them with sporozoites

  • Sporozoites go to liver and infects cells

    • Matures into schizont

  • Shizont ruptures —> merozoites

  • Merozoites infects RBCs —> Ring stage (mature trophozoite)

    • Matures into schizont —> ruptures to merozoites

      OR

    • Differentiates into sexual erythrocytic stage (gametocyte)

    • Gametocytes ingested by Anopheles mosquito during blood meal to continue life cycle

• Clinical Manifestations

  • Symptoms of uncomplicated malaria may be mild and go undiagnosed

  • Untreated malaria may progress to severe forms —> may be rapidly fatal

  • Symptoms: fever, chills, headache, myalgias, arthralgias, weakness, vomiting, diarrhea

  • Other clinical features: splenomegaly, anemia, thrombocytopenia, hypoglycemia, pulmonary or renal dysfunction, neurologic changes

  • Varies, depending on infecting species, level of parasitemia, and patient’s immune status

  • P. falciparum —> may be fatal, affecting CNS or causing renal failure, severe anemia, or respiratory distress

  • P. vivax —> splenomegaly

  • P. malarie —> nephrotic syndrome

• Paroxysm

  • Part of the body’s allergic response to:

    • Development of schizonts

      AND

    • Circulating parasitic antigens following release of merozoites

  • Characterized by

    • Chills

    • Fever

    • Sweating and Fatigue

  • Periodicity of paroxysms varies

    • P. Vivax —> Every 48 hours

    • P. ovale —> Every 48 hours

    • P. malariae —> every 72 hours

    • P. falciparum —> every 36-48 hours

• Laboratory Diagnosis

  • Multiple sets of Giesma-stained peripheral blood smears

    • Thick smears for screening

    • Thin smears for differentiation of Plasmodium spp

    • Use oil immersion (100x)

  • Timing of blood collection

    • Collect between paroxysms

  • Antigen testing

  • Molecular testing (PCR)

• Morphologic Forms

  • Ring Forms (early trophozoites)

    • Ring-like structure that develops after invasion od RBC

    • Blue cytoplasmic circle with red chromatin dot(s) on Giemsa stain

    • Area within ring vacuole

  • Developing Trophozoites

    • Appearance varies by Plasmodium species

    • Ring form is more pleomorphic and ameboid

    • Circle of ring and chromatin dot(s) may still be present

    • Brown pigment may be present

    • Infected young, pliable RBCs are usually larger due to growing parasite

  • Schizonts

    • Parasite occupies more space within RBC

    • Fully developed merozoites emerge (asexual sporozoa trophozoites)

    • Number and arrangement of merozoites vary

    • Characterized by presence of multiple contiguous chromatin dots

  • Gametocytes

    • Sexual erythrocytic stage

    • Macrogametocytes

    • Mircogametocytes

    • Shape varies

  • Inclusions that may be present in red blood cells infected with Plasmodium species

    • Plasmodium vivax — Schuffner’s dots

    • Plasmodium ovale — Schuffner’s dots

    • Plasmodium malariae — Ziemann’s dots

    • Plasmodium falciparum — Maurer’s dot

• Plasmodium vivax

  • Tertian fever

  • Cycle of paroxysm — 48 hours

  • Relapse possible due to reactivation of dormant infected liver cells (hypozoites)

  • Most widely distributed of Plasmodium species

    • Tropical, subtropical, and temperate regions

  • Symptoms start after 7-10 days

  • Requires Duffy antigen

  • Tend to invade young, pliable RBCs or reticulocytes

  • Infected cells are pale and enlarged

  • Schuffner’s dots (fine red granules) may be present

  • Cells may become distorted/ameboid

  • Mature ring forms tend to be large and coarse

  • Developing ring forms tend to be large and coarse

  • Developing ring forms frequently present

  • Mature schizonts have up to 24 merozoites

  • All stages may be observed on peripheral blood smear

• Plasmodium ovale

  • Tertian fever

  • Cycle of paroxysm — 48 hours

  • Clinically similar to P. vivax, but less severe

  • Widely distributed in West Africa

    • Uncommon elsewhere

  • 60% infected cells assume an oval shape

  • 20% infected cells have ragged edges

  • Infects young RBCs and reticulocytes

  • RIngs forms and gametocytes difficult to distinguish from P. vivax

  • Schuffner’s dots, when present, may be prominent

  • Mature schizonts have an average of 8 merozoites

  • All stages may be observed on peripheral blood smear

• Plasmodium malariae

  • Quartan fever

  • Cycle of paroxysm — 72 hours

  • Found in sub-saharan africa

  • Persisting low grade parasitemia for many years

  • Spontaneous recovery can occur

  • May lead to nephrotic syndrome

  • Infects mature RBCs

  • Very little RBC enlargement or distortion

  • Ring stage very brief (may not see)

    • Squarish appearance

    • Chromatin dot may be on inside of ring

  • Trophozoite often forms bands/elongated, stretching across cell

  • Mature schizonts may have typical daisy head appearance with 8 merozoites

  • Ziemann’s stippling: fine, pink, pale dots

• Plasmodium falciparum

  • Black water fever

  • Deadliest form of malaria —> affects all of the organs

    • Large amount of toxic cellular debris

    • May attack organs — kidneys, liver brain

  • Cycle of paroxysm — 36-48 hours

    • Flu-like symptoms early in infection

  • Confined to tropics

    • Predominate in Africa, New Guinea, Hai

  • Cerebral malaria

    • Most common

    • Disorientation, violence, coma

  • Black water fever

    • Sudden, intravascular hemolysis

    • Causes dramatic change in color of urine

  • Invades all cell stages

    • May infect >50% cell

    • RBCs retain original size

  • Schizogany occurs in organs rather than blood

  • Typically, only ring forms and gametocytes of peripheral blood

  • Ring forms:

    • Appear fine and delicate

    • May have two chromatin dots

    • May see multiple rings in a cell

  • Accole forms occasionally seen

• Prevention

  • Personal protection

    • Netting, screening, protective clothing, repellents

  • Prophylactic treatment

    • Based on geographical location

    • Length of exposure

  • Vaccine still in clinical trials

• Treatment

  • Antimalarial medications

    • Quinine, quinadine, chloroquine, amodiaquine, primaquine, etc…

    • Each drug effects the parasite in different ways, depends on the morphologic life cycle stage at time of administration

    • Drug-resistant malaria is on the rise

• Babesia spp

  • Vector — ticks (ixodes)

  • Babesia microti — most common in US (northwest and midwest)

  • Babesia divergens — Europe, splenectomized patients

  • Babesia duncani — Washington and California

• Clinical Manifestations

  • Incubation period 1-4 weeks

  • Generally, self-limiting

  • Some are asymptomatic

  • Fever, headache, chills, sweating, myalgia, fatigue

  • May last several weeks

  • More severe in immunocompromised patients

  • Coinfection with other tick-borne diseases (lyme disease, human granulocytic erlichosis)

• Laboratory Diagnosis

  • Giemsa-stained peripheral blood smears

    • Thick smears for screening

    • Thin smears for differentiation of Babesia spp. from Plasmodium spp. Use oil immersion

    • Timing of blood collection not critical

    • Antibody testing

    • IFA (indirect fluorescent antibody test)

    • Molecular testing (PCR)

• Babesia spp.

  • Organisms infect RBCs

  • Trophozoites:

    • Pleomorphic ring-like structures resembles Plasmodium falciparum

    • Absence of : Schuffner’s dots, Ziemann’s dots or Maurer’s dots

    • No pigmentation

  • Merozoites

    • Tetrad formation referred to as “Maltese Cross“ appearance

LAB 10 — Miscellaneous Protozoa

• Balantidium coli trophs

  • Largest protozoan

  • 40-200 micrometers

  • Cilia on cell surface

  • Kidney-shaped macronucleus

  • Smaller, less conspicuous micronucleus

• Cystoisopora belli

  • Oocysts visualized on wet mounts with brightfield microscopy or UV fluorescence

  • May e stained with modified acid-fast stain

  • 25-30 micrometers long

  • Immature oocysts

    • Long, oval

    • One sporont

    • Divides to form two sporoblasts

  • Mature oocyst

    • 2 mature sporocysts

    • 4 sporozoites inside each sporocyst

• Cryptosporidium spp.

  • 4-5 micrometers

  • spherical

  • contains 4 sporozoites

  • Modified acid-fast stain

• Cyclospora cayetanensis

  • 8-10 micrometers

  • Wrinkled appearance

  • Oocyst contains 2 sporocysts, each containing 2 sporozoites

  • Modified acid-fast stain

  • Epifluorescence (UV)

• Blastocystis hominis

  • Spherical to oval cyst-like structures

  • Vary in size

    • 5-30 micrometers

  • Typically consist of central body surrounded by thin rim of cytoplasm containing 6 nuclei

• Toxoplasma gondii

  • 3×7 micrometers

  • Tachyzoites

  • Giemsa-stained slide

  • Crescent-shaped

  • Prominent, centrally placed nucleus

  • One end may appear more rounded