LBBBI18 - Medical Parasitology Lab Ray Paolo Montes

Ms. Joyce de Guia BS BioMed

Specimen Collection: Blood Specimen Type of sample

● Venous blood
● Medical parasitology is not only limited to fecal samples ○ Obtained by venipuncture
■ Always label collection tubes and pre-cleaned slides
with:
Safety
● Patient name
● Follow universal precautions for the prevention of bloodborne ● Date
pathogens when working with human serum and other bodily fluids ● Time of collection
○ Wear PPE ■ Clean site with alcohol and allow to dry
○ Cover cuts and abrasions with adhesive dressing ■ Store in vacuum tube with anticoagulant or collect
○ Use needles and lancets once and discard in the sharps blood in syringe and transfer to a tube with
container anticoagulant and mix
○ Remove gloves, dispose properly, and disinfect hands after ■ Prepare at least 2 thick and thin smears as soon as
completing tasks possible after collection
○ Sufficient for variety of diagnostic tests
Timing ○ Con is that for some parasitic diseases, anticoagulants may
● Whenever possible, collect specimen before treatment interfere with the morphology and staining characteristics
● If suspecting malaria or babesiosis, prepare blood smears and ● Capillary blood
examine immediately ○ Obtained by fingerstick
○ Parasitemia may fluctuate if not examined immediately ■ Label precleaned slides with:
● Microfilariae exhibit periodicity depending on species ● Name,
○ Collection is critical ● Date,
■ Loa loa: midday (10am-2pm) ● Time of collection
● African eyeworm causing congestion and light ■ Clean site with alcohol and dry
sensitivity ■ Prick 3rd or 4th finger’s end
■ Brugia or Wuchereria spp. : after 8pm ■ Wipe away first drop of blood
● Microfilariae are only actively circulating in ■ Prepare at least 2 thick and thin smears as soon as
the blood at night possible after collection
■ Mansonella spp.
■ Onchocerca spp.
● Best time for collection depends on the target parasite
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

Shipment ○ Wear PPE

○ Cover cuts and abrasions with adhesive dressing
● Shipping Blood smear for microscopic examination
○ Use needles and lancets once and discard in the sharps
● Shipping whole blood for parasite isolation
container
○ Place labeled tube of anticoagulated blood in absorbent
○ Remove gloves, dispose properly, and disinfect hands after
material and place in sealed plastic bag of 50mL screw cap
completing tasks
centrifuge tube
○ Pack bag cushioned to protect from breakage
Stool Collection

Disposal ● Collect stool in a dry, clean, and leak-proof container and is not
contaminated by foreign matter
● Human blood sample is treated with 10% bleach solution for 30 mins
● Fresh stool should be examined, processed,or preserved immediately
before disposing
● Specimens must be kept refrigerated if no preservatives
● Slide samples with unfixed specimens are disposed in sharps
○ Specimens stored this way are only suitable for antigen
container
testing
● Slide samples with fixed specimen must be in broken glass box
● Distribution of protozoa in relation to stool consistency

Specimen Collection: Stool Specimen

● Stool specimen is used to diagnose parasitic protozoans and

helminths
○ TC
■ Trophozoites and Cysts
● Preserve specimen as soon as possible
Safety ○ Follow instructions if using commercial collection kit
● Potential risks include ingestion of eggs or cysts, skin penetration by ○ If not available, divide and store in two different preservatives
infective larvae, and infection by nonparasitic agents ■ 10% formalin
○ Can be minimized by adopting universal precautions as well ■ PVA (polyvinyl-alcohol)
as standard microbiological laboratory practices ○ Add one volume of stool specimen to three volumes of
● Follow universal precautions for handling fecal matter (Biosafety preservatives
level 2) ● Ensure specimen is mixed well with the preservative
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○ Break down formed stool Examination of Fresh Specimens

● Ensure specimen containers are sealed well
● Allows observation of motile trophozoites without delay
○ Reinforce with parafilm or other suitable material
● Liquid specimens should be examined within 30 minutes of passage
● Certain drugs and compounds will render the stool specimens
● Soft specimens should be examined within one hour of passage
unsatisfactory for examination
● Specimens preserved in formalin
● Specimen collection may need to be repeated if the first examination
○ Can be examined directly or concentrated prior to further
is negative
testing
○ Ideally, three specimens passed at 2 to 3-day intervals should
○ Can undergo wet mount, immunoassay, chromotrope stain, UV
be examined
fluorescence

Preservation of Stool Specimens

Shipment of Stool Specimens
● Commonly used preservatives
● Preserved specimens
○ 10% Formalin
● Unpreserved specimens
■ Preserves eggs, cysts, and larva
○ Can be requested to isolate a known or suspected pathogen
○ Polyvinyl-alcohol
■ Specimen must be placed in a clean container
■ Preserves cysts and trophozoites
● Ideally, divide specimen and preserve in both preservatives
● Specimens can be stored for months given that commercial two-vial Decontamination and Disposal of Specimens and Infectious materials
kits are used ● Decontamination and disposal of specimens and infectious materials
● Methods of sterilization or decontamination
○ Incineration
○ Autoclaving
■ Used with heat-stable items
○ Boiling in detergent
■ Does not kill spores or inactivate certain viruses
■ Boiling with detergent for 30 mins
○ Burial
■ Prevents material from being a hazard but does not
decontaminate
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○ Two anteriorly placed nuclei and eight, rarely seen flagella

Parasitic Protozoans
Diagnostic Techniques
● Duodeno-jejunal aspiration
Giardia lamblia
○ Fluid is aspirated from the duodenum for a smear analysis
Information ○ Examined in a slide
● Giardiasis is the disease caused by G. lamblia and G. duodenalis ● Direct Fluorescent Body Assay
○ Cyst is the dormant, resting stage ○ Highest sensitivity and specificity
■ Both the infective and diagnostic stage of Giardia ○ Considered the gold standard
■ Plays a huge factor in the prevalence of the disease ○ In commercial DFA kits, concentrated stool specimen are
■ Undergoes excystation when ingested and multiplies needed to increase the probability of detecting low numbers
● Trophozoites go to colon to rehydrate and go of cysts
back into circulation and infection through the ○ Does not provide a permanently stained slide that can be
fecal-oral route of infection archived
○ Trophozoite is a diagnostic stage of the parasite ● Enzyme Immunoassay
■ Trophozoite dies when exposed to external ○ Does not rely on microscopy
environment ○ Borderline positive and questionable negatives must be
confirmed further by DFA
○ Antigens of Giardia are detected in feces
Diagnosis
■ Specimen must not be concentrated prior to testing
● Stool samples are taken ○ Special equipment such as microplate reader and
○ Multiple samples must be tested negative before concluding commercially available test kits are required
a negative test result
● Wet mount (cysts)
○ In bright field microscopy, cysts appear ovoid to ellipsoid in
shape
○ Usually measuring 11-14 micrometers
○ Immature and mature cysts and 2 and 4 nuclei respectively at
one end with median bodies
○ Contains retracted flagella and a tough hyaline wall
● Wet mount (trophozoites)
○ Pear-shaped organism
○ Measures 12-15 micrometers
● Rapid Immunochromatographic Cartridge Assay
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○ Used with preserved specimens Entamoeba histolytica

○ Quick and easy to perform
○ Antigens of Giardia are detected in feces Information
■ Specimen must not be concentrated prior to testing ● Causes amebiasis
○ No special equipment needed ○ Diagnostic guidelines
■ Add 2 drops of sample treatment buffer and ■ Multiple stool samples must be tested before a
centrifuged stool specimen to the tube negative result is reported
■ Add 2 drops of Giardia capture ■ Samples must be concentrated and preserved with
■ Add 2 drops of detection reagent formalin-ethyl acetate
■ Test in test device and wait for result ■ Diagnostic techniques depends on the available
equipments, reagents, experience, time, and cost
Basic Guidelines ○ Life Cycle
● Maximize recovery of cysts
○ Samples in formalin or other fixatives must be concentrated
before microscopic examination
■ Formalin-ethyl acetate concentration procedure
● Choice of diagnostic technique depends on available equipment,
reagents, experience, time and cost

Treatment
● Metronidazole is generally the standard medication used in treating
Giardiasis

Prevention and Control

● Sanitary disposal of human excreta
● Normal water chlorination will not affect cysts; proper water
treatment is enough

■
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

● Chromatoidal bodies are elongated with

Diagnosis rounded ends
■ Mature cysts contains a diffused or absent glycogen
● Diagnostic stages
■ Immature cysts contain clumped glycogen
○ Cyst and trophozoite
● Trichrome staining
■ Mature cysts have 4 nuclei
○ Trophozoites
● Cysts and trophozoites are passed in the feces
■ Measure at about 15-20um
● Fecal-oral route of transmission
■ Single nucleus
○ Ingestion of infested food and water
■ Cytoplasm is finely granular
● Trophozoites reproduce by binary fission
● At times, ingested bacteria or debris may be
● After excystation, trophozoites have three types of colonization”
found
○ Asymptomatic
■ Diagnostic characteristic feature of E. histolytica is the
■ Cysts are only passed through
presence of erythrocytes in the cytoplasm
○ Invasive
● Enzyme Immunoassays (EIA)
■ Intraintestinal infection
○ Commercially available kits for detecting E. histolytica
■ Causes amoebic colitis
○ Requires fresh or frozen stool specimens
○ Amoebic
■ Preserved specimens are not compatible with this
■ Causes liver abscess
diagnostic technique
■ Extraintestinal infection
○ Wells contain immobilized polyclonal antibodies
■ Antibodies bind to the adhesive of Entamoeba
Diagnostic Techniques ■ Conjugate = monoclonal or polyclonal antibody
● E. histolytica and E. dispar are morphologically identical species conjugate specific for E. histolytica
● Diagnostic techniques include wet mounts and trichrome staining of ○ Aliquot of fecal specimen is emulsified in the diluent of the
samples assay
● In wet mounts: ■ Adhesive of Entamoeba binds to the conjugate
○ Cysts polyclonal antibody conjugate specific for E.
■ Appear spherical measuring at about 12-15um histolytica
■ Mature cysts contain 4 nuclei ○ Washing step removes any unbound material
■ Immature cysts contain 1-3 nuclei ○ Substrate is added
■ Peripheral chromatin is fine, uniform, and evenly ■ Color will develop from the enzyme-antibody-antigen
distributed complexes formed by the adhesive
● Rapid Immunochromatographic Cartridge Assay
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○ Detects antigens of E. histolytica/dispar ○ Trophozoite may replicate by promitosis

■ Unable to distinguish between the two ● Trophozoite develops into a temporary nonfeeding flagellated form
○ Stool samples must be fresh or frozen and not concentrated and eventually return back into a trophozoite
○ Borderline positives and questionable negatives must ● Found in freshwater, soil, thermal discharges of power plants,
undergo a different test for confirmation geothermal wells, and poorly chlorinated water
○ Quick and easy ● Thermophilic in nature
■ No special equipment required ● Moves in a linear forward direction through pseudopods
○ Procedure: ● Does not form cysts in human tissues
■ Stool sample is suspended in an extraction buffer;
allowed to precipitate Diagnosis
■ Aliquot of supernatant is put in the test field
● Diagnostic and infective stage
■ If positive, red and blue bands are visible
○ Trophozoites
■ If negative, blue bands only are visible
● Known as the brain-eating amoeba
■ If no bands are present, result is invalid
○ Trophozoite enters the nasal mucosa and migrate to the brain
via olfactory nerves
Treatment ○ Causes the fatal primary amebic meningoencephalitis
● Metronidazole ■ 75% of diagnosis are made after death
○ Used for invasive amebiasis ● Two forms of trophozoites can be seen through microscopy:
● Diloxanide furoate ○ Ameboid
○ Used for asymptomatic cyst passer ■ Cytoplasm is granular and contains a lot of vacuoles
○ Also given after a course of metronidazole ■ Contains a large, centrally located nucleus
● Presence of a large, dense karyosome
Prevention and Control ● Lacks peripheral chromatin
○ Ameboflagellate
● Proper sanitation and clean drinking water
■ Rarely found in humans; usually found in the CSF
● Proper washing of hands
● Sanitary disposal of human feces
Diagnostic Techniques

Naegleria fowleri ● Diagnosis are done by detecting the following through CSF, biopsy, or
tissue specimens
Information ○ Naegleria fowleri organisms
● Naegleria cyst becomes a trophozoite ○ Naegleria fowleri nucleic acid
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○ Naegleria fowleri antigen

● Direct visualization of the tissue Trypanosoma spp.
○ Done through microscopic examination and staining with
hematoxylin and eosin, periodic acid-Schiff, trichrome, Information
Giemsa, or Wright-Giemsa of brain biopsies ● Trypanosoma brucei gambiense
● Direct visualization of the CSF ○ Causes chronic African Trypanosomiasis
○ Done through microscopic examination of fresh, untreated ■ West African sleeping sickness
CSF ● Trypanosoma brucei rhodesiense
○ Actively moving trophozoites may be seen from a ○ Causes acute African Trypanosomiasis
freshly-centrifuged CSF sediment through a wet mount ■ East African sleeping sickness
○ CSF smears or cultures are used and stained by the ○ Cattles and wild ungulates act as reservoir
aforementioned stains ● Both subspecies are morphologically indistinguishable
● Polymerase Chain Reaction ● Tsetse Fly (Glossina spp.) is the only known vector
○ DNA samples are obtained from CSF and unfixed tissue ● Life Cycle
samples and are amplified ○ Mammalian Cycle
○ Can use either conventional or real-time PCR ■ Fly takes a blood meal and injects the infective
● Immunohistochemical staining metacyclic trypomastigotes
○ Done as a confirmatory test ■ Metacyclic trypomastigotes transform into
○ Indirect immunofluorescent and Immune alkaline bloodstream trypomastigotes and migrate to other
phosphatase staining sites
■ Uses specific antibodies for Naegleria fowleri followed ■ Trypomastigotes multiply by binary fission
by microscopy ● During acute phase, circulates in the blood
● Undetectable in latent phase
Treatment ■ Fly takes a blood meal and ingests trypomastigotes
○ Tsetse Fly Cycle
● Symptoms or PAM are indistinguishable from bacterial meningitis
■ Trypomastigotes transform into procyclic
● Most infected persons die before treatment
trypomastigotes in the midgut of the Tsetse fly
● Initial CSF results are suggestive of bacterial etiology
● Multiplies by binary fission
○ Most patients are treated with antibiotics that are useless
■ Procyclic trypomastigotes leave the midgut and
against Naegleria
transform into epimastigotes
● When diagnosed early, a treatment composed of a combination of
■ Epimastigotes transform into metacyclic
Amphotericin B and clotrimazole is synergistic and are successfully
trypomastigotes in the salivary glands of the fly
used to treat PAM
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

● Trypomastigotes are polymorphic ○ Blood cells and trypomastigotes are more clearly visible
○ Slender, flattened, and fusiform in shape
Diagnostic Techniques
Basic Guidelines ● Antibody detection
● Trypanosomes can be demonstrated through the microscopic ○ Rapid tests
examination of the following: ■ Used for T.b. gambiense
○ Chancre fluid, lymph node aspirates, blood, bone marrow, ■ Screens whole blood
and/or CSF ○ Performance depends on endemicity
● Wet preparation should be examined for motile trypanosomes ○ Some kits are commercially available
● Blood smear should be fixed and stained with Giemsa ■ Dipstick and card agglutination formats
● Concentration techniques can be used for microscopic examination ● Molecular detection
○ Blood ○ No tests are currently validated
■ Buffy coat
■ Mini anion-exchange / centrifugation Treatment
■ Quantitative Buffy Coat technique
● Treatment depends on the stage of the disease
○ Spinal fluid
○ Early Stage
■ Centrifugation
■ Intravenous suramin sodium
○ Serial examinations
● For both subspecies of Trypanosoma
■ Used in varying levels of parasitemia
■ Intramuscular pentamidine
● Gambian form
Diagnosis ○ CNS Stage
● Trypomastigote is the only stage found in patients ■ Intravenous melarsoprol
○ Length ranging from 14 to 33um ● If treatment fails, Nitrofurazone is
● Thick Blood Smear administered
○ Trypomastigotes appear with the following ○ Hemolymphatic Stage
■ Small kinetoplast at the posterior end ■ Elfornithine
■ Centrally located nucleus ● Effective only against T.b. gambiense
■ Undulating membrane ● For best result, combination therapy using multiple drugs are done
■ Flagellum along the undulating membrane through ○ Drugs to be used depends on the status of the patient
the anterior end
● Thin Blood Smear
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

Prevention and Control ■ Schizonts rupture and release merozoites

■ Merozoites penetrate myocytes and develop into cysts
● Vector control
with bradyzoites
○ Tsetse fly trap
○ Cysts with bradyzoites are ingested through undercooked
■ Blue exterior attracts flies
meat
■ Black interior contains insecticide
○ In the human definitive host:
● Use of insecticides and protective clothing
■ Motile bradyzoite from ruptured cyst enter intestinal
○ Prevents contact with the insect vector
cells
● Regulation and treatment of reservoir hosts
■ Microgamete and macrogamete produce immature
○ Transmission prevention
oocyst
■ Oocyst exit the host through feces
Sarcocystis spp. ○ Sarcocystis hominis and Sarcocystis suihominis are
Information morphologically undistinguishable

● Found in skeletal and cardiac muscles

● Causes the disease Sarcocystosis Diagnosis
○ Intestinal type ● Spread through the oral-fecal route
■ Diarrhea, fever, vomiting ● Sporocysts, oocysts, and cysts with bradyzoites are the diagnostic
■ Caused by Sarcocystis hominis and Sarcocystis and infective stages of Sarcocystis
suihominis ● Cattles are the intermediate host of Sarcocystis hominis
■ Humans serve as the definitive host ● Swines are the intermediate host of Sarcocystis suihominis
○ Muscular type ● Intestinal sarcocystosis are diagnosed through the observation of
■ Muscle pains, transitory edema, fever oocysts and sporocysts in stool samples
■ Caused by non-human species of Sarcocystis ● Non-human Sarcocystis species are diagnosed through sarcocysts in
■ Humans serve as the accidental, intermediate hosts tissue specimens
○ Oocysts contain two sporocysts
■ Sporocysts contain four sporozoites Diagnostic Techniques
● Life Cycle
● Stool examinations done on separate days during infection is required
○ Sporocysts passed in the feces are ingested by cows and pigs
for identification of sporocysts in feces
○ In the intermediate hosts:
● Wet mount
■ Sporocysts rupture, releasing sporozoites
○ Oocysts measure at about 15-20um long by 15-20um wide
■ Sporozoites enter endothelial blood cells and undergo
○ Sporulated oocysts and sporocysts are shed in the feces
schizogony
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○ Sporocysts are known to autofluoresce under UV light ● Corticosteroids are administered to provide symptomatic relief in
● Negative staining cases of muscular sarcocystosis
○ Uses carbol fuchsin stain under microscope ● Currently no proven routine treatment for sarcocystosis
● Polymerase Chain Reaction
○ Amplification of 18s rRNAs allow molecular differentiation of Prevention and Control
several sarcocysts and oocysts Sarcocystis species
● Freezing meat at -5°C for several days to kill sporocysts
■ S. hominis
● Thoroughly cooking of meat
■ S. fusiformis
● Boiling potentially infected water
■ S. cruzi
● In animals with widespread infestation of visible cysts, whole carcass
○ Allows amplification and identification of species-specific
must not be consumed and discarded properly
gene sequences from as few as 7 to 50 sporocysts stored in
● In animals with lighter infestation, unaffected parts may be consumed
potassium dichromate
● Tissue visualization
○ Uses hematoxylin and eosin stain Trichomonas vaginalis
○ Confirmatory staining with periodic acid-Schiff staining Information
○ Walls of sarcocysts are used in species diagnosis
● Causes trichomoniasis
■ S. hominis
● Found in the urogenital tract
● 6um thick wall
○ Females
● 7-9um long bradyzoites
■ Usually isolated in the vagina but are also found in the
■ S. suihominis
renal pelvis
● 4-9um thick wall
○ Males
● 15um long bradyzoites
■ Found in the urethra, prostate, and the epididymis
● Often described as the most prevalent non-viral sexually transmitted
Treatment infection in humans
● Muscle sarcocystosis can present with symptoms of myositis with or ● No other known stage than a trophozoite
without fever ● The trophozoite in the vaginal and prostatic secretions is the
● Infected patients have elevated levels of eosinophil and creatinine diagnostic stage
kinase without explanation ● The trophozoite in the vagina or urethral orifice is the infective stage
● Muscle biopsy can be performed with conventional histologic staining ● Multiplies by binary fission
but may not demonstrate classic sarcocysts
● Albendazole showed efficacy in one case of muscular sarcocystosis
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

Diagnosis Techniques ● Cultures contain different salts by maintaining

osmotic equilibrium by controlling the
● Females are usually symptomatic
permeability of the specimen
● Males are asymptomatic
● Charcoal neutralizes materials that are toxic
● Diagnostic methods include
to sensitive pathogens
○ Saline preparation / wet mount
● Inoculated cotton swabs are immersed into
■ Traditional diagnostic method
the tubed mediums
■ Quickest and most inexpensive method
■ InPouchTV
■ Visualizes motile T. vaginalis parasites
● Allows direct inoculation, culture, and
● Sample must be examined within 10mins.
microscopic examination
○ Culture
● Can also be used in transporting specimen
■ Accepted gold standard with 100% specificity and
● Advantageous if immediate transport to the
75-96% sensitivity
laboratory is not available
■ 2-5 days before test result
● An oxygen-resistant plastic with two
■ Diamond’s modified medium
chambers
● Selective cultivation of Trichomonas spp.
○ Top pouch
● Cultured tubes are removed from -20°C
○ Bottom pouch
● Tubes are incubated at around 37°C for 1 to 2
○ Papanicolau smear
hours
■ Also known as Pap Smear
● Sample is directly swabbed into the cultures
■ Usually used to screen cervical cancer in females
● Incubated vertically at 37°C in 5% carbon
■ Low sensitivity for detecting trichomonads
dioxide
■ Specificity of 95% for trained technicians
● Binocular or inverted microscope are used
○ Rapid antigen test
after being prepared into a wet mount slide
■ OSOM Trichomonas Rapid Test
● If slides turn out negative, incubate and
● Uses immunochromatographic capillary-flow
examine again before concluding a negative
enzyme immunoassay dipstick test
test result
● Performed in vaginal secretions
■ Amies gel medium
● Results in 10mins.
● Semisolid medium used for samples needing
● Presence of blue test line in the dipstick
transport to maintain specimen viability for up
indicates a positive test result
to 24 hours in room temperature
○ Nucleic acid amplification tests
● Recommended for qualitative procedures
■ Most sensitive test available for T. vaginalis
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

■ In vitro test for detection of Trichomonas RNA ■ Isoenzyme analysis

■ APTIMA Trichomonas vaginalis Assay ■ Molecular methods
● FDA-approved more accurate and reliable ■ Monoclonal antibodies
testing compared to other diagnostic
procedures for trichomoniasis Diagnosis
● Life Cycle
Treatment ○ Human cycle
● Metronidazole ■ Sandfly takes a blood meal injecting the promastigote
● Tinidazole into the skin
■ Promastigotes are phagocytized by macrophages
Prevention and Control ■ Promastigote transforms into amastigotes
■ Amastigotes multiply in cells and infects other cells
● Reduce risk exposure
○ Sandfly cycle
○ Limiting sexual partners
■ Sandfly takes a blood meal and ingests amastigotes
● Usage of protective devices properly
through macrophages or other infected cells
● Simultaneous treatment of sexual partners
■ Amastigotes transform into promastigotes in the
○ Prevents recurrent infections or “ping-pong”
sandfly gut
● Prompt follow-up check ups of patients and their contacts
■ Promastigotes divide through binary fission and
● Sex and health education about sexually transmitted diseases
migrate to the proboscis
● Diagnostic stage
Leishmania spp. ○ Amastigotes
Information ● Infective stage
○ Promastigote
● Causes leishmaniasis
■ Flagellated
○ A vector-borne disease
● Tissue specimens are the primary samples due to cutaneous
■ Sandfly as the biological vector
leishmaniasis
● Phlebotomus papatasi
○ One of the most common clinical manifestation of
● Genus Lutzomyia
leishmaniasis
● Obligate intracellular protozoan
○ Skin lesions with raised edges and central crater
● 15 species cause clinical manifestations in humans
○ Other clinical manifestations include:
○ Morphologically indistinguishable
■ Mucocutaneous leishmaniasis
○ Only differentiated by other diagnostic methods
● Involves mucous membranes
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

■ Visceral leishmaniasis ○ Conventional diagnostic approach for Leishmania species

● Proliferation of parasites in the bone marrow, identification
spleen, and liver ○ Isolation is done through a biphasic medium
■ Includes a solid blood agar base with defibrinated
Diagnostic Techniques rabbit blood
● Serology
● Diagnosis is based on microscopic demonstration of Leishmania
○ Antibody detection
○ Lesion and tissue scrapings
○ Useful technique for detection of visceral leishmaniasis
○ Aspirates or biopsy
● Molecular diagnosis
● Blood tests that can detect antibody to the parasite can be helpful for
○ Based on PCR amplification
diagnosis of visceral leishmaniasis
■ Uses generic primers amplifying a segment of the
● Microscopy
rRNA internal transcribed spacer 2 (ITS2) for multiple
○ Amastigotes of Leishmania are:
Leishmania species
■ Spherical to ovoid and measure 1 to 5um long by
■ DNA sequencing analysis is performed on the
1-2um wide
amplified fragment for species identification
■ Possesses a large nucleus, prominent kinetoplast, and
a short axoneme that is rarely visible by light
microscopy Treatment
■ Amastigotes are visualized with Giemsa and ● Treatment approach depends on host and parasite factors
Hematoxylin and Eosin stains ○ Some treatment protocols are effective only against certain
■ Amastigotes reside in macrophages and other Leishmania species and only in particular geographic regions
mononuclear immune cells ○ Special groups may need different medication and dosage
■ In biopsy specimen, regimens
● Appear as ovoid and typically lines the walls ● Sodium stibogluconate and n-methyl-glucamine
of vacuoles ○ People living with HIV are at risk for treatment failure and
● Isoenzyme analysis relapse
○ Isoenzyme ● Amphotericin B
■ Group of enzymes catalyzing the same reaction but ○ Used for cutaneous and visceral leishmaniasis
have different forms and catalytic efficiencies ● Pentamidine
■ Species identification through electrophoresis ○ Administration is limited due to side effects and the
● Each band corresponds to a specific marker development of drug resistance
for species identification
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

Prevention and Control Diagnosis

● Personal protective measures ● Life Cycle
○ Minimizing nocturnal outdoor activities ○ Sporulated oocysts exits the host and contaminates water
○ Protective clothing and food
○ Insect repellent ○ Contaminated water and food is ingested by the host
● Insecticide-treated clothing and fine-mesh bed nets ○ In the host intestine,
● Regulation of reservoir host ■ Oocyst excysts into a sporozoite
○ Insecticide-treated dog collars ■ Sporozoite embeds into the intestinal tract and
○ Mass testing of domestic dogs transforms into a trophozoite
■ Trophozoite develops into a Type I Meront in which
Cryptosporidium spp. merozoites are produced
● Asexual cycle
Information ○ Meront becomes a trophozoite again
● Causes cryptosporidiosis ● Sexual cycle
● Protected by an outer shell, allowing it to survive outside the host for ○ Merozoites develop into Type II
a period of time Meronts
● The presence of an outer shell makes it tolerant for chlorine ○ Merozoites hatch out of the meront
disinfection and becomes an undifferentiated
● Multiple stool specimens should be tested before reporting a gamont
negative results ■ Gamont may become
● Specimens should be concentrated to maximize the recovery of microgamonts and
oocysts macrogamonts
○ Except for EIA or Rapid Immunochromatographic Cartridge ○ Microgamonts produce microgametes
Assay which fertilizes a macrogamont to
■ Concentration procedure destroys antigens vital for produce a zygote
the aforementioned tests ○ Zygote develops into an oocyst and
● Thick walled sporulated oocysts are both the diagnostic and infective exits the body
stage
● Cattles are the major hosts for Cryptosporidium parvum
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

Diagnosis Techniques ○ Used with preserved specimens

○ Quick and easy to perform
● Depends on the available equipment and reagents, considerations of
time and cost
● Direct visualization through Wet Mount Treatment
○ Using differential interference contrast (DIC) ● Infected individuals with a healthy immune system may recover from
■ Oocysts appear as small round structures similar to cryptosporidiosis without serious symptoms and treatment
yeasts ● Nitazoxanide
■ Does not autofluoresce ● Azithromycin
● Direct visualization through Modified Acid-Fast Stain
○ Oocyst walls stain light pink to bright red Prevention and Control
○ Resolving infections may have colorless oocysts
● Maximize the synergistic effect of multiple disinfectant and water
■ Called ghosts
treatment processes
○ Mature oocysts may have visible sporozoites
○ Reduces the risk of C. hominis in drinking water
● Direct Fluorescent Antibody Assay (DFA)
● Natural water and swimming pool water should not be swallowed
○ Highest combination of specificity and sensitivity
● Prevention of water contamination through proper feces disposal and
○ Gold standard
sanitation
○ Does not provide a stain that cannot be archived
○ Requires fluorescence microscope and test kits
■ Test kits are commercially available Toxoplasma spp.
● Safranin stain Information
○ Used to detect Cryptosporidium spp.
● Defined as a zoonotic infection caused by the parasite Toxoplasma
■ Must be confirmed by other diagnostic techniques
gondii
○ Oocysts often stain bright-reddish orange color
● Wide range of clinical manifestations in humans
○ Stain is usually advocated for Cyclospora spp. as
● Infective stages:
Cryptosporidium oocysts may not stain properly
○ Oocysts
● Trichrome stain
○ Tachyzoite
○ Inadequate for definite diagnosis as oocysts appear unstained
○ Bradyzoite
● Enzyme Immunoassay (EIA)
● Diagnostic stages (in humans and other intermediate shots)
○ Does not rely on microscopy
○ In humans and other intermediate shots
○ Highly sensitive and specific
■ Tachyzoite
○ Useful for screening multiple specimens
■ Bradyzoite
● Rapid Immunochromatographic Cartridge Assay
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○ In domestic and wild felids ■ Tachyzoites can also infect through organ
■ Oocysts transplantation
■ Tachyzoite ○ Bradyzoites reside in tissue
■ Bradyzoite ■ Bradyzoites can be transmitted through ingestion of
● Tachyzoites and Bradyzoites are the only stages found in humans infected tissue
● Tachyzoites localize in neural and muscle tissue ● Cysts
○ Further develops into tissue cysts bradyzoites ○ Toxoplasma cysts range in size from 5 to 50 um in diameter
● Routes of infection include: ○ Appear spherical in the brain
○ Ingestion of undercooked meat ■ Appears elongated in cardiac and skeletal muscles
○ Consuming food or water contaminated with cat feces or ○ Commonly found in the brain and skeletal and cardiac
environmental samples that were in contact with cat feces muscles but can be found in various sites around the body
○ Blood transfusion, organ transplantation, and transplacental ● Sporulated oocysts
infections are possible ○ Only shed in the feces of domestic and wild felids—the
definitive hosts
Diagnosis ○ Sexual reproduction occurs in the felid intestinal epithelium
○ Sporulation takes about 48-72 hours and becomes infective
● Life Cycle
○ Mature oocysts contain two sporocysts and measure at about
○ Oocysts are passed out in the feces of the cat
10-12 um in diameter
■ Unsporulated
○ Human infection occurs through the ingestion of sporulated
○ Oocysts are ingested by the host through contaminated food
oocysts or trophozoite-infected meat
or water
● Tachyzoites
○ Oocysts migrate to the intestines of the host and releases
○ Tapered anterior end; blunt posterior end
four sporozoites
○ Contains a large nucleus
○ Sporozoites migrate to the lymphatic system and other organ
○ May be found in various sites throughout the body
tissues and fluids
○ Sporozoites transform into tachyzoites
○ Tachyzoites multiply and give rise to bradyzoites Diagnosis Techniques
■ Bradyzoites are slow-multiplying ● Microscopy
■ Tachyzoites can be transmitted human-to-human ○ Tissue samples are prepared and stained with Giemsa or
through granulocyte blood transfusion Hematoxylin and Eosin stains
■ Tachyzoites can pass through the placental barrier in ● Serodiagnostics
pregnant women ○ Used to detect antibodies against T. gondii in the blood serum
● Polymerase Chain Reaction
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○Successfully used in the diagnosis of toxoplasmosis using ■ Dominant species in the Philippines accounting to
samples from the patient 70% of the cases
■ Serum ■ Most virulent and have the highest rate of mortality
■ Amniotic fluid ■ Exhibits resistance to antimalarial medicines such as:
■ Cerebrospinal fluid ● Chloroquine, sulfadoxine-pyrimethamine, and
■ Bronchoalveolar lavage mefoxin
● Parasite Isolation from blood or bodily fluids ○ P. vivax
○ Intraperitoneal inoculation into mice ■ Causes 30% of malaria cases
○ Tissue culture ■ Mostly benign and rarely leads to mortality
○ P. malariae
Treatment ■ Found worldwide
■ <1% of the cases
● Pyrimethamine
■ Species known to only infect monkeys
○ 25-100 mg daily
● P. knowlesi
● Sulfadiazine
○ Species infecting monkeys but also
○ 1-1.5 g, 4x daily
recently caused human malaria
○ P. ovale
Prevention and Control ■ Not present in the Philippines
● Proper handwashing
● Protection from cat feces Diagnosis
● Meat and eggs must be well-cooked
● Life Cycle of Plasmodium spp.
● Usage of gloves when gardening
○ Involves two hosts:
● Cat litters should be changed daily
■ Human
■ Biological vector (mosquito)
Plasmodium spp. ○ Mosquito takes a blood meal and injects sporozoites
Information ○ Once bloodstream is infected, sporozoites infect liver cells
○ Sporozoites develop into schizont and contain merozoites
● Causes Malaria
○ Schizonts burst and release merozoites
○ Parasitic disease contracted through mosquito bites
○ Merozoites enter bloodstream and infect RBCs
(Anopheles spp.)
○ Infected RBCs are infected with immature trophozoites called
● Species of Plasmodium infecting humans
the ring stage
○ P. falciparum
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

■ Mature trophozoite goes into the erythrocytic cycle ■ Shade

and turn into schizont again, continuing growth and ■ Exposure to sunlight
multiplication cycle ○ Each Anopheles species has its
■ Develop into gametocytes which the mosquito ingests specific requirements
after taking a blood meal
○ Gametocytes fuse and form zygote in the mosquito and Diagnostic Techniques
develops into an Ookinete in the gut of the mosquito
● Basic Guidelines for Malaria
○ Ookinete develops into oocysts which proliferates in the
○ Capillary blood obtained by fingerstick or venous blood
salivary gland of mosquito and ruptures as it takes a blood
through venipuncture
meal, injecting the sporozoites into the bloodstream
○ At least two thick and thin blood smears
● Transmission of Malaria
■ Thin spear is for species-level identification
○ Plasmodium is transmitted by a female Anopheles mosquito
■ Thick smears are used for detection of the presence
bite
of parasites
■ Malaria transmission depends on the interrelationship
○ Gold standard for diagnosis is Microscopy
of 3 factors
○ Giemsa stain is most ideal for determination of certain
● Agent (Plasmodium)
morphologic features
○ Plasmodium needs to infect humans
○ Plasmodium immature trophozoite rings
● Host
■ Delicate cytoplasm with one or two small chromatin
○ Young age, malnutrition, and
dots; RBCs are enlarged and multiple infection are
pregnancy increase susceptibility to
prevalent in P. falciparum infections (P. falciparum)
infection
■ RBCs can be normal or enlarged; rings have large
○ Anopheles flavirostris
chromatin dots and cytoplasm may look amoeboid (P.
● Natural environment
vivax)
○ Influences Anopheles environment and
○ Plasmodium mature trophozoite
density
■ Rarely seen in peripheral blood smears; May appear
○ Factors that define the suitability of
slightly amoeboid in shape; More compact cytoplasm
breeding for Anopheles
(P. falciparum)
■ Altitude
■ Amoeboid cytoplasm with large chromatin dots (P.
■ Topography
vivax)
■ Rainfall
○ Plasmodium schizonts are rarely seen except in severe cases
■ Humidity
■ Temperature
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

■ When seen, contains 8-24 merozoites; Mature ● Personal protection against bites
schizont usually fills about ⅔ of the infected RBC (P. ● Chemoprophylaxis
falciparum) ● Prophylactic drugs
■ Large schizonts with 12-24 merozoites, with one
chromatin dot (P. vivax) Babesia spp.
○ P. falciparum gametocytes are crescent or sausage shaped
■ Diffuse chromatin are microgametes Information
■ Compact chromatin are macrogametes ● Causes Babesiosis
■ Gametocytes can undergo exflagellation ○ A hemolytic disease
● Commonly found in mosquito gut ○ Also known as Tick/splenic/redwater/Texas cattle/Nantucket
○ P. vivax gametocytes are round to oval fever
■ Microgametocytes are the same size as infected RBC ● Babesia spp.
with pale blue cytoplasm ○ Intraerythrocytic and heteroxenous parasites causing
■ Macrogametocytes are round to oval and usually fill babesiosis
the host cell, brown cytoplasm ○ Exhibits host specificity
○ Infects humans and animals
Treatment ■ Humans commonly infected by Babesia microti
● Treatment of Malaria per Phase of Infection
○ Prophylactic Diagnosis
■ Prevents establishment of parasite in the liver ● Life Cycle
○ Blood Schizonticidal ○ Tick makes a blood meal and introduces sporozoites into the
■ Attacks parasites in the RBC host mouse
○ Gametocytocidal ○ Sporozoite in mouse undergoes budding into merozoite and
■ Destroys sexual forms of the parasite gametes
○ Hypnozoitocidal ○ Gametes are ingested by tick and are fertilized in the tick gut
■ Kills dormant form of parasite in the liver ○ Fertilized zygote turn into ookinete and enters the salivary
○ Sporonticidal gland to develop into sporozoites
■ Inhibits development of oocysts in mosquitos ○ Sporozoites are injected into the human host which develops
into trophozoite and merozoites
Prevention and Control ○ Human to human transmission is done through blood
transfusion
● Control of breeding sites of Anopheles flavirostris
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

Diagnostic Techniques Treatment

● Diagnostic Methods for Babesiosis ● Combination of clindamycin and quinine or azithromycin and
○ Direct microscopy atovaquone
■ Giemsa-stained blood smear ● Antipyretics
■ Must rule out misdiagnosis of malaria ● Vasopressors
○ Pleomorphism of Babesia
■ Ring-form Prevention and Control
■ Pear-shaped form
● Avoid tick infested areas
■ Tetrad form
● Immediate removal of attached ticks
○ Immunofluorescent Assay Test
● Bug repellents
■ Widely used in acute cases and epidemiological
● Control of rodent population
studies
■ Drawbacks
● Non Differentiation of active and past Notes
exposure
● Malaria is caused by Plasmodium spp. and has to be examined
● Possibility of cross reactivity between
immediately as it is prone to degradation outside the body
antigens of closely related species
● Microfilariae is the diagnostic stage of filarial nematodes
● Subjective quantification
● Preferred anticoagulant - EDTA
○ PCR
■ The gold standard for Babesia detection
● Highly specific Parasitic Platyhelminthes
● Expensive and time-consuming
● Requires DNA extraction and gene sequencing
for strain comparison Clonorchis sinensis
○ ELISA ● Found across Asia
■ Less subjective but specific ○ Also known as Chinese liver fluke
■ Needs larger amounts of antigen samples ● Causes clonorchiasis
■ Less practical in the field ○ A liver fluke parasite infection
○ Immunochromatoraphic test ● Life cycle
■ Simple, quick, reliable, sensitive, and inexpensive ○ Different organisms involved
diagnosis for acute and latent Babesia infections ■ Snail is the intermediate host; Bithynia sp.
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

■ Freshwater fish is the definitive host; C. sinensis ● Prevention and Control

encysts in fish skin ○ Health education for the promotion of cooked fish
■ Human ingests cyst from fish; C. sinensis excysts and ○ Stool examination and treatment of positive cases
develops to adult form; defecates embryonated egg ○ Make fish safe for human consumption through irradiation
which will be ingested by the snail
● Embryonated egg develops Fasciola spp.
○ Miracidia - sporocyst - rediae -
● Causes Fascioliasis
cercariae
○ Fasciola hepatica (Temperate liver fluke)
● Snail intermediate hosts
○ Fasciola gigantica (Tropical liver fluke)
● Fish intermediate hosts
● Life cycle
○ Family Cyprinidae
○ Ruminants are the definitive hosts containing eggs; eggs are
● Diagnosis
excreted with the feces
○ Egg
■ When eggs are in contact with water, it becomes
■ 27-35 x 11-20 micrometers
embryonated
■ Oval shaped convex operculum found on shoulders
○ Snail intermediate host ingests embryonated egg
■ Abopercular knob—a small knob or hook-like
■ Develops into
protrusion
sporocysts-rediae-cercariae-metacercaria
○ Adult
■ Snail host of F. hepatica
■ Flattened and lanced shape
● Amphibious and found in the mud
■ 10-25 x 3-5 mm in size
■ Snail host of F. gigantica
■ Oral and ventral suckers (acetabulum) are small
● Aquatic snails living in slow-flowing water
● Diagnostic methods
■ Snail hosts in the Philippines
○ Cholangiography
● L. philippinensis
■ Radiographic examination of the bile duct
● L. auricula rubiginosa
○ Immunodiagnosis
○ Vegetation is the secondary intermediate hosts
■ ELISA
■ Cercaria attaches to vegetation
■ PCR
■ Ipomea obscura
■ EIA
■ Watercress
● Treatment
○ Vegetation is ingested by ruminants, herbivores, or humans
○ Praziquantel
■ Metacercaria develops into adult form; adult is a
○ Albendazole
macro specimen
○ Praziquantel-albendazole
■ Adult produces eggs
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

● Diagnosis ○ Egg becomes embryonated when in contact with water

○ Egg identification in stool and duodenal contents/bile through ○ Embryonated egg develops into sporocysts-rediae-cercaria
microscopy ○ Cercaria infects a crustacean
■ Parasite eggs however are not found in the feces until ○ Human gets infected after ingesting infected crustacean
3 to 4 months after infection
■ Low sensitivity in low intensity infections Schistosoma japonicum
■ Eggs are ellipsoidal and operculated measuring 120 to
● Cercariae are bifurcated
150 micrometers long by 60 to 90 micrometers wide
● Mode of transmission is skin penetration
○ Adult worms through surgical exploration
● Life cycle
○ Immunodiagnosis
○ Copulated adults and eggs migrate to the human rectum and
■ Elisa
gets excreted with the stool
■ Western blot
○ Eggs hatch and develop into miracidia
● Confirms the protein bands from the sera of
○ Miracidia penetrates snails—intermediate host
animals that are positive for Fasciola
■ Miracidia develops into sporocytes in the snail
○ Radiological examinations
○ Sporocytes develop into cercariae and are released into the
■ Sonography
water
● More useful and easily available method
○ Cercariae (free-swimming) penetrates human skin and
■ CT Scan
becomes schistosomula
● Treatment
○ Triclabendazole
○ Bithionol Taenia saginata/solium
■ Not commercially available ● Resides in pig/cow muscles
● Control and Prevention ● Also called tapeworm
○ Thorough washing and cooking of vegetables and boiling ● Depending on what is ingested:
water in areas where infection is endemic ○ Causes taeniasis or cysticercosis
○ Elimination os snail intermediate host ● Life cycle
○ Vaccination of animals ○ Egg or gravid proglottids passes into the environment through
infected feces
Paragonimus westermani ○ Oncosphere hatch and penetrate intestinal walls and migrate
to muscles
● Also called lung fluke
○ Oncospheres develop into cysticerci
● Clinical manifestation is similar to tuberculosis
■ Can develop in the eyes, brain, or the muscles
● Life cycle
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

○ Oncospheres are ingested through infected meat and ○ Female

develops into adult form in humans ■ Pointed, long, slender posterior end
○ Adult parasite attaches itself (through scolex) in the intestine
○ Adults in the intestine produces eggs which are then excreted Ancylostoma duodenale / Necator americanus
through the feces
● Caused by Ancylostoma duodenale and Necator americanus
● Eggs hatch into infective larvae when placed into soil
Parasitic Nematodes ○ Rhabditiform larvae
○ Filariform larvae
● Larvae infects through skin penetration
Trichinella spiralis
● Adult worms
● Causes the disease trichiniasis ○ Male
○ Caused by infection of Trichinella spiralis ■ Anterior end shows cutting teeth in A. duodenale
○ Ingested from cyst-infested undercooked meat, specifically ■ Anterior end shows cutting plate in N. americanus
from skeletal muscles ○ Female
○ Diagnostic stage are encysted larvae

Trichuris trichiura
Enterobius vermicularis
● Embryonated egg is the diagnostic stage
● Humans are the only hosts ● Embryonated egg develops further in the soil
● Infection happens via self-inoculation through the fecal-oral route or ● Infection is through the ingestion of embryonated eggs
aerosol ● Anterior end of adults contains whip-like structures
● Eggs are found in perianal folds; may also become airborne ● Eggs are barrel-shaped, like a lemon, and contains polar plugs on
● Diagnostic methods are fecal sample (very rare) or scotch tape test each end
● Highly contagious ● Adult form
● Eggs are ingested by humans and develop to larva after ingestion ○ Male
● Both sexes may have the same anterior end morphology ■ Posterior end is coiled
● Gravid female migrates to perianal region at night to lay eggs and die; ○ Female
males die after mating ■ Posterior end is straight
● Adult worms
○ Male
■ Anterior end contains the esophagus divided into Capillaria philippinensis
muscular and bulbous portions ● Causes intestinal capillariasis caused by the nematode Capillaria
■ Blunt posterior end philippinensis endemic to the Philippines
LBBBI18 - Medical Parasitology Lab Ray Paolo Montes
Ms. Joyce de Guia BS BioMed

● Embryonated thick shelled eggs are passed in the feces and develop
in the water.
● Larva infects through the ingestion of raw, infected, undercooked fish
and lives in the intestines