Professional Documents
Culture Documents
E. Histolytica, E. Dispar and E. Moshkovskii Are Morphologically Undistinguishable E. Histolytica Was Distinguished From The Two by
E. Histolytica, E. Dispar and E. Moshkovskii Are Morphologically Undistinguishable E. Histolytica Was Distinguished From The Two by
Subphylum Sarcodina -> Superclass Rhizopoda -> class Lobosea -> family Entamoebidae -> genus
Entamoeba
A pseudopod
E. histolytica, E. dispar and E. moshkovskii are morphologically undistinguishable
E. histolytica was distinguished from the two by:
o Isoenzyme analysis Polymerase Chain Reaction
o Restriction Fragment Length Polymorphism (RFLP)
o Typing with monoclonal antibodies
Most invasive Entamoeba parasite
Case manifestations:
o Asymptomatic
Most E. histolytica cases in endemic communities
o Amebic colitis
Abdominal pain
Diarrhea w/ or w/o blood and mucus in the stool
Fever (rare)
Children may develop fulminant colitis with all clinical manifestations
Most serious complication
Perforation
Secondary bacterial peritonitis
o Amebic Liver Abscess (ALA)
Most common extra-intestinal form of amebiasis
Right upper quadrant (RUQ) pain
Hepatomegaly in acute cases (50% of cases)
Incubation period of 8 to 10 days
Most serious complications
Rupture into pericardium
Rupture into pleura
Super infection
o Ameboma
<1% of intestinal infections
Mass-like lesion with abdominal pain and a history of amebic dysentery
Can be mistaken for carcinoma
Two stages:
o Infective cyst
Quadrinucleated cyst is the diagnostic stage and is resistant to gastric acidity and dessication
o Vegetative (invasive) trophozoite
Mode of transmission is through:
o ingestion of fecally-contaminated material
o venereal transmission through fecal-oral contact
o Direct colonic inoculation
Excystation occurs in the small or large vowel and undergoes nuclear then cytoplasmic division to
form eight trophozoites
E. histolytica trophozoite:
o 12-60um diameter (20um avg) o Hyaline pseudopodia
o Progressive and directional movement o Ingested RBC in the cytoplasm
E. histolytica cyst:
o Spherical o 1-4 nuclei
o 10-20um o Rod-shaped (cigar-shaped)
o Highly refractile hyaline cyst wall chromatoidal bars
Trophozoites multiply by binary fission
Trophozoite encyst to produce uninucleate cyst which will undergo two nuclear division to form
quadrinucleated cysts
E. histolytica at one time may have contained mitochondria
Differences in biochemical pathways from higher eukaryotes and E. histolytica:
o Lack of glutathione metabolism
o Use of pyrophosphate instead of ATP at several steps in glycolysis
o Inability to synthesize purine nucleotides de novo
Mechanisms for virulence:
o production of enzymes or other cytotoxic substances
o Contact-dependent cell killing
o Cytophagocytosis
Amebic killing of target cultivated mammalian cells:
o Receptor-mediated adherence of ameba to target cells
o Amebic cytolysis of target cells
o Amebic phagocytosis of viable target cells
Trophozoites adhere to the colonic mucosa through Galactose-inhabitable adherence lectin (Gal
lectin)
Amebae kill mucosal cells by activation of their caspase-3
PCR has confirmed that there is a higher prevalence of E.dispar than E. histolytica
Activated T-cells kill E. histolytica by:
o Directly lysing trophozoites
o Producing cytokines
o Providing helper effect for B-cell antibody production
Cytokine studies revealed that Interferon (IFN) and Interleukin (IL-2) may have a role in activating
macrophages for amebicidal activity
Principal Antibody dependent cell-mediated cytotoxicity (ADCC) did not work against amebae
Bacillary dysentery vs Amebic dysentery
o Fever and significantly elevated leukocyte count are less common in amebic colitis
Differential diagnosis of ALA include pyogenic liver abscess, tuberculosis of the liver and hepatic
carcinoma
Differential diagnosis of genital amebiasis include carcinoma, tuberculosis, chancroid and
lymphogranuloma venereum
Standard method of parasitologic iagnosis is microscopic detection in stool samples (examine w/in
30 minutes; minimum of three stool specimen collected on different days)
Trophozoite motility can be observed using DFS with saline solution
Using saline and methylene blue, Entamoeba species will stain blue
Using saline and iodine will help us differentiate E. histolytica from non-pathogenic amebae
Charcot-Leyden crystals can be seen in stools with amebiasis
FECT and MIFC are more sensitive than DFS for detection of cysts
Robinson’s and Inoki medium is more sensitive than stool microscopy, but is not routinely available
Differentiation of E. histolytica and E. dispar can be done by
o PCR (gold standard)
o Enzyme –linked immunosorbent assay (ELISA) (gold standard)
o Isoenzyme analysis
In ALA, trophozoites are missed because they are located in the periphery of the abscess
Serological tests for amebic disease
o Indirect hemagglutination (IHAT) (can detect antibodies as long as 10 years ago) (gold standard)
o Counter immunoelectrophoresis (CIE)
o Agar gel diffusion (AGD)
o Indirect fluorescent antibody test (IFAT) (gold standard)
o ELISA
Noninvasive and sensitive methods in the early detection of ALA
o Ultrasound
o MRI
o CT scan
Objectives of the treatment of amebiasis
o Cure invasive diseases at both intestinal and extraintestinal sites
o Eliminate the passage of cyst from the intestinal lumen
Drugs for treatment of invasive amebiasis
o Metronidazole
o 5-nitroimidazole derivatives
Secnidazole
Tinidazole
o Diloxanide furoate (for asymptomatic cyst passers)
Prevalence of amebiasis is approx. 1 to 5% worldwide
It is the 3rd most important parasitic disease (next to malaria and schistosomiasis)
E. dispar is prevalent in immigrants, travelers from endemic countries, homosexual males, HIV
patients and institutionalized people
Prevention and control
o Proper sanitation
o Safe water supply
o Good personal hygiene
o Proper food preparation
o Vaccines
COMMENSAL AMEBAE
Significance of the presence of commensal amebae in stool
o Be mistaken for the pathogenic E. histolytica
o Indication of fecal contamination of food or water
Must be differentiated from pathogenic E. histolytica to avoid unnecessary treatments
Entamoeba Endolimax Iodamoeba
Spherical nucleus Vesicular nucleus Large, chromatin-rich
Nuclear membrane lined w/ Large, irregularly shaped karyosome surrounded by a
chromatin granules karyosome anchored to the layer of achromatic globules
Small karyosome near the nucleus by achromatic fibrils Anchored to the nuclear
center of the nucleus membrane by achromatic
Trophozoites usually have fibrils
only one nucleus
Entamoeba hartmanni
Morphologically similar to E. histolytica apart from its smaller size
Trophozoites measure from 3 to 12um in diameter
Mature cysts measure from 4 to 10um
E. hartmanni does not ingest RBC
Entamoeba coli
Cosmopolitan in distribution
More common than other human amebae
E. coli trophozoites:
o 15 to 50um in diameter o Broader and blunter pseudopodia used
o More vacuolated or granular more for feeding
endoplasm with bacteria and debris o Sluggish, unidirected movements
o Narrower, less differentiated ectoplasm o Thicker, irregular peripheral chromatin
o Large, eccentric karyosome
E. coli cyst:
o 10 to 35um in diameter
o Up to eight nuclei
o Granular cytoplasm
o Splinter-like chromatoidal bodies
Iodine staining reveals dark-staining, perinuclear masses, which are actually Glycogen
Entamoeba gingivalis
Found in the mouth
E. gingivalis trophozoite:
o 10 to 20um
o Moves quickly
o Numerous blunt pseudopodia
o Food vacuoles that contain cellular debris and bacteria are numerous
Abundant in cases of oral disease
NO CYST STAGE
Transmission is most probably direct
Swab between the gums and teeth is examined for trophozoites
Endolimax nana
Occurs with the same frequency as E. coli
E. nana trophozoites
o 5 to 12 um
o Sluggish movement
o Blunt pseudopodia
o Large, irregular karyosome
o Food vacuoles in the cytoplasm contain bacteria
Cysts are about the same size as trophozoites, and are quadrinucleated when mature
Iodamoeba butschlii
I. butschlii trophozoite
o 9 to 14 um in diameter
o Large, vesicular nucleus
o Large, central karyosome
o Surrounded by achromatic granules
o No peripheral chromatin granules on the nuclear membrane
I. butschlii cyst
o 9 to 10um in diameter (6 to 16um)
o Uninucleated
o Large glycogen body
o Stains dark brown with iodine
Naegleria spp.
Two vegetative forms: ameba (trophozoite form), and a flagellate (swimming form)
A dormant cyst forms when conditions are not favorable
Thermophilic – they thrive best in warm aquatic environments
Naegleria fowleri
Two forms of trophozoites: ameboid (only one found in humans)and ameboflagellate
N. fowleri ameboid trophozoite (INFECTIVE STAGE)
o When rounded are usually 10 to 15um in diameter (may get over 40um in culture)
o Granular cytoplasm
o Many vacuoles
o Large single nucleus
o Large, dense karyosome
o Lacks peripheral chromatin
Only species to consistently cause disease
Three stages: cysts, trophozoites (replicate by promitosis) and flagellated forms
Can turn into temporary nonfeeding flagellated forms
Trophozoites infect by penetrating the nasal mucosa and migrating to the brain via the olfactory
nerves
Trophozoites are found in CSF and tissue
Flagellate forms are occasionally found in CSF
Cysts are not seen in brain tissue
Causative agent of Primary Amebic Meningoencephalitis (PAM)
PAM
Rare, but fatal
Usually occurs in healthy adults with a history of swimming
In contrast to Acanthamoeba, which is largely an opportunistic organism, N. fowleri is considered a
true pathogen
Symptoms of PAM are indistinguishable from bacterial meningitis
PAM presents as
o Fever
o Nausea
o Headache
o Nuchal rigidity
o Mental status changes
o Rapid progression to coma and death
Characteristic CSF findings
o Elevated WBC with neutrophilic predominance, high protein, and low glucose
Postmortem examination of infected brain
o Hemorrhagic necrosis
o Congestion and edema of neural tissue
o Leptomeninges are inflamed and congested
Diagnosis
o Introduced into bacteria-seeded agar culture medium to exhibit active trophozoites
o PCR and immunostaining
o ELISA is less useful
Treatment
o Amphotericin B with clotrimazole
o Azithromycin
o Voriconazole
Local Naegleria – N. philippinensis (isolates from a thermally polluted stream)
Prevention and control
o Avoid accidental inhalation of water
o N. fowleri is easily killed by chlorination of water at 1ppm or higher
Giardia duodenalis
Worldwide distribution
Discovered by Antonie van Leeuwenhoek
First described as Cercomonas intestinalis
Causes giardiasis. Significant, but not life-threatening
Lives in the duodenum, jejunum, upper ileum
Simple Asexual life cycle
Has trophozoites and quadrinucleated infective cyst stages
Classified as either A or B genotype
G. lamblia Trophozoite:
o 9 to 12um long by 5 to 15um wide
o Pyriform or teardrop shaped, pointed posteriotly
o Pair of ovoid nuclei
o Bilaterally symmetrical with a medial line called axostyle
o Tumbling motion, falling leaflike
o Four pairs of flagella
o Divide by longitudinal binary fission
o Antigenic variation results in the entire surface of the parasite being covered with variant-
specific surface proteins (VSPs)
o May be found in the jejunum
G. lamblia cysts:
o Ovoid
o 8 to 12um long by 7 to 10um wide
o Young cysts have two nuclei; mature cysts have four
o Flagella retracted into axonemes
o Median or parabasal body
o Deeply-stained curved fibrils surrounded by a tough hyaline cyst wall
o Excyst in the duodenum
Infection through ingestion of contaminated food or water
It has an adhesive sucking disc on its ventral side
Able to avoid peristalsis by trapping itself within the villi or within the intestinal mucus
1-4 weeks incubation
Half of the patients with giardiasis may be asymptomatic
Signs and symptoms of giardiasis
o Abdominal pain described as cramping
o Diarrhea (most common symptom, followed by malaise and flatulence)
o Excessive flatus with an odor of “rotten eggs” due to hydrogen sulfide
o Other features
Abdominal bloating
Nausea
Anorexia
o Chronic infection is characterized by steatorrhea
o Weight loss
o Profound malaise
o Low-grade fever
Diagnosis
o DFS for trophozoites
o Concentration techniques for cysts in stools
o Antigen detection tests
o Immunochromatographic assays detect the presence of Giardia antigen in stool
o Cyst Wall Protein 1 (CWP1) is the antigen used for the test
o Direct fluorescent antibody assays is considered as the gold standard in standard diagnosis
Treatment
o Metronidazole
o Tinidazole
o Furazolidone
o Albendazole (equally effective as metronidazole at the above doses)
o Nitazoxanide (in drug-resistant cases)
86% of the isolated genotypes belong to assemblage B
Direct oral-anal sexual contact among men with men increases the chance of giardiasis
Relative resistance to chlorine facilitates the transmission of Giardia
For prevention and control, normal water chlorination will not affect cysts, but usual water
treatment modalities should be adequate
Trichomonas vaginalis
Causes a sexually-transmitted disease called trichomoniasis
Most prevalent nonviral STI (trichomoniasis)
ONLY IN THE TROPHOZOITE STAGE
o Pyriform shape
o 7 to 23um
o Four free anterior flagella that appears to rise from a simple stalke (kinetosome)
o Fifth flagellum embedded in the undulating membrane
o Median axostyle
o Single nucleus
o Multiply by binary fission
Found in the urogenital tract
Mode of transmission is by sexual intercourse
Requires vaginal, urethral and prostatic tissues to survive
Causes desquamation of the vaginal epithelium followed by leukocytic inflammation of the tissue
layer
Results in liquid vaginal secretion, greenish or yellow in color
Vaginal secretions may cause intense itchiness and burning sensations
Symptoms of trichomoniasis
o Vaginal discharge
o Vulvitis
o Dysuria
o Hemorrhages of the cervix (strawberry cervix)
o In males (almost always asymptomatic), recurring urethritis may happen. Prostatitis is the most
common complication
Diagnosis
o Saline preparation of vaginal fluid
o Gold standard is culture
o Unstained wet drop may be fixed and stained by
Giemsa
Papanicolau
Romanowsky
Acridine orange stains
o Can be cultured using
Diamond’s modified medium
Feinberg and Whittington culture medium
o PCR assays are available, but not widely used locally. PCR assays appears to detect more cases in
men, tho
Treatment is Metronidazole or Tinidazole 2g
Local isolates of T. vaginalis show low genetic polymorphism
Prevention by limiting the number of sexual partners, use of protective sexual devices and having
good sex education
NON-PATHOGENIC FLAGELLATES
Trichomonas hominis
Occurs ONLY AS TROPHOZOITE
o Pyriform shape
o 7 to 13um
o Five anterior flagella
o Posterior flagellum projecting from an undulating membrane
o Cytostome and nucleus at the anterior end
o Axostyle extends from anterior to posterior along the mid-axis
Transmission through fecal contamination of food and drinks
Habitat is the cecal area of the large intestine
Non-invasive
Prevalence in the ph is less than 1%
Trichomonas tenax
Pyriform flagellate
ONLY IN THE TROPHOZOITE STAGE
o 5 to 12 um
o Smaller and more slender than T. vaginalis
o Four free equal flagella and a fifth one on the margin of an undulating membrane
o Single nucleus
o Cytostome
o Multiplies by binary fission
Harmless commensal living in the human mouth
Quite resistant to changes in temperature
Exposure through direct contact (like gingivalis)
Diagnosis through swabbing (like gingivalis)
Drug of choice is Metronidazole
Chilomastix mesnili
Inhabits the cecal region of the large intestine
Well-defined trophic and cyst stages
C. mesnili trophozoite:
o Asymmetrically pear-shaped
o Spiral groove extending through the middle half of the body
o 6 to 10um
o Boring and spiral forward movement
o Three anterior free flagella and a more delicate one within the prominent cytostome
C. mesnili cyst
o Pear or lemon-shaped
o Broadly rounded and somewhat bluntly conical at the other end (nipple-shaped)
o Has knob-like protuberance
o H&E films demonstrate the single large vestibular nucleus and the cytostome
o Good preparations reveal a fibril
Transmission occurs through ingestion of cysts
Prevalence is less than 1%
No treatment indicated
Dientamoeba fragilis
Identified in all regions of the worlds
Iron-hematoxylin stains have been carefully examined
Originally described as an amoeba, but is actually a flagellate
ONLY THE TROPHOZOITE STAGE IS KNOWN
o 7 to 12um
o One or two rosette-shaped nuclei
o No peripheral chromatin
o Karyosome consists of four to six discrete granules
o Resembles Trichomonas
Lives in the mucosal crypts of the appendix, cecum and the upper colon
Exact life cycle is unknown
Transmission is probably fecal-oral or via transmission of helminth eggs particularly that of
Enterobius vermicularis
Stools from macaques and gorillas are found to carry this
Does not invade tissues, but its presence in the intestine produces irritation of the mucosa with
secretion of excess mucus and hypermotility of the bowel
Usually asymptomatic
Onset of infection is usually accompanied by
o Loss of appetite o Flatulence
o Abdominal pain o Anal pruritus
o Intermittent diarrhea with excess o Chronic infection may cause an Irritable
mucus bowel syndrome (IBS)
o Abdominal tenderness
o Bloating sensation
Diagnosis
o Observation of binucleate trophozoites in multiple fixed and stained fresh stool samples
o May be misdiagnosed as other amebae
o PVA or Schauddin’s fixative has been found to be helpful
Treatment
o Antimicrobial therapy
o Iodoquinol
o Metronidazole
As high prevalence rates in developed countries