Protozoa: Phylum Sarcomastigophora (Amoebae and Flagellates) Notes
Introduction
Medically important protozoans under Phylum Sarcomastigophora will be discussed, along with their life cycle, morphological characteristics, epidemiology, and clinical diagnosis and treatment.
Includes intestinal/lumen-dwelling amoebae, free-living amebae, atrial flagellates, and hemoflagellates.
Commensals and species of questionable pathogenicity are present.
Generally of worldwide distribution; the prevalence of intestinal protozoa correlates with the level of sanitation.
Identifying Intestinal Protozoa
Numerous characteristics assist in identifying intestinal protozoa.
Motility patterns are classically described for many amebae and the ciliate Balantidium coli, appreciated via wet mount examination of fresh sample.
Size, shape, nuclear and cytoplasmic characteristics are used for identification in wet mounts of concentrated specimens and permanently stained preparations.
Size is important; a calibrated ocular micrometer is essential for routine diagnostic use.
Flagellates are elongated and tapered, with nucleus/nuclei at one end; amebic trophozoites are rounded to oval with occasional pseudopod projections.
Phylum Sarcomastigophora
Amoeboid and flagellated parasites under Kingdom Protozoa cause infections in humans.
Amoebae are classified under sub-phylum Sarcodina, characterized by foot-like pseudopodia.
Flagellates are classified under sub-phylum Mastigophora, characterized by whip-like flagella.
Intestinal and Urogenital Protozoa
Protozoa inhabiting the intestinal tract of humans include amebae, flagellates, ciliates, and coccidia; many are nonpathogens.
Most intestinal infections are acquired by fecal-oral contamination directly from food handlers or indirectly via contaminated water.
Identification can be difficult; organisms are small, and pathogenic species must be differentiated from nonpathogenic species, inflammatory cells, epithelial cells, yeasts, pollen, and other objects.
Morphologic Features
Number and size of nuclei and pattern of chromatin distribution are important, best seen in permanent stained preparations, along with cytoplasmic structures like fibrils in flagellates, ingested materials in amebic trophozoites, and glycogen masses and chromatoid bodies in amebic cysts.
Trophozoites typically predominate in liquid stools but degenerate within 30 minutes to 1 hour after passage if not fixed.
Cysts typically predominate in formed stool and are more resistant to degeneration.
Intestinal Amoeba
Three genera of amebae inhabit the human intestinal tract: Entamoeba, Endolimax, and Iodamoeba.
Cysts are ingested and excyst in the small intestine; resulting trophozoites proliferate by binary fission in the colon lumen.
Both cysts and trophozoites may be passed in feces, but only mature cysts are infective.
Entamoeba histolytica is the only ameba in which the trophozoites can invade tissues and cause clinical disease.
Genus Entamoeba
Characterized by chromatin on the nuclear membrane; includes E. histolytica (etiologic agent of amebiasis), E. dispar (nonpathogenic), E. hartmanni and E. coli (commensal), and Entamoeba polecki (occasionally found in people who have contact with pigs or primates).
E. moshkovskii and E. bangladeshi have been identified in human stool specimens and are morphologically indistinguishable from E. histolytica.
Entamoeba Species
E. histolytica, E. dispar, and E. moshkovskii are morphologically similar and can only be differentiated by isoenzyme analysis, restriction fragment length polymorphism, typing with monoclonal antibodies, and polymerase chain reaction.
E. hartmanni is considered to be the “small race” of E. histolytica due to its small size.
E. gingivalis does not have a known cyst stage and inhabits the oral cavity of people with poor oral hygiene.
Endolimax nana and I. buetschlii are nonpathogenic species.
Generalities of Entamoeba
All entamoeba are lumen-dwelling protozoans except E. gingivalis.
All are non-pathogenic except E. histolytica.
Cystic stage is the infective stage except E. gingivalis.
Entamoeba Histolytica
First described by Losch, from a patient with diarrheic stool.
Considered to cause amoebic dysentery and liver abscess worldwide.
Mostly found in tropical and subtropical areas with poor sanitation and overcrowding.
Genetically distinguished from the commensal E. dispar and E. moshkovskii.
Entamoeba Histolytica: Trophozoites
Size: 10-60 µm in diameter, with nonpathogenic forms usually 15-20 µm and invasive forms >20 µm.
Active progressive motility via rapidly formed hyaline pseudopodia with sharp demarcation between endoplasm and ectoplasm; unstained nuclei are not easily visible.
In invasive disease, some trophozoites contain ingested erythrocytes, diagnostic of E. histolytica infection.
Trophozoites (Stained)
Peripheral nuclear chromatin is often evenly distributed along the nuclear membrane as fine granules.
The karyosome is small and often centrally located, with fine fibrils not visible, attaching it to the nuclear membrane.
In degenerating organisms, the cytoplasm may become vacuolated, and nuclei may show abnormal chromatin.
Diagnostic Feature
Presence of red blood cell inclusion from patient with dysentery is diagnostic for E. histolytica.
Trophozoites with ingested RBCs are termed “hematophagous” trophozoites.
In preparation for cyst formation, trophozoites extrude all ingested material and assume a rounded form.
This precyst stage has a single rounded nucleus, absence of ingested material, and lacks a cyst wall; nuclear morphology is often confusing at this stage; rely on trophozoites or cysts for specific ID.
Trophozoite Appearance
The nucleus of the unstained trophozoite is usually not visible.
Bacteria may be ingested, or seen in the cytoplasm if the amoeba is degenerating.
Death/degeneration leads to vacuoles in the cytoplasm – a "Swiss cheese" appearance – and degenerate forms cannot be accurately identified.
If kept too long at room temperature before fixing, the finer nuclear structures undergo change, which are important for specific identification.
Cysts
Recognized by the presence of a hyaline cyst wall, highly refractile in unstained preparations.
Yellow-stained glycogen mass (reddish-brown with iodine stain) and highly refractile chromatoidal bars with smoothened and rounded edges are typically seen in immature cysts (precystic stage).
Mature Cysts
Size: 10-20 µm in diameter (usually 12-15 µm).
Usually spherical but may be ovoid or irregular in shape.
Nuclei: quadrinucleated mature cyst; contain from 1-4 (or rarely more) nuclei.
May appear as small, refractile spheres within the cytoplasm of the unstained cyst; often not visible.
Peripheral chromatin ring may appear thicker and less uniform in size.
Karyosomes appear small and compact; centrally located, usually eccentric.
Cyst Cytoplasm
Glycogen mass disappears.
Chromatoidal bars stain with hematoxylin like the chromatin of the nucleus--elongated, with rounded or squared ends but may be ovoid.
Chromatoidals are more frequently encountered in the mono- and binucleate cysts; and a large proportion of mature quadrinucleate cysts do not possess them.
With hematoxylin, the chromatoidals take the same bluish black stain as the chromatin material of the nucleus, and with trichrome they stain bright red.
Life Cycle
Infective E. histolytica cyst is ingested by the host.
Excystation occurs in the small intestine and requires neutral or slightly alkaline pH levels; no excystation occurs in an acidic environment.
Once activated, the ingested cyst will separate into four trophozoites.
E. histolytica trophozoites inhabit and undergo binary fission in the large intestine.
Some trophozoites invade the intestinal lumen and mucosal crypts, feeding on red blood cells and forming ulcers.
Ulceration of the intestinal wall may give rise to amoebic dysentery.
Invading amoeba may enter capillaries and be transported via the bloodstream to the liver or other organs, where abscesses form.
Encystation
Formation of non-labile, non-motile protozoan.
Cyst formation is possible when the gut is dehydrated and only happens in the intestinal tract; once passed in stool, cyst formation will no longer be possible.
When hypermotility occurs in the intestinal tract, the amoeba in the lumen may pass out in liquid or semi-formed stool as trophozoites.
With normal motility, they will “round up” and differentiate and develop into the four-nucleated resistant cyst stages.
Symptoms & Pathogenesis
Entamoeba histolytica infection may be asymptomatic or cause various clinical diseases, most commonly amebic dysentery, amebic colitis, and amebic liver abscesses.
Amebic dysentery: acute disease characterized by bloody diarrhea with abdominal pain and cramping.
Trophozoite invasion of the intestinal mucosa occurs, producing ulceration that may lead to perforation and peritonitis.
Disease Forms
Amebic colitis: may mimic ulcerative colitis and other inflammatory bowel diseases.
Amebomas: masses of granulomatous tissue, producing a napkin-ring lesion that could be mistaken as carcinoma.
E. histolytica trophozoites invade the intestinal wall, may enter the bloodstream, and disseminate via the portal blood supply to the liver and other organs.
Amebic Liver Abscess
Most common form of extraintestinal amebiasis.
Symptoms: fever and right upper quadrant pain.
Diagnosed by radiographic scans, ultrasound, and parasite-specific host antibodies.
Amebic abscesses in other organs (lungs, brain, or skin) occur by hematogenous spread from the intestine or by contiguous spread from a liver abscess.
In brain involvement, trophozoites must be differentiated from free-living amebae by careful examination of trophozoite morphology, associated host response, and clinical history.
Virulence
Production of enzymes and cytotoxic substances.
Cysteine proteinase: degrades host proteins; enables attachment to the gut by degrading mucus debris; stimulates host cell proteolytic cascades.
Contact-Dependent Cell Killing
Gal/GalNAc-binding lectin: responsible for cellular adherence, contact-dependent toxicity, resistance to host complement, and cell endocytosis
Pathogenic strains of E. histolytica may be resistant to complement mediated via inhibition of membrane attack complex formation.
Amebapore: forms ion channels in phagocytized bacteria and eukaryotic cells.
Cytophagocytosis
Trophozoites of a virulent strain of E. histolytica can kill normal human polymorphonuclear neutrophils, monocytes, and macrophages in vitro.
Activated macrophages can kill the same virulent amoebae through contact-dependent, antibody-dependent mechanisms.
Clinical Classification from WHO Report on Amoebiasis (1969)
Asymptomatic infections
Symptomatic infections
Intestinal amoebiasis
Dysenteric
Nondysenteric colitis
Extraintestinal amoebiasis
Hepatic
Acute nonsuppurative
Liver abscess
Pulmonary
Other extraintestinal foci (very rare)
Asymptomatic Infections
Cysts are present in stool among patients with negative or weak antibody titer.
Occurs in patients infected with pathogenic or nonpathogenic species.
E. histolytica/ E. dispar trophozoites may be recovered in stool; however, phagocytized red blood cells are absent in the cytoplasm.
Isoenzyme analysis may be particular for the non-pathogenic isolates – E. dispar.
Symptomatic Infections
Symptoms accompanied by little local reaction and often no recognizable symptoms: diffuse inflammation, indistinguishable from nonspecific inflammatory lesions of other types of colitis.
May provoke mild diarrhea with only a few loose stools daily, perhaps alternating with periods of constipation.
In normal stools, careful examination of the feces may reveal flecks of blood-tinged mucus, often containing numbers of motile E. histolytica.
Patients with more acute illness may have a dozen or more explosive liquid stools daily, containing much blood and mucus, perhaps accompanied by abdominal cramps.
Tenesmus, painful spasms of the anal sphincter, is a sign of rectal ulceration.
Intestinal Infections
Attachment of E. histolytica trophozoites in the colonic mucosa is mediated by an amoebal galactose-inhibitable adherence lectin.
Intestinal secretory IgA against the parasite Gal/GalNac lectin is associated with immunity to reinfection.
After attachment, the parasite kills the host cell in an extracellular process that involves activation of host cell Caspase-3, leading to apoptotic death and engulfment of the host cell.
Amoebic Colitis
Fever is not characteristic of uncomplicated amoebic colitis.
Mild leukocytosis may be seen, probably a response to the secondary bacterial infection frequently present.
White blood cell count seldom rises above 12,000 per microliter.
In moderately severe attacks of diarrhea or dysentery, spontaneous subsidence or alteration with periods of constipation is common.
Abdominal palpation may reveal tenderness of the cecum, transverse colon, or sigmoid.
Amoebae Penetration
Penetration of muscularis mucosae into the submucosa results in flask-shaped ulceration and intraluminal bleeding.
If large numbers of ulcers are produced, they may coalesce by means of intercommunicating submucosal sinus passages.
The undermined mucosa may remain fairly normal in appearance.
If the undermining is extensive and there is secondary bacterial infection, necrosis and sloughing of large portions of the intestinal wall may occur.
An intestinal cast may appear, but rarely in stools.
Sigmoidoscopic Examination
Almost normal mucosal pattern or indistinguishable from those seen in ulcerative or granulomatous colitis.
Scattered ulceration may range from a few millimeters in diameter (10-12 mm) with raised edges, but with normal-looking mucosa elsewhere.
Used to differentiate amoebic from bacillary dysentery, where the entire mucosa is involved in bacillary dysentery.
As the amoebic infection progresses, coalescence of the ulcers may produce irregularly wandering ulcer trenches, sometimes with hairlike remnants of the more resistant supportive structures projecting from their bases (“buffalo skin” or “Dyak hair ulcers”).
Amoebic Ulcer Perforation
Accompanied by the usual signs of peritoneal irritation or infection.
Slow leakage to the abdominal cavity through a severely diseased colonic wall may be more common, marked by distention, ileus, and gas in the peritoneal cavity.
Surgical intervention may be feasible in the case of acute perforation but not in the chronic type or in amoebic appendicitis.
Chronic Granulomatous Lesion
Known as an amoeboma, develops most frequently in the cecal or rectosigmoid region.
May produce a so-called napkin ring constriction of the bowel wall indistinguishable on x-ray examination from an annular carcinoma, or may give rise to a characteristic conical configuration of the cecum.
Radiologic findings are similar to those seen in inflammatory bowel disease, although seldom with involvement of the terminal ileum.
Extraintestinal Infections
Hepatic enlargement: a toxic response to intestinal infection, unrelated to the local presence of amoebae.
Amoebic hepatitis: a diffuse early stage of liver infection, without abscess formation.
Hepatic infection: characterized by liver tenderness and enlargement, fever, weight loss, and sometimes a cough with evidence of pneumonitis involving the right lower lung field.
Hepatomegally and tenderness may occur in amoebic colitis without any evidence of hepatic infection; the right leaf of the diaphragm may be elevated and fixed in position.
Hepatic Infection Progression
Multiplication of amoebae in the liver may lead to the development of single or multiple abscesses; a majority of amoebae that reach the liver are probably destroyed there and do not produce abscesses.
A single large abscess may arise from the coalescence of multiple smaller ones.
With abscess formation, hepatic pain becomes more severe and continuous; pain may also be referred to the right or left shoulder, depending on the position of the abscess.
Leukocytosis of 15,000 – 35,000 per microliter without a characteristic differential, fever, and night sweats may also occur.
Liver Abscess Symptoms
Fever occurs usually in the afternoon, reaching a peak of about 102°F, and is accompanied or followed by profuse sweating.
Aspiration of an amoebic abscess usually yields a thick, reddish-brown fluid, which rarely contains amoebae.
Pleuro-pulmonary Amoebiasis
Pulmonary amoebiasis occurs in 2-3% of patients with invasive infections and is the second most common extraintestinal infection caused by pathogenic E. histolytica, frequently associated with the erosion of a hepatic abscess through the diaphragm into the lung.
Pleurisy, with or without effusion or pleural rub, or right lower lobe pneumonitis may signal a subdiaphragmatic abscess without actual rupture into the pleural space.
If the abscess is localized in the left lobe of the liver, it may involve the left lung.
If hepatic spread of the infection extends to involve a bronchus, amoebae may be found in the sputum.
Primary pulmonary amoebiasis is associated with infection resulting from blood leakage from the colon as the primary site rather than from the liver.
Extraintestinal Involvement
Such as the brain, pericardium, and spleen, are uncommon; when they occur, they are most often accompanied by amoebic liver abscess.
Amoebic infection of the skin is rare but may produce extensive gangrenous ulcerations of the perineal tissues or affect the skin surrounding a colonostomy or draining hepatic abscess.
Vaginal, urethral, and clitoral infections have been reported.
Amoebiasis of the penis is seen following intercourse with a partner who has vaginal amoebiasis and also as a consequence of anal intercourse.
Diagnosis
A laboratory diagnosis of E. histolytica infection, unless confirmed by visualization of ingested red blood cells in the trophozoite, should be substantiated by:
Presence of red blood cells in the stool
Serum antibody titer
Stool E. histolytica antigen titer.
Routine Diagnosis
Microscopic examination of stool is useful.
Traditional stool ova and parasite exam is not sensitive and lacks specificity in identifying the organism.
Antigen and Antibody Detection
The best way to diagnose E. histolytica infection is by a combination of stool antigen detection and serology.
Indirect hemagglutination (IHA) and enzyme-linked immunosorbent assay (ELISA)
Enzyme immunoassays (EIA) are available to detect Entamoeba-specific antigen in feces.
Monoclonal ELISA kit detects E. histolytica Gal/GalNac lectin in stool and distinguishes E. dispar.
Molecular Diagnosis
Polymerase chain reaction (PCR) amplification of genomic DNA, hybridization of cDNA clones, and rRNA probes provided additional evidence for the separation of the invasive E. histolytica from noninvasive E. dispar.
Subsequent research using RNA and DNA probes also indicated differences between invasive and noninvasive strains.
The inability to morphologically distinguish E. histolytica from nonpathogens E. dispar and E. moshkovskii underscores the importance of modern diagnostic tests.
Liver Scans
Reveal areas of nonvisualization, most frequently single and in the right lobe, less often multiple or in other locations.
Sonography, magnetic resonance imaging, and computed tomography offer convenient means for evaluating the development and resolution when refined imaging techniques lead to early diagnosis and treatment.
Liver function tests are of little value in the differential diagnosis of amoebic abscess.
Organisms are confined to the hepatic tissue of the abscess walls; diagnosis by response to therapy is the only practical approach; modern techniques such as real-time PCR are highly sensitive for detecting E. histolytica DNA in the abscess.
Tissue Examination
Processing may include special histopathological staining techniques.
Periodic acid-Schiff (PAS) staining is used to locate organisms within tissues, where a trophozoite appears bright pink in a green-blue background.
Hematoxylin and eosin staining allows accurate morphological demonstration of trophozoites.
Epidemiology
Sporadic cases of E. histolytica infections occur worldwide but are mostly prevalent throughout Southeast Asia, Southeast and West Africa, and Central and South America.
The prevalence of amoebic infection varies with the level of sanitation and is higher in the tropics and subtropics than in temperate climates.
Enteric infections caused by pathogenic amoeba remain one of the causes of mortality worldwide (2017).
In the Philippines, amoebiasis has been noted among children alongside other diarrheal disease etiologies (rotavirus, cholera, salmonella, and enterotoxigenic organisms).
Factors Influencing Disease Severity
The severity of the disease and the incidence of complications may be greater in the tropics, reflecting the higher incidence of infection.
The severity of infection is associated greatly with malnutrition.
E. histolytica and E. dispar infection is observed in men who have sex with men.
Asymptomatic patients are of utmost importance in the transmission of the disease caused by:
Water contamination
Poor food handling
Food contamination by flies and possibly cockroaches
The use of human feces as fertilizer
Cysts are relatively resistant but are killed by drying, temperatures >55°C, and by superchlorination or the addition of iodine to drinking water.
Treatment
All patients with amebiasis should be treated with suitable antiparasitic agents; asymptomatic individuals are treated with iodoquinol, paromomycin, or diloxanide furoate to eliminate the luminal stage of intestinal infection and prevent environmental shedding of infectious cysts.
Patients with invasive or disseminated disease are first treated with metronidazole or tinidazole (good systemic penetration) followed by either iodoquinol or paromomycin to eradicate any intestinal parasites.
Invasive Disease Treatment
Metronidazole or tinidazole.
Intestinal (luminal) infection.
Paromomycin
Diloxanide furoate (Furamide) is restricted to patients who only pass cysts.
Metronidazole is effective against anaerobic or microaerophilic organisms; it is activated by reduction of ferredoxin, generating a reactive radical.
Metronidazole Use
Recommended for acute amoebic colitis.
Side effects include nausea, diarrhea, metallic taste, and headache.
Patients should abstain from alcohol during treatment.
Approved for use during the last two trimesters of pregnancy.
Tinidazole is generally better tolerated than metronidazole and requires a shorter course of treatment.
Treatment Failures, Emetine Usage
In cases of treatment failures, metronidazole treatment should be followed with a luminal agent (paromomycin or diloxamide) to eliminate intestinal colonization and prevent relapse.
Emetine was formerly used for treatment of amoebiasis, but cardiac toxicity has precluded its routine use.
Metronidazole and tinidazole are first-line agents in the treatment of hepatic abscess.
Draining of larger abscesses may be necessary in exceptional circumstances.
Prevention
Amoebiasis is acquired through fecal contamination of food and water; prevention involves measures to break the chain of infection.
Water can be readily disinfected by boiling and treatment with iodine.
Regulations regarding food handlers vary, but a minimum criterion for cure should be a series of negative stool examinations (preferably at least three examinations) taken at least 1 month after completion of treatment.
Avoid the usage of human feces as fertilizers.
Avoid eating food sold in streets and consumption of salads and fruits from unreliable food handlers in developing countries.
Non-Pathogenic Amebae
Entamoeba Dispar and E. Moshkovskii
Emile Brumpt (1925) proposed that E. histolytica is a complex species in which E. histolytica is the invasive species, and E. dispar and E. moshkovskii are morphologically identical noninvasive ones.
Redescription of E. histolytica confirmed Brumpt’s hypothesis.
E. dispar (dispar = different) and E. moshkovskii are synonymous with formerly designated nonpathogenic E. histolytica.
Diagnosis of these parasites
Microscopy alone for the unequivocal detection of E. histolytica infection is not reliable; E. histolytica, E. dispar, and E. moshkovskii are morphologically indistinguishable.
Microscopic identification of E. histolytica can only be made if ingested erythrocytes are present in its trophozoites.
Regardless of symptoms, the presence of what appear to be E. histolytica-like forms in the stool, along with a positive serologic response, indicates the presence of true E. histolytica.
A negative serologic test and E. histolytica-like amoebae in the stool indicate E. dispar.
Reporting Procedures
Morphologically distinguished organisms must be reported as “E. histolytica/E. dispar” using routine microscopic analysis of stool without immunoassay procedures.
Reliable detection procedures for Entamoeba antigens and antibodies are commercially available, as are commercially produced serological test kits that detect a fecal Entamoeba antigen or antibody common to both species.
Molecular studies have flourished in the past decades; attempts at genetic encoding have identified techniques that will differentiate various parasitic species.
Detection rates and specificity have been greatly improved by PCR assays.
Characteristics Valuable in Identification of E. histolytica/E. dispar/E. moshkovskii Species Complex
Trophozoites (Unstained): Progressive motility; hyaline pseudopodia; no ingested bacteria; nuclei not visible. Ingestion of red blood cells.
Trophozoites (Stained): Clear differentiation of ectoplasm and endoplasm; no ingested bacteria. Fine, uniform granules of peripheral chromatin and small central karyosome in the nucleus; ingested red blood cells; average size 12µm.
Cysts, unstained: Four nuclei; rod-like chromatoidals
Cysts, stained: Maximum of four nuclei having both karyosome and peripheral chromatin; diameter over 10µm. Typical nuclear structure; chromatoidal bars with rounded or squared ends; diameter over 10µm.
Entamoeba Hartmanni
Morphologic differences between E. histolytica/E. dispar/E. moshkovskii and E. hartmanni only lie in size.
E. hartmanni has now attained general acceptance as the name for the amoebae formerly designated as “small race” E. histolytica.
Studies of prevalence in which E. hartmanni has been differentiated from E. histolytica indicate roughly similar incidence and distribution for the two.
E. hartmanni is considered to be nonpathogenic; do not treat this infection.
Morphology of Entamoeba Hartmanni
Rounded trophozoites measure from 3–12 µm in diameter; cyst size range is from 4–10 µm.
Nuclear structure shows the same variations seen in E. histolytica and there is no consistent difference between the two species in nuclear-cytoplasmic ratio.
Chromatoidal material assumes a similar rod or cigar-like form in the two species.
Ingest bacteria but not red blood cells.
Entamoeba Coli
Nonpathogenic amoeba that closely resembles E. histolytica.
Initially considered as pathogens, but were proven to be nonpathogenic by 1913.
Morphology of Trophozoites
Size: 15–50 µm in diameter (average slightly more than 20 µm)
Motility: sluggish, nondirectional, and nonprogressive.
Move by means of blunt and short pseudopodia; never long and fingerlike as they may be in E. histolytica.
Pseudopodia are extruded slowly and are not hyaline, and there is no striking differentiation of the cytoplasm into ectoplasm and endoplasm.
Nucleus of Trophozoites
The karyosome is large, irregular in shape, usually eccentric in position, and surrounded by a halo of non-staining material.
Granules of chromatin may be seen scattered between the karyosome and the peripheral chromatin, and sometimes a linin network is visible.
The peripheral chromatin in E. coli is irregular both in size and arrangement on the nuclear membrane; definitely more abundant than in usual E. histolytica.
A ring of refractile granules, representing the peripheral chromatin, encloses another eccentric refractile mass, the karyosome.
Cytoplasm of Trophozoites
Granular, containing many vacuoles.
Red blood cells are not ingested by this amoeba except under the most unusual circumstances.
Bacteria are regularly seen in vacuoles in the cytoplasm.
Precystic forms are seen, but the morphology is not very distinctive.
Cyst Morphology of Trophozoites
The spherical cyst wall is highly refractile, and the cytoplasm is granular in appearance.
Food vacuoles are absent.
Size: 10 to nearly 35 µm in diameter; the average diameter is greater than in cysts of the pathogenic species.
Cyst Nuclei
Usually readily observed; they vary in number from 1-8.
A mature cyst contains 8 nuclei; hypernucleate forms with 16–32 nuclei are observed.
The eccentric position of the karyosome can be distinguished, even in unstained amoebae.
Cytoplasm: very granular; areas occupied by glycogen before fixation are marked by empty spaces in the cytoplasm of the fixed and stained cysts.
Glycogen may occur in the cysts of E. histolytica; the perinuclear disposition of this material is more characteristic of E. coli.
Chromatoidal Characteristics
Less common in E. histolytica but occasionally may be observed as clear, thin lines or rods of refractile material in the cytoplasm.
Less frequently observed than E. histolytica/E. dispar.
The chromatoidals are seen to be composed of splinter-shaped or rarely ribbon or threadlike bodies.
Heavier bodies with irregular ends are also observed.
With an iodine stain, glycogen may be seen in the cysts of E. coli; often, masses of this dark-staining material completely surround the nuclei, which are entirely obscured.
Identification of Intestinal Parasites
Trophozoites, unstained: Sluggish, nondirectional motility; short, granular pseudopodia; ingested bacteria; visible nucleus
Trophozoites, stained: Granular cytoplasm without much differentiation into ectoplasm and endoplasm; bacteria in food vacuoles and Nucleus with irregular clumps of peripheral chromatin; large, irregular, eccentric karyosome
Cysts, unstained: Eight nuclei; glycogen mass surrounding nuclei (iodine stains).
Cysts, stained: Maximum of eight nuclei, having karyosome and peripheral chromatin and Typical nuclear structure; splinter-shaped or irregular chromatoidals
Entamoeba Gingivalis
Bears a close morphologic resemblance to E. histolytica.
Isolates are often found in pyorrheal pockets between teeth and gums and in the tonsillar crypts.
First parasitic amoeba described in humans.
Has been reported to multiply in bronchial mucus and appear in the sputum, where it might be mistaken for E. histolytica from a pulmonary abscess.
E. gingivalis was found in 59% of 113 dental patients and in 32% of 96 control subjects with good oral hygiene.
Few cases of Entamoeba infection of the uterus have been described in patients with no intestinal infection who lived in areas of low endemicity.
Morphology of Entamoeba Gingivalis
Forms no cysts.
Size: 5-15µm in diameter.
The cytoplasm may contain bacteria, occasional red cells, but is most frequently filled with portions of ingested leukocytes.
Lobose pseudopodia may be seen to be long and blunt.
Nuclear fragments from the leukocytes are recognizable in stained specimens and serve to identify the amoeba, as E. gingivalis is the only species that ingests these cells.
Mononucleated with a centrally located small karyosome.
Entamoeba Polecki
First reported as an intestinal parasite of pigs and monkeys and occasionally found in humans.
A few reports describe patients with diarrhea apparently caused by infection with this parasite.
Pig-to-human transmission is considered the most likely route of human infection; the possibility of human-to-human transmission exists where the prevalence of infection is high.
Morphology of Trophozoites
E. polecki resemble E. coli in their motility, in the granularity and degree of vacuolization of their cytoplasm, and in the ingestion of bacteria.
Directional motility such as seen in E. histolytica occurs sporadically.
Pseudopodia are usually formed slowly but occasionally may be thrust out in the explosive manner characteristic of E. histolytica.
The nucleus is occasionally visible in the unstained trophozoite.
In stained preparations, the nuclear structure appears somewhat intermediate between those of E. histolytica and E. coli.
Nucleus and Peripheral Chromatin
The karyosome is small in trophozoites and is usually centrally located; it may be spherical or stellate in shape, or it may consist of a group of small granules.
The peripheral chromatin is generally seen in the form of fine granules evenly distributed on the nuclear membrane; normally compact but occasionally dispersed.
Cyst Characteristics
Characterized by a single nucleus; very rarely, it is binucleate or quadrinucleate.
Chromatoidal material resembling that seen in E. histolytica is formed and is often abundant.
The ends of chromatoidals are angular and sometimes pointed, rather than regularly rounded or squared off as in E. histolytica; threadlike chromatoidals have also been reported.
Differentiation of Cysts
Unstained cysts cannot be differentiated with any certainty from mononucleate cysts of the other two species of Entamoeba, though the presence in a formed stool of large mononucleate cysts, and the near absence of cysts with greater numbers of nuclei, is suggestive.
Iodine-stained cysts are likewise not distinctive: The inclusion mass does not take the dark stain characteristic of glycogen and is not seen clearly.
Diagnosis
Routine microscopic analysis of fresh stool remains the traditional method.
Morphological characteristics of E. polecki are difficult to distinguish from Entamoeba spp.
Molecular techniques, including polymerase chain reaction and nucleotide sequencing, are widely utilized for accurate diagnosis of Entamoeba species and subtypes.
Some doubt is cast on the validity of this species by isoenzyme studies of a number of isolates showing the morphologic characteristics of E. polecki; the isoenzyme pattern fell within one or another of what are now