SECTION 1: INTRODUCTION TO PARASITOLOGY Flashcards
Organisms may develop unique relationships due to their (?) with one another. These relationships are very important to their survival.
habitual and long associations
is the living together of unlike organisms
Symbiosis
may involve protection or other advantages to one or both partners
Symbiosis
Different forms of symbiosis may be distinguished on the basis of whether or not the association is (?) to one of the two partners.
detrimental
is a symbiotic relationship in which two species live together and one species benefits from the relationship without harming or benefiting the other.
Commensalism
is a symbiosis in which two organisms mutually benefit from each other.
Mutualism
is a symbiotic relationship where one organism, the parasite, live in or on another, depending on the latter for its survival and usually, at the expense of the host.
Parasitism
Majority of animal parasites are (?) which are harmful and which frequently cause mechanical injury to their hosts.
pathogens
A (?) harbors a particular pathogen without manifesting any signs and symptoms.
carrier
is the process of inoculating and infective agent
Exposure
connotes the establishment of the infective agent in the host
infection
is the period between infection and evidence of symptoms. It is sometimes referred to as the clinical incubation period.
incubation period
also known as the biologic incubation period
pre-patent period
is the period between infection or acquisition of the parasite and evidence of demonstration of infection
pre-patent period
results when an infected individual becomes his own direct source of infection.
Autoinfection
happens when the already infected individual is further infected with the same species leading to massive infection with the parasite
superinfection or hyper infection
There are various sources of parasitic infections. The most common sources are
contaminated soil and water
Lack of (?) and the use of (?) or human excreta as fertilizer allow the eggs to get in contact with the soil.
sanitary toilets
night soil
Another possible source of infection is (?), which contain the infective stage of the parasite.
water and food
Consumption of (?) can result in several intestinal and liver fluke infections.
undercooked or raw fresh water fish
can also transmit infection
Arthropods
are vectors of malaria and filaria parasites
Mosquitoes
Other animals, whether (?), may also harbor the parasite.
wild or domesticated
Other sources include (?).
another person, his beddings and clothing, the immediate environment he has contaminated, or even one’s self
An (?) may be transmitted from its natural reservoir to a susceptible host in different ways.
infectious agent
There are different classifications for modes of transmission:
- Direct
- Indirect
contact w/ an infected person or animal, directly from the source to the susceptible host without involving an intermediate object
Direct
a) Droplet spread
Direct
b) Sexual intercourse
Direct
c) Kissing
Direct
d) Holding hands
Direct
e) Transplacental / Vertical - mother to fetus
Direct
a) Ingestion of contaminated food & drink
Indirect
b) Contact w/ contaminated soil
Indirect
c) Bite of an infected arthropod (vector)
Indirect
d) Through fomites
Indirect
Since the most common source of parasitic infection is contaminated food and water, the most likely portal of entry is the (?).
mouth
Majority of infections among cestodes, trematodes, and intestinal protozoans are
foodborne
from eating food harboring the infective larval stages
Taenia solium, Taenia saginata, and Diphyllobothrium latum
from drinking water contaminated with cysts
Entamoeba histolytica and Giradia lamblia
through ingesting raw or improperly cooked freshwater fish containing the parasitic larvae
Clonorchis, Opistorchis and Haplorchis
is another route of transmission
Skin penetration
enter via exposure of skin to soil
Hookworms and Strongyloides
species enter skin via water.
Schistosoma
also serve as vectors and transmit parasites through their bites
Arthropods
Examples are agents of malaria, filariasis, leishmaniasis and trypanosomiasis.
Arthropods
can cross the placental barrier during pregnancy
Toxoplasma gondii trophozoites
In transmammary infection with (?), the parasites may be transmitted through the mother’s milk.
Ancylostoma and Strongyloides
Other ways of acquiring the infection include inhalation of airborne eggs of (?), and sexual intercourse as in the case of (?).
Enterobius
Trichomonas vaginalis
Although parasitic life cycles range from simple to complex, they all have three common components
mode of transmission
a morphologic form
one (or more) forms
invades humans, known as the infective stage
a morphologic form
can be detected via laboratory retrieval methods, known as the diagnostic stage
one (or more) forms
Some parasites require only a (?), whereas others also require one or more (?).
definitive host
intermediate hosts
A parasitic life cycle consists of two common phases
One phase involves the route a parasite follows when (?).
in or on the human body
This information provides an understanding of the symptomatology and pathology of the parasite.
in or on the human body
Insights about the best the method of diagnosis and selection of appropriate antiparasitic medication may also be determined.
in or on the human body
The other phase, the route a parasite follows (?), provides crucial information pertinent to epidemiology, prevention, and control.
independently of the human body
A (?) may affect the entire body or body areas associated any of its parts.
parasitic disease
The major with such processes include the following:
(1) the gastrointestinal (GI) and urogenital (UG) tracts; other (2) blood and tissue; (3) liver, lung, and major organs; and (4) miscellaneous locations, eye, skin, and extremities
A wide variety of representative (?), may occur when a parasite infects a human host.
symptoms
Some persons remain (?), whereas other parasites produce severe symptoms and may result in death.
asymptomatic
The most commonly observed symptoms include (?).
diarrhea, fever, chills, abdominal pain, and abdominal cramping
Other symptoms, such as (?), may develop.
elephantiasis, anemia, vitamin deficiency, bowel obstruction, edema, enlargement of major organs, skin lesions, and blindness
an enlargement of areas such as the breast, leg, and scrotum caused by a parasite’s presence
elephantiasis
may be taken against every parasite infective to humans.
Prevention and control measures
designed to break the transmission cycle are crucial for successful parasite eradication.
Preventive measures
Examples of such measures include the following: (?).
education programs, use of insecticides and other chemicals, protective clothing, protective netting, proper water treatment, good personal hygiene, proper sanitation practices, proper handling and preparation of food, and avoidance of unprotected sexual relations
The vast capital expenditures required to accomplish these measures are not available in many endemic countries in the world. The problem of eradicating parasites is an ongoing process and is a key goal of international health groups such as the
World Health Organization (WHO)
The following are the specimens needed to properly diagnose the presence of parasites inside the human body.
1.Stool
2.Urine
3.Sputum
4.Blood
5. Cerebrospinal fluid
6. Tissue aspirates
7. Orifice swab
8. Tissue Biopsy
- for intestinal protozoans, nematodes and helminthes
Stool
- for the recovery of Trichomonas vaginalis and Schistosoma haematobium
Urine
Urine Collection:
mid-stream catch
- Paragonimus westermani, larvae of nematodes
Sputum
Sputum
Must be digested using
4-5% sodium hydroxide
- for malarial parasites, filarial worms, Leishmania and Trypanosoma
Blood
- Acanthamoeba species
Cerebrospinal fluid
Cerebrospinal fluid Collection:
lumbar tap
Tissue aspirates:
a) Liver aspirate
b) Duodenal aspirate
c) Broncho-alveolar lavage
hydatid cyst and liver amoebic abscess
a) Liver aspirate
Giardiasis and Strongyloidiasis infection
b) Duodenal aspirate
Paragonimus westermani
c) Broncho-alveolar lavage
Duodenal aspirate Collection:
endoscopy
: duodenal contents collected for Giardia and Strongylodes
Duodenal drainage or “String test”
: Schistosomiasis, Amoebiasis, Balantidiasis and Shigellosis (Large intestines)
Sigmoidoscopy
Orifice swab
a) Vaginal swab
b) Perianal swab
Trichomonas vaginalis
a) Vaginal swab
Enterobius vermicularis and Taenia
b) Perianal swab
Tissue Biopsy
a) Muscle
b) Rectal
Trichinella spiralis
a) Muscle
granulomas secondary to Schistosomiasis
b) Rectal
There are parasites where the method of isolation and identification is through processing of (?).
blood
❖ Blood Films
- Fresh water smears
- Thin Dry smears
- Thick Dry smears
for diagnosis of Trypanosomes and microfilaria
- Fresh water smears
for the study of the morphology of the parasites and the blood cells
- Thin Dry smears
used for malaria survey among patients with chronic infections or who are undergoing anti-malaria therapy
- Thick Dry smears
❖ Stains
- Giemsa
- Rapid stains
- Permanent stains & Other stains
Rapid stains:
a) Wright’s stain
b) Leishmann stain
c) Field’s stain
d) Acridine orange
e) Jaswant Singh Battacharya (JSB) Stain for thick and thin films
Permanent stains & Other stains:
a) Iron Hematoxylin Stain
b) MIF Fixative Stain (Merthiolate iodine formaldehyde)
c) Chlorazol Black E
d) Modified Kohn’s
e) Wheatley Trichrome
f) Methenamine Silver
g) Fluorescent Staining
most preferred
Giemsa
Giemsa Composition
Stock solution 1 ml
Buffered water (pH 7.0 – 7.2) 49 ml
Giemsa Staining time:
30 minutes
Too dark :
acidic pH
Too red:
alkaline pH
– It is used to stain blood smears in the detection of blood parasites.
Wright’s stain
The stain distinguishes easily between blood cells and became widely used for performing differential white blood cell counts, which are routinely ordered when infections are expected.
Wright’s stain
The stain contains a fixative, methanol, and the stain in one solution.
Wright’s stain
Thin films of blood are fixed with methanol to preserve the red cell morphology so that the relationship between parasites to the red cells can be seen clearly.
Wright’s stain
Wright’s stain
✓ Fix with 1-2 drops of (?)
✓ Cover the film with (?): 5 minutes
✓ wash with (?), drain, dry and examine
methanol
10% Giemsa stain
distilled water
It is a mixture of Methylene blue, and Eosin dye, prepared in Alcohol medium and diluted with buffer or distilled water during staining procedure.
Leishmann stain
is a differential stain that is used to variably stain the various components of the cells and it can be used to study the adherence of pathogenic bacteria to the human cells
Leishmann stain
It differentially stains the human and bacterial cells and appeared as purple and pink colored bodies respectively.
Leishmann stain
is one of the best stains for routine blood stain to stain the Peripheral blood smear for the examinations of blood film under the microscope and is satisfactory for malaria and other blood parasites
Leishmann stain
Leishmann stain
✓ Add (?) of the stain: (?)
✓ Add (?) of buffered distilled water
✓ Mix thoroughly, let stand (?)
✓ Rinse, drain, dry and examine
7-8 drops; 1-2 minutes
12-15 drops
4-8 minutes
It is a histological method for staining of blood smears.
Field’s stain
It is used for staining thick blood films in order to discover malarial parasites.
Field’s stain
Field’s stain consists of -
two parts
Field’s stain A is
methylene blue and Azure 1 dissolved in phosphate buffer solution
Field’s stain B is
Eosin Y in buffer solution.
is used as a fluorescent staining agent to detect the presence of malaria parasite in blood cultures and other bodily fluids
Acridine orange
is a fluorochrome dye that can interchalate into nucleic acid.
Acridine orange
standard method
Jaswant Singh Battacharya (JSB) Stain for thick and thin films
laboratories under the National Malaria Eradication Programme in India
Jaswant Singh Battacharya (JSB) Stain for thick and thin films
- used for most of the original morphological descriptions of intestinal protozoa found in humans
Iron Hematoxylin Stain
- diagnosis of Trichomonas
MIF Fixative Stain (Merthiolate iodine formaldehyde)
- An acid dye, used as a fat and general tissue stain, and to stain protozoa in fecal smears or in tissues.
Chlorazol Black E
- modification of the chlorazol black E staining technique
Modified Kohn’s
- all-purpose (amoebae, flagellates)
Wheatley Trichrome
- cyst
Methenamine Silver
- Microsporidium
Fluorescent Staining
Infected erythrocytes are counted in relation to a predetermined number of WBCs
Thick Blood Film
Standard: average of 8000/µl
Thick Blood Film
All parasite species and forms (sexual and asexual) are counted
Thick Blood Film
Thick Blood Film Formula:
If parasites are ≥10, 200 leukocytes are counted:
of parasites/µl =# of parasites counted x 40
If parasites are <10, 500 WBCs should be counted:
of parasites/µl = # of parasites counted x 16
Earle and Perez method
of asexual parasites/ 5µl
thick film is used
Thick Blood Film
used only in research studies
Thick Blood Film
% of parasitemia: P. falciparum
Thin Blood Film
of infected red cells in 1000 RBCs
Thin Blood Film
smear is scanned carefully, one ‘row’ at a time
Thin Blood Film
total number of red cells and the number of parasitized red cells are tabulated separately
Thin Blood Film
If 1000 red cells are counted:
• divide the number of parasitized red cells by 10 to get the percentage
• less than 1000 red cells counted
• # of parasitized cells/# of RBCs X 100
less precise
“plus system”
variation in the thickness of the film
“plus system”
results in variation in parasite count
“plus system”
= 1–10 per 100 thick fields
+
= 11-100 per 100 thick fields
++
= 1–10 per thick field
+++
= >10 per thick field
++++
(?) of fecal sample is essential for the isolation and proper diagnosis of infection.
Proper collection
The following should be considered:
- Container
- Avoid contamination with urine, water and soil
- Label
- Handle carefully
Container (for physical examination of the specimen)
sterile, disposable, wide mouth with tight-fitting lid, transparent
Handle carefully because it is a potential source of infection unsuitable samples from patients receiving:
a. Barium
b. Oil
c. Bismuth
d. Kaolin
e. Antibiotics
TYPES of FECAL SPECIMEN
- Liquid
- Soft
- Formed
Liquid –
either diarrheic or saline-purged
PRESERVATION a) Physical:
- Room temperature
- Refrigeration
PRESERVATION b) Chemical
- Polyvinyl Alcohol (PVA)
- 5-10% formalin
- Merthiolate-Iodine-Formalin (MIF) or Thimerosal Iodine Formalin (TIF)
- Schaudinn’s Fixative
used for the preservation of stained fecal+ smears; preservation of trophozoites
Polyvinyl Alcohol (PVA)
▪ For concentration techniques or direct fecal smear
5-10% formalin
▪ For cysts, helminthes and larvae
5-10% formalin
▪ Not sufficient for preparing permanent stained fecal smears
5-10% formalin
▪ Good for all stages
Merthiolate-Iodine-Formalin (MIF) or Thimerosal Iodine Formalin (TIF)
▪ For liquid stools
Merthiolate-Iodine-Formalin (MIF) or Thimerosal Iodine Formalin (TIF)
▪ For bulk feces, the solution is mixed in the proportion of 9.4 ml MIF and 0.6 ml Lugol’s for a gram of fecal material
Merthiolate-Iodine-Formalin (MIF) or Thimerosal Iodine Formalin (TIF)
▪ For fresh materials and those recovered from intestinal mucosal linings
Schaudinn’s Fixative
▪ For permanent staining
Schaudinn’s Fixative
IMPORTANCE OF FECALYSIS
- To detect the presence of intestinal parasites
- For the detection of evidence of malfunction of some parts of the GIT, liver and pancreas
- For the detection of GIT bleeding
- For the detection of excessive fats in stool (steatorrhea)
- Used as a clue in medical and surgical diagnosis.
- Physical Examination
❖ Form and Consistency: Normal:
soft to formed
– seen in diarrhea and administration of saline cathartic
a) Very soft and watery
– constipation due to lack of mucus
b) Excessively hard and scybalous
– cholera
c) Rice water stool
– early typhoid
d) Pea soup stool
– syphilis, spastic colitis and obstruction at the lower portion of the colon
e) Flattened or ribbon like
- fibrocystic disease of the pancreas
f) Butter like
– excessive carbohydrate fermentation
g) Gaseous and fermentative
Color Normal:
light brown to dark brown due to stercobilin
- due to administration of santonin and senna
a) Yellow
– seen after ingestion of Barium meals
b) Light clay or putty color
- bleeding in the lower GIT; undigested beets and tomatoes
c) Reddish or bloody
- bleeding in the upper GIT
d) Dark red/chocolate brown
- associated with digestion of blood due to bleeding in the upper GIT
e) Black/tarry
– amoebiasis
f) Greenish
Odor Normal:
foul to offensive due to indole, skatole and butyric acid
– found in ulcerative and malignant tumor of the lower bowel
a) Putrid odor
- indicates gas formation, fermentation of carbohydrate, unabsorbed fatty acids
b) Sour/rancid odor
– usually in alkaline stools, putrefaction of undigested protein.
c) Extremely foul odor
– blood which is not visible to the naked eye and can only be detected by chemical means only
Occult blood
Laboratory Examination for Occult Blood:
a) Benzidine test
b) Guaiac;s test
c) Hematest
The (?) (due to peroxidase contained in the heme portion of hemoglobin) decomposes (?) to produce (?).
peroxidase-like activity of blood
H2O2
water and oxygen
The (?) causes the oxidation of the colorless chromogen into a colored compound (blue)
liberation of oxygen
Hematest Patient Preparation:
meat free diet for 3-5 days prior to the test
The following structures may be seen microscopically:
a) Trophozoites and cysts of amoeba
b) Helminth eggs and larvae
c) RBC
d) Macrophages present in bacterial or parasitic infection
e) WBC
f) Fungi
g) Plant cells, pollen grain and spores
h) Epithelial cells
i) Calcium oxalate and triple phosphate crystals
j) Bacteria
k) Plant fibers, root hairs and animal cells ( similar to helminth ova)
l) Charcot-layden crystals
indicative of inflammation
WBC
- due to hemorrhagic disorder, ulcers and contamination
RBC
TECHNIQUES
– simplest and most frequently used
DIRECT FECAL SMEAR
- for the observation of the motility of trophozoites
a) 0.85% sodium chloride/NSS
– for protozoan cysts and helminth ova
b) D’antonis solution/Lugol’s Iodine
KATO THICK SMEAR (KTS)
▪ Reagents:
Distilled water 100 ml
Glycerin 100 ml
3% malachite green 1 ml
Use cellophane as cover slip
KATO THICK SMEAR (KTS)
- used in the detection of a small number of parasites which are not detected using DFS
CONCENTRATION TECHNIQUES
– uses either specific gravity or centrifugation
Sedimentation Method
– concentrates helminth eggs, larvae and protozoan cysts
Formalin-Ether techniques
– used for formed stools; applicable for helminth eggs and larvae
Acid-Ether Sedimentation
– time-consuming
Simple sedimentation
- for concentration of microfilariae’ utilizes venous blood as specimen
Knott Concentration Technique
Allows the separation of helminth eggs, protozoan cysts and larvae (1.05 – 1.15) using chemical solutions of higher specific gravity (1.12-1.21)
Flotation method
Eggs and cysts float to the surface of the solution while fecal materials sink to the bottom
Flotation method
Superior to sedimentation for concentrating cysts and eggs other than operculated, schistosomal and infertile Ascaris eggs
Flotation method
- valuable method for concentrating cysts and eggs
Zinc Sulfate centrifugal flotation technique
- specific gravity: 1.18 (hydrometer); 1:20 formalinized feces
Zinc Sulfate centrifugal flotation technique
- Cryptosporidium (rounded oocysts)
Sugar Flotation Technique/Sheather’s Sugar Flotation
- uses sodium chloride solution
Wilie’s Brine
Lane’s Direct Centrifugal Technique
Magnesium Sulfate Techniques
• D’Antonis
Temporary Staining
1% Isotonic Eosin Solution & 2% Brilliant Cresyl Blue
Temporary Staining
Eosine in saline
Temporary Staining
Buffered Methylene Blue (ex. Nairs Methylene Blue)
Temporary Staining
1% Isotonic Eosin Solution & 2% Brilliant Cresyl Blue
- Trophozoites-
- Background-
blue green
pink
Permanent Staining
Gomori’s Trichrome Stain
Lawless’ Permanent Mount Stain
Modified Acid-Fast stain
Iron Hematoxylin Stain
MIF Fixative stain ( Merthiolate iodine formaldehyde)
Chlorazol Black E
Modified Kohn’s
The trichrome technique of Wheatley for fecal specimens is a modification of Gomori’s original staining procedure for tissue.
Gomori’s Trichrome Stain
It is a rapid, simple procedure which produces uniformly well stained smears of the intestinal protozoa, human cells, yeast cells, and artifact material in about 45 min or less.
Gomori’s Trichrome Stain
- Wheatley’s modification
Gomori’s Trichrome Stain
- For intestinal protozoa
Gomori’s Trichrome Stain
A rapid permanent-mount stain technic for the diagnosis of the intestinal protozoa
Lawless’ Permanent Mount Stain
Rapid method of staining trophozoites and cysts
Lawless’ Permanent Mount Stain
Protozoa – blue to purplish color
Lawless’ Permanent Mount Stain
- Cryptosporidium, Isospora and Cyclospora
Modified Acid-Fast stain
- Two separate stains: Carbol fuchsin and methylene blue
Modified Acid-Fast stain
- lacks specificity
Modified Acid-Fast stain
- Use of the modified Ziehl-Neelsen stain for faecal smears has already been established for coccidian protozoa, in particular, oocysts of Cryptosporidium species, but it is also useful to confirm the presence of oocysts of Isospora belli and Cyclospora cayetanensis
Modified Acid-Fast stain
- used for most of the original morphological descriptions of intestinal protozoa found in humans.
Iron Hematoxylin Stain
- On oil immersion power (1,000 x ), one can examine the diagnostic features used to identify the protozoan parasite.
Iron Hematoxylin Stain
can be used with either fresh, SAFpreserved, or PVA-preserved specimens.
Iron Hematoxylin Stain
diagnosis of Trichomonas
MIF Fixative stain ( Merthiolate iodine formaldehyde)
An acid dye, used as a fat and general tissue stain, and to stain protozoa in fecal smears or in tissues.
Chlorazol Black E
modification of the chlorazol black E staining technique
Modified Kohn’s
Enterobius vermicularis and Taenia
Cellulose Tape Preparation/Graham Scotch Tape Method
Cellulose Tape Preparation/Graham Scotch Tape Method Collection:
morning before the patient washes or defecates
Commercial collection kits are available
Cellulose Tape Preparation/Graham Scotch Tape Method
provides an estimate of worm burden
Egg Count Technique
Determines the degree of infection (light, moderate or heavy)
Egg Count Technique
For recovery of hookworms, Ascaris and Trichuris
Egg Count Technique
Egg Count Technique Methods:
- Direct Smear Egg Count (Method by Beaver)
- Dilution Egg Count
- Thick Smear Egg Count
- Dilution-Filtration Egg Count
Method by Beaver
Direct Smear Egg Count
= eggs per gram (epg)
- 1.5 mg feces (667)
= epg
- 2.0 mg feces (500)
– uses 0.1 N NaOH
Dilution Egg Count
- Stoll’s Egg Counting Technique
Dilution Egg Count
– KTS for schistosomes
Thick Smear Egg Count
– Schistosomes
Dilution-Filtration Egg Count
is a laboratory tool used to determine a given parasite’s resistance to extant drug therapy.
Egg hatch assay (EHA)
Fresh eggs are incubated from the parasite of interest and serial dilutions of the drug of interest are applied.
Egg Hatching Technique
The percentage of eggs that hatch or die is determined at each concentration and a drug response curve may be plotted.
Egg Hatching Technique
The data can then be transformed and analysed to give further statistics such as an ED50.
Egg Hatching Technique
This technique is labour intensive, expensive and can take some time, however an egg hatch assay will give and accurate and reliable result.
Egg Hatching Technique
✓ For accurate detection of the organisms as a supplement to other methods
CULTURE METHODS
✓ For obtaining a rich yield of organisms to be used as antigen in immunologic diagnosis
CULTURE METHODS
✓ For in-vitro screening of drugs
CULTURE METHODS
✓ For investigating the physiology of organisms
CULTURE METHODS
✓ As a source for inoculating susceptible experimental animals
CULTURE METHODS
CULTURE METHODS for PROTOZOA
a) Balamuth’s Monophasic Medium
b) Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw
c) Cleveland & Collier’s
d) Trussel and Johnson’s medium
e) Axenic Culture Method
- All liquid medium commonly used for the isolation and maintenance of Entamoeba histolytica and some other intestinal protozoa.
Balamuth’s Monophasic Medium
- It is satisfactory for Balantidium coli, although the quality and amount of starch may need to be adjusted
Balamuth’s Monophasic Medium
- Use of egg yolk
Balamuth’s Monophasic Medium
- Egg slant
Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw
- E. histolytica
Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw
- Concentrate from saline centrifugal sedimentation of feces rather than the feces is recommended.
Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw
- Use of whole hen’s eggs
Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw
- This medium is highly specific for E. histolytica , which grows luxuriantly on this medium.
Cleveland & Collier’s
Other intestinal amoebae do not grow readily on this medium.
Cleveland & Collier’s
The technique of overlaying the medium with fresh sterile horse serum-saline mixture, as reported by Cleveland and Collier, was reported to be the best method of isolation of E. histolytica .
Cleveland & Collier’s
Proteose peptone and infusion from liver provide amino acids and other nitrogenous substances that support growth of E. histolytica .
Cleveland & Collier’s
Sodium chloride maintains the osmotic balance of the medium and sodium a-glycerophosphate acts as a phosphorous source.
Cleveland & Collier’s
Isolation of Trichomonas vaginalis
Trussel and Johnson’s medium
- histolytica
Axenic Culture Method
- Axenic cultivation has been an essential component of numerous immunological and biochemical studies
Axenic Culture Method
- Monophasic clear liquid for rapid growth of dense populations of amebae
Axenic Culture Method
CULTURE METHOD for LEISHMANIA & TRYPANOSOMES
a) Novy, MacNeal and Nicole (NNN) Diphasic
b) Cellular Media
- For Trypanosoma cruzi and Toxoplasma
b) Cellular Media
- Use of sterile defibrinated rabbit blood
Novy, MacNeal and Nicole (NNN) Diphasic
- Best medium
Novy, MacNeal and Nicole (NNN) Diphasic
IMMUNOLOGIC or SEROLOGIC TESTS
a) Indirect Hemagglutination (IHA)
b) Flocculation or Agglutination
c) Indirect Fluorescent Antibody (IFA)
d) Gel Diffusion (Ouchterlony) and Countercurent Electrophoresis (CEP)
e) Enzyme -linked immunosorbent assay (ELISA)
f) Sabin-Feldman Dye Test
g) Circumoval Precipitin Test (COPT)
h) Radioimmunoassay (RIA)
i) Radioallergosorbent Test (RAST)
j) Immunoblotting/Western Blot
k) Complement Fixation (CF)
involves antigen-antibody reactions
IMMUNOLOGIC or SEROLOGIC TESTS
PRINCIPLE: Patient serum and commercial sheep blood cells coated with the appropriate antigen are required. The serum and cells are combined and allowed to react. The test sample is then examined for the presence of red blood cell agglutination
Indirect Hemagglutination (IHA)
USES: amoebiasis, Chaga’s disease, malaria, toxoplasmosis, cystisercosis, hydatid cyst, filariasis, schistosomiasis
Indirect Hemagglutination (IHA)
PRINCIPLE: The patient serum is added to known cultured parasites. This mixture is observed for the presence of parasite clumping.
Flocculation or Agglutination
USES: Leishmaniasis, Chaga’s disease
Flocculation or Agglutination
PRINCIPLE: Microscopically visible parasites fixed on a slide are allowed to react with test serum. The slide is washed and treated with anti-human globulin with fluorescein (sandwich method)
Indirect Fluorescent Antibody (IFA)
PRINCIPLE: Patient serum and known antigens are placed in adjacent wells located on the agar gel. The antigen and antibody are allowed to migrate across the agar towards one anther. Specific patterns of precipitate ( precipitin bands) result between the inoculated wells and are then interpreted.
Gel Diffusion (Ouchterlony) and Countercurent Electrophoresis (CEP)
USES: Detect amebiasis and fascioliasis infections
Gel Diffusion (Ouchterlony) and Countercurent Electrophoresis (CEP)
PRINCIPLE: Test serum is allowed to react with an antigen (coat the surface of plastic well). Anti-human IgG antibody conjugated with enzyme is added. If specific antibody was present in the test serum, the anti-human IgG antibody labeled with an enzyme attaches to the antigen-antibody complex. The addition of a suitable substrate generates a visible color reaction.
Enzyme -linked immunosorbent assay (ELISA)
USES: T. gondii, T. vaginalis, leishmaniasis, malaria, schistosomiasis etc.
Enzyme -linked immunosorbent assay (ELISA)
✓ Modification of the standard ELISA for increased sensitivity and specificity in detecting antibodies of the IgM class to Toxoplasma
Antibody Capture or Double Sandwich ELISA
✓ Plastic well are coated with anti-human IgM antibodies
Antibody Capture or Double Sandwich ELISA
✓ After washing, the test serum is added followed by further washing and addition of the test antigen
Antibody Capture or Double Sandwich ELISA
✓ The final reagent is an antigen-specific antibody to which the color reagent has been conjugated
Antibody Capture or Double Sandwich ELISA
✓ The plates are read spectrophotometrically
Antibody Capture or Double Sandwich ELISA
✓ This method can be used for assay of parasite-specific IgE antibodies by starting with anti-human IgE
Antibody Capture or Double Sandwich ELISA
is based on the fact that live Toxoplasma tachyzoites can actively take up methylene blue dye from the culture medium, whereas parasites that are killed because of complement-mediated lysis do not take up the dye and remain colorless.
Sabin-Feldman Dye Test
Specifically designed for the diagnosis of Toxoplasmosis
Sabin-Feldman Dye Test
is a serological test used for diagnosis of schistosomiasis japonica.
Circumoval Precipitin Test (COPT)
Soluble egg antigens of Schistosoma japonicum block the formation of the circumoval precipitin by serum from infected humans.
Circumoval Precipitin Test (COPT)
Consequently, circumoval precipitin inhibition was used to monitor purification of the soluble egg antigens of S. japonicum.
Circumoval Precipitin Test (COPT)
PRINCIPLE: Radioimmunoassay (RIA) is a very sensitive in vitro assay technique used to measure concentrations of antigens by use of antibodies. As such, it can be seen as the inverse of a radiobinding assay, which quantifies an antibody by use of corresponding antigens
Radioimmunoassay (RIA)
is a radioimmunoassay test to detect specific IgE antibodies to suspected or known allergens for the purpose of guiding a diagnosis about allergy
Radioallergosorbent Test (RAST)
In this test, the parasite antigen is first bound to an inert sorbent or matrix to which the test serum is allowed to react. After washing, radio-labeled antibody (IgE or IgG) is added to the sorbent. After another washing to remove excess labeled antibody, the remaining radioactivity is a measure of antigen-specific IgG or IgE in the test serum
Radioallergosorbent Test (RAST)
PRINCIPLE: CSF and serum may be used. By using electrophoresis technique, the DNA containing portion of the patient sample is transferred to a polyacrylamide gel. This process results in the separation of protein antigens in the gel. A blotting technique is employed to transport the proteins onto a paper.
Immunoblotting/Western Blot
USES: giardiasis
Immunoblotting/Western Blot
PRINCIPLE: Three ingredients is necessary to for performing the first phase: patient serum, a commercial antigen source, known amount of complement. The complement is “fixed” by the antibodies present in the serum. In the 2nd phase, sensitized sheep’s rbc is added to the mixture. The absence of free complement prevents lysis of these cells ( positive). If the patient sera does not contain Ab, complement is free to lyse ( negative)
Complement Fixation (CF)
USES: leishmaniasis, Chaga’s disease, pneumocystosis, paragonimiasis
Complement Fixation (CF)
Animal Used: hamster
ANIMAL INOCULATION TESTS
Leishmania
Specimen: aspirates from biopsy materials from cutaneous ulcers, lymph nodes, spleen, liver
ANIMAL INOCULATION TESTS
Leishmania
Animal Used: Guinea pigs or white rats (Trypanosoma gambiense and T. rhodesiense)
ANIMAL INOCULATION TESTS
White mice (Trypanosoma cruzi)
ANIMAL INOCULATION TESTS
An experimental bug (vector) is allowed to take a blood meal from the patient
XENODIAGNOSIS
The intestinal contents of the bug is assessed for the presence of diagnostic stages of the parasite
XENODIAGNOSIS
Over the years, parasites which were once considered commensal have evolved to become human pathogens.
During this time, tremendous knowledge was obtained of the epidemiology, parasite-host relationships, life cycles, disease processes and symptoms, treatment, and prevention and control of parasites.
In addition, parasites are classified based on their individual characteristics.
Traditional as well as new methodologies for parasite identification allow for accurate laboratory diagnosis.
Parasitology is an interesting and exciting field of the clinical laboratory sciences.
The continued development of high-tech, highly sensitive parasite test methodologies provides the key to the future of parasitology.
Because it is highly unlikely that parasites will totally be eradicated in the near future, competent practitioners educated in the field of parasitology are essential to ensure proper parasite identification.
