Parasitology Part 3 Flashcards
1
Q
Parasitic Protozoa
A
- Reproduction in amebae, flagellates and ciliates usually asexual, by multiple fission
- Most intestinal amebae, flagellates and the sole ciliate have both trophozoite and cyst stage
- Cyst stage is resistant stage to survive environment and is usually infective stage
2
Q
Protozoa: Intestinal pathogens
A
- Balantidium coli
- – Blastocystis hominis (maybe)
- Cryptosporidium*
- Cyclospora
- Cystoisospora belli
- Dientamoeba fragilis
- Entamoeba histolytica
- Giardia intestinalis*
- Microsporidium
3
Q
Protozoa: Intestinal nonpathogens
A
- Chilomastix mesnili
- Entamoeba dispar
- Entamoeba hartmanni
- Entamoeba coli
- Entamoeba polecki (possibility pathogenic)
- Endolimax nana
- Iodamoeba buetschlii
- Pentatrichomonas hominis
- Retortomonas hominis
4
Q
Protozoa: pathogens other sites
A
- Acanthamoeba
- Babesia
- Balamuthia
- Leishmania
- Naegleria
- Plasmodium
- Trichomonas vaginalis
- Toxoplasma gondii
- Trypanosoma
5
Q
When to collect?
A
- 2-3 specimens collected every other day or
- 3 specimens within 10 days
6
Q
Preservatives
A
- 10 % buffered neutral formalin
- Zinc PVA – zinc preservative with PVA (polyvinyl alcohol)
- Copper PVA – copper preservative with PVA
- SAF – Sodium acetate-Acetic acid-Formalin
- MIF – Merthiolate-Iodine-Formalin
- Various commercial kits containing proprietary formulations
7
Q
Match stain and preservative
A
- Mercuric-chloride based PVA - trichrome or iron hematoxylin stain (gold standard)
- Zinc-based PVA – trichrome stain
- SAF - iron hematoxylin stain
- UNIFIX or Z-PVA - trichrome stain
- ECOFIX and ECOSTAIN
8
Q
What is the purpose of the permanent stain smear?
A
- To provide contrasting colors for both background debris and parasites present
- Allows for detail examination at x1000
- Designed to allow recovery and identification of the intestinal protozoa
- How much of the permanent stained smear should be examined?
- At least 300 oil immersion fields (x1000)
9
Q
Permanent stained smears
A
- Preferred:
- • Fresh stool in Schaudinn’s fixative
- • PVA-preserved stool
- Not preferred but will work:
- • SAF-preserved stool
- • MIF-preserved stool
10
Q
*What is the purpose of the iodine dish in the
trichrome stain protocol?*
A
- • Smears from some proficiency testing agencies may still prepare fecal specimens that have been preserved in mercury-based fixatives
- • To remove mercury
- • It is a chemical substitution of iodine for mercury
- • The iodine is removed during the next two alcohol rinses
- • Not needed for zinc sulfate-based PVA because zinc sulfate-based PVA is water soluble
11
Q
*What role does acetic acid play in
the trichrome stain?*
A
- Both trichrome and iron hematoxylin stains are considered regressive stains
- • The fecal smears are overstained and then destained
- • Acetic acid in the 90% alcohol rinse step in the trichrome stain removes some of the stain and provides better contrast
12
Q
What causes the xylene (or xylene substitute) dehydration solution to turn cloudy?
A
- The xylene (or xylene substitute) dehydration solution turns cloudy when a slide from the previous alcohol dish is moved forward into the xylene dish
- • If there is too much water carryover from the last alcohol dish the xylene solution may turn cloudy
- • Replace the 100% alcohol dishes and back up the slide into 70% alcohol
- • Allow to stand for 15 minutes then move slide to the 100% alcohol step
13
Q
What to look for in a protozoan trophozoite?
A
- generally larger
- Look for the nucleus if no nucleus disregard object
- some organisms have multiple nuclei
- 2 for giardia
- • Trophozoite
- Size
- Nucleus
- Cytoplasm appearance
- Inclusions
- Number of flagella
14
Q
What to look for in a protozoan cyst?
A
- Just one nuclei than a trophozoite
- Cysts
- Size
- Shape
- Number of nuclei
- Nuclear peripheral chromatin
- Karyosome
- Chromatoidal bodies only in cysts
- Glycogen
15
Q
What to look for in others?
A
-
Ciliate- Balantilium coli
- Size
- Cilia
- Shape of nucleus
- Macro kidney-shaped nucleus and a micro circular one
- Coccidia- Smaller in size 4 to 10 um
- Oocyst
- Modified acid-fast stain
- Sporocyst
- Size
- Microsporidia* not discussing
- Size
- Stripe
- Vacuole
16
Q
What causes air bubbles
A
- Large clumps of stool
- Any water left in the material will cause air bubbles to form
- Return to xylene and repeat alcohol dehydration
- Allow to stand for 15 mins
17
Q
Organisms that have 2 nuclei in their trophozoite form
A
- Dientameboa fragilis- clover leafed karyosome
- Giardi lamblia
18
Q
E. histolytica v. E. coli
A
- Centric karyosome for histolytica
- Acentric karyosome for coli
19
Q
Flagellates
A
- Giardia lamblia* (intestinalis, duodenalis)
- Chilomastix mesnili
- Trichomonas vaginalis*
- Pentatrichomonas hominis
- Enteromonas hominis
- Retortamonas intestinalis
- Dientamoeba fragilis*
- *Pathogenic in humans
20
Q
Giardia lamblia (intestinalis)
A
- Giardia lamblia Trophozoites
- 10-20 µm in length and 5-1 µm in width (“falling leaf” motility observed in fresh sp.)
- 4 pairs of flagella
- 2 nuclei visible
- 2 axonemes
- Stained with trichrome
- Giardia lamblia Cysts
- Trichrome staining
- 11-14 µm and 7-10 µm in width
- Cytoplasm retracting from the cell wall
- Nuclei
- Axonemes
- Giardiasis Symptoms
- Symptoms usually begin 1 to 3 weeks after a person has been infected (incubation
- Signs and symptoms may vary and can last for 1 to 2 weeks or longer
- Self-limiting within 2 weeks in immunocompetent
- Acute symptoms include:
- Diarrhea (foul-smelling, no blood)
- Greasy, light-colored stools that tend to float (steatorrhea)
- Gas/flatulence
- Stomach or abdominal cramps
- Nausea/ vomiting
- Dehydration (loss of fluids)
- Chills, low-grade fever
- Giardiasis Diagnosis
- Identify cysts and/ or trophozoites in stool
- Cyclic shedding, ideally 3 stool specimens collected (on separate days) increase test sensitivity
- Fecal immunoassays
- Molecular testing (e.g., polymerase chain reaction) can be used to identify the subtypes of Giardia
- Antigen detection-EIA, ELISA,DFA,Rapid, cartridge (lateral flow)
- Giardia lamblia Epidemiology
- Worldwide distribution (Traveler’s diarrhea), backpacking, camping
- Infects both humans and animals
- More prevalent in children
- Infects 7% of adults in developed countries worldwide
- 5-50% people in developing countries have had giardiasis
- Giardia lamblia Transmission
- Fecal oral route
- Ingestion of Giardia cysts found in contaminated food or water (10-100 cysts are sufficient for infection)
- Acquired from unwashed hands
- May be passed person to person or even animal to person
- Oral-anal contact during sex
- Giardiasis Prevention
- Hand washing
- Filtering water, iodine treatment, boiling
- Avoid water (drinking and recreational) that may be contaminated
- Avoid eating food that may be contaminated
- Clean up after ill pets and people (hard surfaces, dishes, toys, clothing)
21
Q
Chilomastix mesnili
A
- Worldwide distribution
- Nonpathogenic flagellate
- Both cyst and trophozoite stage
- Resides in the cecum/ colon of the infected human where the organism feed on bacteria and debris
- Ingestion of cyst - fecal-oral route
- contaminated water
- food
- Chilomastix mesnili trophozoite
- Single nucleus with eccentric karyosome
- End tapers to point
- Length: 6-24 µm; stained with trichrome
- Cytostome
- Chilomastix mesnili cyst
- Length 6-10 µm; Trichrome 1000x
- Curved fibril “shepherd’s crook”
- Diagnosis and Prevention
- Demonstration of trophozoites and cysts in feces stage
- No treatment is necessary
- Improve personal hygiene
22
Q
Trichomonas vaginalis
A
- Worldwide, cause trichomoniasis
- Cosmopolitan distribution within all racial groups
- Throughout all socioeconomic levels
- Infects urogenital tract in both males and females
- Most common pathogenic protozoan of humans in industrialized countries
- Higher prevalence with multiple sexual partners
- Trichomonas vaginalis Trophozoite Features
- Ovoid, round or pear-shaped.
- Three to five flagella originating at the anterior end and one at the posterior end.
- No trailing flagellum at the posterior end.
- Jerky and rapid movement in urine and vaginal secretions.
- Undulating membrane extending half of body length.
- Prominent axostyle which curves around nucleus and granules may be seen along the axostyle
- One ovoid nucleus with granular karyosome
- Trichomoniasis Symptoms
- Often asymptomatic
- Women: vaginitis, cervicitis, urethritis, endometritis, infertility
- Onset with diffuse, malodorous, yellow-green vaginal discharge with intense vaginal and vulvar pruritus
- Infection has been associated with premature rupture of membrane, premature birth, low birth weight baby
- Men: urethritis, epididymitis, and prostatitis, mostly asymptomatic
- Asymptomatic carriers serve as a reservoir for transmission and remain at risk for developing the disease
- Trichomonas vaginalis
- Diagnosis Microscopy
- Culture
- Antigen detection
- DFA
- Latex agglutination test
-
PCR
- Detection of organisms from vaginal swabs
- More sensitive than culture for males
- Point of care diagnostics test
- T. vaginalis Epidemiology
- Primarily a STD
- Frequently coexistent with another infection candidiasis, gonorrhea, syphilis or HIV infection
- Non venereal transmission possible,
- 180 million cases worldwid
- US infections
- Estimate of new cases 7.4 million annually
23
Q
Pentatrichomonas hominis-Trichomonas hominis
A
- Worldwide
- Both warm and temperate climates
- Nonpathogenic
- Identified in individuals with diarrhea
- Trophozoites live in large intestine
- No known cyst stage
- Feed on bacteria, not invasive
- More often in children
- Pentatrichomonas hominis
- Nonpathogenic
- GI tract/ cecum
- Trichomonas vaginalis
- Pathogenic
- Urinary Genital system
- Trophozoite Features
- Pear shaped
- Trophozoites (5-15 µm in length) in stool specimens, stained with trichrome
- Three to five anterior flagella and one extending from the posterior end going beyond undulating membrane
- Jerky movement in stool specimens
- Undulating membrane extending full body length
- Axostyle extends beyond posterior end of body
- One nucleus with a small central karyosome without peripheral chromatin
- Conical cytosome cleft at anterior end opposite to undulating membrane
-
Diagnosis
- Freshly passed stool specimens motility may be visible
- In wet preparation look
- for the flagellar movement from the undulating membrane
- the presence of the axostyle
- Permanent stained smear recommended
- Prevention
- No treatment necessary
- Improved personal hygiene and sanitary conditions
- Length of undulating membrane
- Entire length
- versus Trichomonis vaginalis 1/2 the length
25
Q
Enteromonas hominis
A
- Found in warm or temperate climates
- Both trophozoite and cyst forms
- Infection acquired through the ingestion of cysts, in fecal contaminated food or water
- Resides in the large intestine, is regarded as commensal
- Cysts may resemble those of E. nana cysts with 1-4 nuclei having central karyosome without peripheral chromatin
- 4 flagella; 3 anteriorly and one posterior
- Cytosome not visible
26
Q
Retortamonas intestinalis
A
- Found in warm or temperate climates
- Infection acquired through the ingestion of cysts, in fecal contaminated food or water
- Resides in the large intestine, is regarded as a commensal
- Both trophozoite and cyst form
- 2 anterior flagella in trophozoite
- One nucleus with small central karyosome and a fine ring of chromatin granules
- Cytosome extending up to half body length starting in the anterior end
- Cysts (4-7 µm long) in a stool specimen, stained with trichrome
- Trophozoites (4-10 µm long) in a stool specimen, stained with trichrome
- In the large intestine, excystation releases trophozoites
27
Q
Dientamoeba fragilis
A
- In the gastrointestinal tract (cecum/ colon) of humans
- Closely related to Trichomonas spp.
- Trophozoites only, lack external flagella, move by sluggish pseudopodia or by Brownian movement
- Clover leaf-shaped clumps (disintegrated appearance) of chromatin in the nucleus
- Harder to stain, blend with the background
- Cysts have not been identified
- May cause dientamoebiasis in institutionalized or MSM
- Known to be transmitted on helminth eggs
- D. fragilis trophozoites (5-15µm long) stained with trichrome
- The trophozoite is the only stage found in the stools of infected individuals
- Symptoms/Disease
- Mostly asymptomatic in 75% of adults
- In 90% of children mostly symptomatic:
- intermittent diarrhea
- abdominal pain
- nausea
- anorexia
- fatigue
- malaise
- poor weight gain
- Dientamoeba fragilis Diagnosis
- Stained smear is the recommended procedure because no cyst stage exists
- Culture
- Antigen, and antibody techniques, are not commercially available
- PCR from fresh unpreserved stool
-
Epidemiology
- Worldwide
- Probably transmitted by fecal-oral route and possible transmission via helminth eggs
Ascaris, Enterobius - Tends to be common in some pediatric populations
- Institutionalized individuals, homosexuals
29
Q
Balantidium coli
A
- Only pathogenic ciliate for humans
- Is the largest of all the protozoa
- Both trophozoite and cyst forms
- Pigs are the primary reservoir for B. coli
- Other reservoirs chimpanzees and monkeys (tropics)
- 50-100 X 40-70 um
- Balantidiasis Symptoms
- Mostly asymptomatic
- Symptomatic infection:
- Persistent diarrhea (weeks to months), occasionally dysentery (similar to amebiasis), abdominal pain, tremendous fluid loss, and weight loss
- Noninvasive
- Severe in debilitated persons
- Balantidiasis Diagnosis
- Detection of trophozoites in
- stool specimens
- tissue collected during endoscopy
- Cysts less frequently encountered
- Passed intermittently and once outside colon rapidly destroyed
- Stools collected repeatedly, and immediately examined or preserved
- Detection of trophozoites in
- Balantidium coli Epidemiology
- Worldwide
- Rare in US
- Human infection balantidiasis
- in warm and temperate climates
- sporadically in cooler areas
- in institutionalized groups
- pig farmers or people in slaughter
houses
35
Q
Entamoeba histolytica
A
- General
- Amebic dysentery = bloody diarrhea
- Liver abscess
- Amoeboma
- histo = tissue
- lytic = destroy
- Transmission
- Fecal-oral
- contaminated food/water
- sexual contact
- risk groups
- travelers
- recent immigrants
- institutionalized populations
- specific sex practices
- Pathways
- noninvasive infection
- asymptomatic
- luminol amebiasis
- intestinal disease
- diarrhea, dysentery
- invasive extraintestinal disease
- right upper quadrant pain
- fever
- Invasive extraintestinal disease
- organisms carried by the bloodstream
- abscesses spread to other organs
- liver most common sites
- right upper quadrant pain
- fever
- noninvasive infection
- Diagnosis
- Microscopy
- Peripheral chromatin uniformly distributed
- Granular or “ground glass” cytoplasm
- Trophozoite
- Size: 15-20 μm (range 12-60 μm)
- Centrally placed karyosome
- Single nucleus
- Cyst
- Size= 12-15um (range 10-20 um)
- 4 karyosome
- Direct exam: stool sigmoidoscopy, biopsy specimens & aspirates
- Serologic testing: EIA, IHA, antigen detection (not on formalin-fixed stool)
- Culture: From fecal specimens - Robinson’s medium, Jones’ medium
- PCR
- Microscopy
36
Q
Entamoeba gingivalis
A
- trophozoites only
- oral transmission
- 10-35um in length
37
Q
Entamoeba hartmanni trophozoite
A
- Trophozoite
- Single nucleus with centrally located karyosome, the bull’s eye
- clean cytoplasm
- size = 8 to 10 um (5-12 um range)
- cysts
- Cytoplasm often clean
- Chromatoidal bodies with round ends
- Size: 6 to 8 μm (range 5-10 μm)
38
Q
Entamoeba coli
A
- Trophozoite
- single nucleus with large eccentric karyosome
- Size: 20-25 μm (range 15-50 μm)
- Peripheral chromatin clumped or uneven
- chromatoidal bodies with splintered ends
- Cytoplasm coarse and vacuolated “dirty” ingests debris, vacuoles
- Cysts
- Chromatoidal bodies with splintered ends
- Cytoplasm may be clean
- 8 nuclei vary in morphology
- Length: 15 to 25 μm (range 10 to 35 μm)
- cysts that don’t stain well
- five nuclei or more confirmation of coli
39
Q
Entamoeba polecki
A
- Trophozoite
- nucleus with small discrete karyosomal chromatin
- even or uneven peripheral chromatin
- size = 15 to 20 um ( 10 to 15 um range)
- cysts
- One or rarely two nuclei with eccentric karyosome
- Length: 11 to 15 μm (range 9 to 18 μm)
- Inclusion mass
- Red chromatoid bodies
40
Q
iodamoeba butschlii trophozoites
A
- trophozoite
- one nucleus with large central karyosome
- refractile achromatic
- Size:12 to 15 μm (range 8 to 20 μm)
- Dirty cytoplasm
- cysts
- uninucleate
- Length: 10 to 12 μm (range 5 to 20 μm)
- Single large nucleus with eccentric karyosome
- Glycogen mass
41
Q
Endolimax nana
A
- Trophozoite
- Single nucleus with large irregular blot-like centric or eccentric karyosome
- Size: 8 to 10 μm (range 6 to 12 μm)
- No peripheral chromatin
- Clean cytoplasm
- cysts
- 4 nuclei with large blot-like karyosome
- Length: 6-8 μm (range 5 to 10 μm)
43
Q
Naegleria fowleri
A
- primary amoebic meningoencephalitis (PAM)
- infection introduced far up the nose
- 1-9 days after swimming
- dead after 5 days of infection
45
Q
Acanthamoeba spp.
A
- keratitis (contaminated contact lens solution)
46
Q
Balamuthia mandrillaris
A
50
Q
Cryptosporidium parvum
A
- Fecal Contamination of water
- Outbreaks of cryptosporidiosis have been reported in several countries
- C. parvum found in both animals and humans
- C. hominis only in humans (common in US)
- A waterborne outbreak in Milwaukee (Wisconsin) in 1993, that affected more than 400,000 people
- Symptoms
- Age and immune status
- Immunocompetent and immunodeficient
- Immunocompetent
- short term
- self-limited diarrhea lasting approximately 2 weeks
- Immunocompromised: CD4 T cell counts <200/µl
- results in a prolonged, life threatening, cholera-like illness
- Procedure
- Examine stool using the routine stool formalin ethyl acetate concentration
- Use one of the modified acid fast stains or
- The newer immunoassay kit reagents
- Oocyst numbers variable
- Test multiple stool samples before reporting
- Increased centrifugation speed or time (500 × g , 10 minutes)
- Wet mount
- Size measure 4-6 μm
- EIA
- Direct FA
- Resistance to Disinfectants
- Transmission
- Numerous potential hosts for zoonotic transmission especially waste water runoff particularly from lambs and calves
- Direct contact person to person contact (fecal material)
- Indirect from laboratory or from contaminated surfaces or raw food or water
- Low infectious dose; as few as 10 oocysts
- Oocysts are resistant to chlorine and ammonia based disinfectants
- More common in summer due to increased recreational water activities
- Prevention
- Handwashing
- Safe sex
- Avoid touching farm animals
- Avoid pet stools
- Wash and cooking food
- Avoid drinking water from lakes and streams
- Bottle water and other drinks may not be safe
51
Q
Cyclospora cayatenensis
A
- More common in tropics and sub tropics; endemic in Nepal, Peru, Haiti; outbreaks reported from US, South America, Europe, India
- Modified acid-fast
- Cyclospora Oocyst
- Bile and trypsin in small intestine trigger the release of sporozoites which invade epithelial cells
- Type 1 meronts (merozoites) infect other intestinal cells and type 2 progress to sexual stage
- Once fertilized, zygote develops into an immature oocyst that’s passed in feces
- Takes 1-2 weeks to mature to become infective
- Appears similar to C. parvum cyst but is larger, 8 -10 um
- After sporulation (maturation), produces 2 sporocysts with 2 sporozoites each
- Wet mount and conventional parasitology stains
- Do not stain with trichrome and iron hematoxylin stains
- Two special stains allow a more reliable diagnosis
- Modified acid fast stain
- Modified safranin stain
52
Q
Cystoisospora belli (Isospora belli)
A
- General
- Infects the epithelial cells of the small intestine
- Least common of the three intestinal coccidia
- Known as C. belli causing the disease isosporiasis
- Worldwide, especially in tropical and subtropical areas
- Epidemiology
- Infects humans and animals
- Infect both adults and children, less frequent than Cryptosporidium
- C. belli has also been implicated in traveler s diarrhea
- Sexual transmission
- Outbreaks in institutionalized groups in the US
- More at risk with Hodgkin disease and lymphoblastic leukemia
- Symptoms
- Diarrhea, which lasts (months to years), weight loss, abdominal colic, fever with nonbloody diarrhea
- Bowel movements (usually 6-10 per day)
- In immunosuppressed patients, infants and children, the diarrhea is severe
- Recurrences common after discontinuing treatment (TMS)
- Eosinophilia may be present
-
Cystoisopora Oocyst
- Bile and trypsin in small intestine trigger the release of sporozoites which invade epithelial cells
- Oocyst is not immediately infectious when shed in stool
- Takes 24-48 hours to mature to become infective
- No thin-walled oocyst to cause autoinfection as in case of Cryptosporidium
- Immature oocyst has a sporoblast which matures to form 2 sporocysts which are walled bodies containing 4 elongated sporozoites each
- Mature oocyst is 20-30 by 10-19 um
-
Detection
- Modified acid fast stain is used for detection
- Do not stain with trichrome and iron hematoxylin stains
- Like Cyclospora cyst, it autofluoresce and appears:
- Bluish at 365 nm
- Bright green at 405 nm
- Prevention
- Ingestion of food and water contaminated with mature, sporulated oocysts
- Sexual transmission
- Oocysts are very resistant to environmental conditions and remain viable for months if kept cool and moist
- Improve personal hygiene and sanitary conditions
