OVERVIEW OF DIAGNOSTIC PARASITOLOGY AND SPECIMEN COLLECTION
Diagnosis of parasitic infections through demonstration of parasite or parasite components (adults, egg, larvae,
cysts, trophozoite)
Specimens received in the Lab:
 Stool – most common
 Aspirates (Duodenal, Liver)
 Blood, Buffy Coat and Lymphatic Fluid
 Urine
• Eye Scrapings, Skin Scrapings
• Biopsy Specimens
• Other body fluids (i.e. CSF, Vaginal Fluid, Amniotic Fluid)
1. Stool
• Most common method
• Demonstration of eggs, larvae, adults, trophozoites, cysts, or oocysts in the stool
• Best collected in:
• clean, wide-mouthed containers made of waxed cardboard or
• plastic with a tight-fitting lid to ensure retention of moisture and to prevent accidental spillage
• Properly Labeled; submitted together with a lab request
• Important Factors to be considered
• Intake of drugs/ medicinal substances
1. Antacids
4. Bismuth
2. Anti-diarrheals
5. Laxatives
3. Barium
•Stool Examination should be deferred
•Amount of Stool Submitted
A routine stool examination usually requires:
• a thumb-sized specimen of formed stool
• about 5 to 6 tablespoons of watery stool.
•Contamination with toilet water, urine, or soil must be prevented since these can destroy protozoan trophozoites. In
addition, soil and water may contain free-living organisms that would complicate diagnosis of infections
•Stool Processing and Handling
• Watery/Diarrheic Stool: examine within 30 minutes from time of passage
• Formed stool: up to 24 hours
• Temporary storage of fecal samples in a refrigerator (3-5°C) is acceptable
• Trophozoites are killed by refrigeration, although helminth eggs and protozoan cysts are usually not damaged.
• NEVER FREEZE STOOL SAMPLES. NEVER KEEP THEM IN INCUBATORS
• Fixatives
• Ratio: 3 parts preservative to 1-part stool specimen
• Fixation Time: 30 minutes
1.Formalin
4.SAF
2.Merthiolate Iodine Formalin
5.Modified PVA
3.PVA (combined with Schaudinn’s)
6. Alternative Single Vial System
Ova and Parasite Examination
1. Macroscopic Examination
2. Microscopic Examination
Techniques
• Direct Fecal Smear
• Kato-Thick Smear
• Concentration Techniques
• Kato-Katz Techniq
• Stoll Dilution Technique
• Preparation of Permanent Stained Smears – confirmation of intestinal protozoan
o Use of Iron Hematoxylin or Trichrome
•Macroscopic Examination
• Physical Characteristics
• Color
• Consistency or Form of Stool
• Gross Abnormalities
Microscopic Examination
3 Distinct Procedures:
• Direct Wet Preparations
• DFS
• Concentrated Wet Preparations
• Sedimentation – ex. FECT, AECT
• Flotation – ex. Zinc Sulfate Flotation (1.18-1.20); Sheather’s Sugar
Flotation
• Permanently Stained Smear
• Trichrome, Iron Hematoxylin, Specialized Stains
• Presence of Cysts, Eggs, Adult forms, larval forms, trophozoite stages
• Fecal Elements that might be mistaken as Parasites
> Leukocytes
> Vegetable Hair
> Muscle Fibers
> Fat Droplets
> Vegetable Cells
> Fungal, Yeast Cells
> Vegetable Spiral
• Charcot Leyden Crystals: eosinophil breakdown product
> Significant
Stool Specimens
• Other Procedures
• Cultures - Harada Mori Technique, Coproculture & Use of Culture Media
• Egg Counting Procedure - Kato-Katz (Cellophane Covered Thick Smear) & Stool Egg Count
Other Specimens from the intestinal tract and urogenital system
• Collection of Perianal Swab - Scotch Tape Swab or Cellulose Tape Swab
• Sigmoidoscopy Material
• Duodenal Contents- Duodenal Drainage & Duodenal Capsule Technique (Entero-Test)
2. Blood
• Thick and Thin Smears - for malaria
• Knott’s Concentration technique – for microfilaria
• Buffy Coat Smear: for hemoflagellates
3. Sputum
Parasites that may be recovered on sputum:
• Migrating larvae: (ASH) Ascaris lumbricoides, Strongyloides stercoralis, and hookworms
• Paragonimus ova
• Echinococcus granulosus hooklets from pulmonary hydatid cysts
• Protozoa such as:
• Entamoeba histolytica trophozoites from pulmonary amebic abscess
• Cryptosporidium parvum oocysts, although very rare
• Non-pathogenic Entamoeba gingivalis and Trichomonas tenax
• First morning specimen best
• Patient cannot expectorate > use inductants (10% sodium chloride or hydrogen peroxide)
4. Urine
• First morning specimen best since there could have been concentration of parasites overnight
• Best for Trichomonas vaginalis
• May also detect Wuchereria bancrofti and Schistosoma haematobium
5. Tissue Aspirates
• Sample aspirated from the ff organs:
• Liver
• Duodenum
• Skin
• Bronchial
• Lymph node
• Liver aspirate: most common in the Philippines
• To rule out hepatic amoebic abscess
• For diagnosis of Echinococcus granulosus in endemic areas
• Duodenal aspirate: uses String test
• Cutaneous or Skin aspirates : For Cutaneous Leishmaniasis (Oriental sore)
• Cerebrospinal Fluid:
•Trypomastigotes of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Trypanosoma brucei
gambiense
• trophozoites of Naegleria
• Parastrongyliasis
• Specimen examined within 20 minutes
• Tissue biopsy : For Trichinella spiralis
• Rectal biopsy : Presence of deposited eggs of Schistosoma japonicum
6. Animal Inoculation/Xenodiagnosis
• Animal Inoculation
• Xenodiagnosis
• Uses arthropod vectors or other hosts as an indicator of infection
• Used in diagnosis of Chagas’ disease and Trichinosis
PROTOZOANS
Amebae
MEDICALLY IMPORTANT PARASITES
• Protozoans
• Helminthes
• Nematodes
• Trematodes
• Cestodes
PROTOZOANS (KINGDOM PROTISTA)
a. Phylum Sarcomastigophora
• Subphylum Sarcodina – Ameba
• Subphylum Mastigophora – Flagellates
b. Phylum Ciliophora – Ciliates
c. Phylum Apicomplexa
• Class Sporozoa
• Suborder Haemosporina
d. Suborder Eimeria
PROTOZOANS (SARCODINA)
Taxonomy of Protozoans
Kingdom Protista
• Phylum Sarcomastigophora
o Subphylum Sarcodina
o Subphylum Mastigophora
• Phylum Ciliophora
• Phylum Apicomplexa
• Phylum Microspora (now fungi)
PROTOZOANS
• Unicellular Organisms
• Vary in shape, size, locomotion
• Reproduce Sexually or Asexually
• Do not possess a cell wall
• Consists of Nucleus and Cytoplasm
• Nucleus: Genetic Material Contains nucleolus or karyosome or endosome
• Cytoplasm 2 Regions: Endoplasm & Ectoplasm
• Trophozoite
a.
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STAGES OF DEVELOPMENT
• Cyst
Subphylum SARCODINA
Possess Pseudopodia for locomotion
Inhabit the large intestine except E. gingivalis
All undergo encystation except E. gingivalis
All undergo Binary Fission as mode of
reproduction
All are commensals except E. histolytica
Sarcodina
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•
Pathogenic: Entamoeba histolytica
Commensals:
o E. dispar
o E. moshkovskii
o E. hartmanni
o E. coli
o Endolimax nana
o Iodamoeba butschlii
1. Entamoeba histolytica
• Entamoeba histolytica – only pathogenic
member
• MOT: Ingestion of Infective Cyst
• Habitat: Large Intestine
• Final Host: Man
Entamoeba coli vs E. histolytica
Categories
Movement
Pseudopodia
Nucleus of Trophozoites
and location of karyosome
E. histolytica
Trophozoite
Unidirectional
One at a time on an explosive
manner
Finger- like Blunt;
E. coli
Sluggish;
Not progressive;
Several at a time but slow
Rounded
Mononucleated Central
karyosome
CYST
Red Blood Cells
(hematophagous)
Mono Eccentric karyosome
Cytoplasm
No. of Nuclei (Cyst)
Clean looking
1-4 or 4
Dirty looking
1-8 or more than 4
Chromidial bars/ bodies
Sausage
shaped; rod-cigar shaped; coffin
with rounded ends
Fine and evenly distributed
Broom stick needles;
splintered glasses;
witch broom
Irregular, clamp, coarse
Inclusions
Peripheral chromatin
PATHOGENESIS
Bacteria, yeast, debris
E. histolytica
Mechanisms for virulence:
1. Asymptomatic – majority of
• production of enzymes
cases
or other cytotoxic
• Excrete cysts
2. Intestinal Disease
substances
• contact-dependent cell
• Incubation Period 1-4 weeks
killing
• Release of enzymes to lyse
• cytophagocytosis
mucosal lining
DISEASE MANIFESTATIONS
• Ameboid Movement
a. Asymptomatic Carrier
• Formation of FLASK SHAPED ULCERS
Clinical Forms of Intestinal Amebiasis
State – majority of
cases
• Dysentery – majority of cases
b. Intestinal Disease
• Amebic colitis (abdominal pain + diarrhea +/- blood &
(amebic colitis,
mucus in the stools )
ameboma)
• Fulminating Colitis (seen in children)
c. Extra-intestinal
• Amebic Appendicitis
Disease – Hepatic
• Ameboma
3. Extra-intestinal Disease
• Ectopic form of amebiasis
• Usually occurs in the Liver >>> Amebic Liver
Abscess
• Cardinal Signs: Fever, Right Upper Quadrant Pain
• Other signs include: tender liver and hepatomegaly
Drainage of a liver abscess
Chronic amebiasis: drainage of a lung abscess
Chronic amebiasis: brain abscess
PATHOLOGY
•
•
Ability to lyse tissues
Attributed to its Virulence Factors:
o Lectin (Gal/GalNAc Lectin)
o Amebapores
o Cysteine Proteinases
LABORATORY
DIAGNOSIS
•
Ova and Parasite Examination
o DFS
o Concentration Techniques
o Permanent Stained Smear (Iron Hematoxylin or Trichrome Stain)
 Charcot Leyden crystals
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Culture
o Boeck’s, Rice Egg Saline, Diamond, Balamuth’s Egg Yolk Infusion
Serology (ELISA)
Molecular Methods (PCR)
Rectal Biopsy
Examination of Liver Aspirates
Ultrasound, CT scan, MRI
EPIDEMIOLOGY
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Worldwide Distribution
More Prevalent in Tropics
High Risk Groups
Issues on the occurrence of a “non-pathogenic” E. histolytica
o Recent identification of a “E. histolytica look-a
Treatment
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Metronidazole: Tx of acute amebic colitis
Diloxanide Furoate: Asymptomatic Cyst Carriers
Iodoquinol
PREVENTION
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Proper Disposal of Waste
Proper Sanitation/personal hygiene
Access to safe water and food
Development of an effective Vaccine
E. histolytica
a. Pathology: Invasiveness and abscess formation are due to amoebic proteolytic
enzymes
b. Immunology: Antibodies are detectable in chronic infections but they are of
questionable protective value
Diagnostic
features
E. histolytica and some non-pathogenic amoebae
Amebiasis Differential diagnosis: Amebiasis is different from giardiasis and bacterial dysentery. (Mucus and blood
in stool, No granulocytosis, No high fever)
2. E. dispar and E. moshkovskii are morphologically similar to E. histolytica but they are non-pathogenic
Other Commensal Amebae: Generally, do not cause disease and their life Cycle is similar to Entamoeba histolytica
3. Entamoeba coli
4. Entamoeba hartmanni is a small Race of E. histolytica (Commensal)
5. Entamoeba polecki is an Ameba of pigs and monkeys (Entamoeba species)
Iodamoeba butschlii is an Ameba of swine (pigs)
Prominent Feature:
• Uninucleated with a large eccentric karyosome; with achromatic granules “Basket of Flowers”
•
Large glycogen vacuole
6.
7. Endolimax nana is the smallest intestinal amebae (as small as a RBC) (Commensal)
8. Entamoeba gingivalis
• Ameba of oral cavity (Gum Line)
• No cystic stage
• MOT: Person to Person
• Infective and Diagnostic Stage: Trophozoite
• Scavengers and eat debris; can ingest WBCs, debris, RBC(rare)
• Non-pathogenic; but seen in patients with pyorrhea alveolaris
FREE LIVING PATHOGENIC AMEBAE
• Found inhabiting lakes, pools, tap water, air conditioning units and heating units
• In Humans: Found in the CNS
o Acanthamoeba
o Balamuthia
o Naegleria
1. Naegleria fowleri
• Free-living Ameboflagellate
• MOT: Entry into the body: Olfactory Epithelium,
Respiratory Tract, Skin and Sinuses
Important stages
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•
Cyst
Trophozoite – infective stage
Ameba – feeding form
Flagellate – swimming form
Entry into the body
Disease
Manifestation and
Pathology
olfactory epithelium, respiratory tract, skin and sinuses
Primary Amebic Meningoencephalitis
• VERY FATAL
• Risk Factor: Swimming in contaminated pools,
lakes and rivers
Pathogenic
determinant
PRESENCE OF Amebostomes
Diagnosis
• Wet Mount Examination of CSF
• Smears stained with Wright’s or Giemsa
• Biopsy
• Culture
• Molecular Methods
Treatment and
Prevention
Amphotericin B with Clotrimazole
2. Acanthamoeba spp.
• Free-living Ameba
• Characteristic Feature: Presence of acanthapodia
• Morphologic Forms:
Cyst
Disease
Manifestation and
Pathogenesis
Disease
Manifestation
Acanthamoeba
• Causes Granulomatous Amebic Encephalitis
Chronic; slow in progression
Poor Prognosis
Amebic Keratitis
• Keratitis, uveitis, Corneal Ulcerations
• Implicated among contact lens users
Trophozoite
Diagnosis
• Usually diagnosed after death (GAE)
• Biopsy
• Corneal Scrapings
• Culture
• Molecular Methods
Treatment
• Very Fatal once cerebral manifestations appear
• Fluorocystine, ketoconazole, amphotericin B
3. Balamuthia
• Granulomatous Amebic Encephalitis (GAE)
• Found in soil and water
• 1st discovered in 1986 in the brain of a mandrill that died in the San Diego Wild Animal Park