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 • Eye Scrapings, Skin Scrapings
• Aspirates (Duodenal, Liver) • Biopsy Specimens
• Blood, Buffy Coat and Lymphatic Fluid • Other body fluids (i.e. CSF, Vaginal Fluid, Amniotic Fluid)
• Urine

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) PROTOZOANS (SARCODINA)

a. Phylum Sarcomastigophora Taxonomy of Protozoans
• Subphylum Sarcodina – Ameba Kingdom Protista
• Subphylum Mastigophora – Flagellates • Phylum Sarcomastigophora
b. Phylum Ciliophora – Ciliates o Subphylum Sarcodina
c. Phylum Apicomplexa o Subphylum Mastigophora
• Class Sporozoa • Phylum Ciliophora
• Suborder Haemosporina • Phylum Apicomplexa
d. Suborder Eimeria • 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

STAGES OF DEVELOPMENT
• Trophozoite • Cyst

a. Subphylum SARCODINA Sarcodina

• Possess Pseudopodia for locomotion
• Inhabit the large intestine except E. gingivalis • Pathogenic: Entamoeba histolytica
• All undergo encystation except E. gingivalis • Commensals:
• All undergo Binary Fission as mode of o E. dispar
reproduction o E. moshkovskii
• All are commensals except E. histolytica 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 E. histolytica E. coli
Trophozoite
Movement Unidirectional Sluggish;
One at a time on an explosive Not progressive;
manner Several at a time but slow
Pseudopodia Finger- like Blunt; Rounded

Nucleus of Trophozoites Mononucleated Central Mono Eccentric karyosome

and location of karyosome karyosome
CYST
Inclusions Red Blood Cells Bacteria, yeast, debris
(hematophagous)

Cytoplasm Clean looking Dirty looking

No. of Nuclei (Cyst) 1-4 or 4 1-8 or more than 4

Chromidial bars/ bodies Sausage Broom stick needles;

shaped; rod-cigar shaped; coffin splintered glasses;
with rounded ends witch broom
Peripheral chromatin Fine and evenly distributed Irregular, clamp, coarse

E. histolytica
PATHOGENESIS Mechanisms for virulence: 1. Asymptomatic – majority of
• production of enzymes cases
or other cytotoxic • Excrete cysts
substances 2. Intestinal Disease
• 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
State – majority of Clinical Forms of Intestinal Amebiasis
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 • Ova and Parasite Examination

DIAGNOSIS o DFS
o Concentration Techniques
o Permanent Stained Smear (Iron Hematoxylin or Trichrome Stain)
▪ Charcot Leyden crystals
 • 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 • 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 • Metronidazole: Tx of acute amebic colitis

• Diloxanide Furoate: Asymptomatic Cyst Carriers
• Iodoquinol

PREVENTION • 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)

6. 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

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 • Cyst
• Trophozoite – infective stage
Ameba – feeding form
Flagellate – swimming form

Entry into the body olfactory epithelium, respiratory tract, skin and sinuses
Disease Primary Amebic Meningoencephalitis
Manifestation and • VERY FATAL
Pathology • Risk Factor: Swimming in contaminated pools,
lakes and rivers

Pathogenic PRESENCE OF Amebostomes

determinant

Diagnosis • Wet Mount Examination of CSF

• Smears stained with Wright’s or Giemsa
• Biopsy
• Culture
• Molecular Methods

Treatment and Amphotericin B with Clotrimazole

Prevention

2. Acanthamoeba spp.
• Free-living Ameba
• Characteristic Feature: Presence of acanthapodia
• Morphologic Forms:

Cyst Trophozoite

Acanthamoeba
Disease • Causes Granulomatous Amebic Encephalitis
Manifestation and Chronic; slow in progression
Pathogenesis Poor Prognosis
Disease Amebic Keratitis
Manifestation • Keratitis, uveitis, Corneal Ulcerations
• Implicated among contact lens users
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
 CILIATES

1. Balantidium coli
• Largest protozoan parasite affecting humans
• Disease: balantidiasis, balantidiosis, or balantidial dysentery
• Normal host : pigs, man • MOT: Ingestion of Cysts
• Habitat: Colon (cecum)

BALANTIDIUM COLI
PATHOGENESIS • Attacks intestinal epithelium Balantidiasis has 3 forms of clinical
• Ulcers: rounded base and wide neck manifestations:
• Ulcerations are due to hyaluronidase 1. Asymptomatic
• Can spread to extraintestinal sites 2. Fulminant balantidiasis/balantidial
• Complications/fatal cases include intestinal dysentery
perforations, intestinal hemorrhage, shock, sepsis, 3. Chronic form
and acute appendicitis
DIAGNOSIS • DFS
• Concentration techniques
• Biopsy
• Bronchoalveolar washings
TREATMENT • Tetracycline or metronidazole
• Alternative treatment: doxycycline and nitazoxanide
EPIDEMIOLOGY Prevalent in:
• areas with poor sanitation
• abbatoir, farms (close contact w/ pigs)
• overcrowded institution
• Warm/humid climates
PREVENTION & • Proper sanitation
CONTROL • Safe water supply
• Good personal hygiene
• Proper food preparation
• Cysts are easily inactivated by heat and 1% sodium hypochlorite

FLAGELLATES

PARTS

• Axoneme SUBPHYLUM MASTIGOPHORA

• Axostyle (Class Zoomastigophora)
• Costa 1. Intestinal and Urogenital
• Cytostome Flagellates
• Parabasal Body/Median Body 2. Hemoflagellates
• Undulating Membrane
• Ventral Sucking Disk

A. INTESTINAL/UROGENITAL FLAGELLATES

Generalities

• All inhabit the large intestine except Giardia lamblia, Trichomonas vaginalis,
Trichomonas tenax
• All undergo encystation except Trichomonas species
• All are commensals except Giardia lamblia, Dientamoeba fragilis and
Trichomonas vaginalis

1. Giardia duodenalis
• Other name: G. intestinalis, G, lamblia, Cercomonas intestinalis
• MOT: Ingestion of infective cysts
• Causative agent of Giardiasis, Traveller’s Diarrhea, Backpacker’s Diarrhea,
Beaver Fever
 GIARDIA DUODENALIS
PATHOGENESIS • Parasite attaches to the intestinal cells via an • Low Infective Dose
adhesive sucking disc located on its ventral side • Alteration of the mucosal lining
• Once attached, the parasites cause villous • Ventral Sucker
flattening and crypt hypertrophy (Malabsorption • Lectin
and Maldigestion) • Presence of VSPs
• Half of the patients may be asymptomatic
• Acute cases: excessive flatus with rotten egg (due
to hydrogen sulfide) odor, diarrhea, malaise,
abdominal pains, anorexia
• Chronic cases: steatorrhea, weight loss, profound
malaise, low-grade fever
GIARDIASIS Giardiasis is different from amebiasis and bacterial dysentery:
DIFFERENTIAL • No mucus, blood or PMN in stool
DIAGNOSIS • No granulocytosis and no fever

GALLERY

DIAGNOSIS • DFS
• Concentrations techniques : cysts
• Duodenal-jejunal aspiration
• Enterotest
• Antigen detection test (CWP1)
• Direct fluorescent Ab test

TREATMENT • Metronidazole
• Alternative drugs : tinidazole, furazolidone, abendazole
EPIDEMIOLOGY • Prevalent worldwide
• Areas with poor sanitation and poor hygiene
• Direct oral-anal sexual contact among MSM increase the risk
• Zoonotic disease
• Reservoir host: beavers
PREVENTION & • Proper or sanitary disposal of human excreta
CONTROL • Normal water chlorination will not affect cysts, but usual water treatment modalities should be
adequate.

2. Chilomastix mesnelii

Cyst
 Chilomastiz mesnelii
• MOT: ingestion of cysts in food and drinks
• Diagnosis: DFS
• No treatment is indicated (commensal parasite)
• Preventive and control measures: improved sanitation &
personal hygiene

Notes:

3. Dientamoeba fragilis
• Originally described as ameba
• Trophozoite and Cyst (newly discovered)
• 1 or 2 nuclei
• Nuclear membrane has no peripheral chromatin
• Karyosome: 4-6 discrete granules
• MOT: fecal-oral route or via transmission of helminth eggs (eg E.
vermicularis)

DIENTAMOEBA FRAGILIS
PATHOGENESIS • Usually asymptomatic
• Common symptoms: intermittent diarrhea w/ excess mucus
DIAGNOSIS • DFS
• Not detected by concentration techniques
• Prompt fixation of fresh stool w/ PVA fixative or Schaudinn’s fixative has been helpful

4. Trichomonas vaginalis
• Causes trichomoniasis
• Habitat: Urogenital Area
• MOT: Intimate Contact; Infant Delivery;
Contaminated Towels and Underwear

TRICHOMONAS VAGINALIS
PATHOGENESIS • Common symptoms: vaginal discharge (frothy green/yellow color), vulvitis, dysuria, postpartum
endometritis
• Lower abdominal pain
• Atypical Pelvic Inflammatory Disease
• Strawberry Cervix 2% of the cases)
• Males: asymptomatic, some cases:
• non-gonoccocal urethritis, epididymitis, and prostatitis can occur.
 • May also cause neonatal pneumonia
VIRULENCE • Binding to vaginal epithelial cells using ADHESINS
FACTOR • Immune evasion – surface coating with host proteins; shedding of parasite proteins,
• Cysteine Proteinases
• Cell Detaching Factor – cytopathic effect
• Alkaline pH
DIAGNOSIS • Saline preparation of vaginal fluid
• Culture: gold standard
(Diamond Modified Medium, Feinberg Whittington, Cysteine
Peptone Liver Maltose, Simplified Trypticase Serum Semen
Culture)
• Pap’s smear
• Ag detection test
• PCR
TREATMENT • Metronidazole
• Tinidazole
PREVENTION & • Reduce the risk of exposure
CONTROL • Proper use of protective devices such as condoms and spermicidal foams
• Simultaneous treatment of infected sexual partners
• Health and sex education

B. NON-PATHOGENIC FLAGELLATES
• Pentatrichomonas (Trichomonas) hominis
• Trichomonas tenax
• Chilomastix mesnili

1. Trichomonas hominis 2. Trichomonas tenax

BLOOD AND TISSUE FLAGELLATES

• Flagellates that are found in the blood and other fluids (CSF) and in tissues
• Vector borne parasites
• Medically important genera
o Trypanosoma
o Leishmania

GENERALITIES

• Only Trypanosoma and Leishmaniainfect humans

• Transmitted by a bite of an infected vector
• There are four morphological forms
o Amastigote – (Donovan Leishman)
o Promastigote – (Leptomonas)
o Epimastigote – (Crithidia)
o Trypomastigote
 • All forms are found in Trypanosoma cruzi infections
• Only the epimastigote and trypomastigote are seen in Trypanosoma brucei infections
• Only the amastigote and promastigote are seen in Leishmania infections
• Diagnostic Stages (found in humans) include amastigote and/or trypomastigote

1. Trypanosoma cruzi
• Causative agent of Chagas disease or American
trypanosomiasis
• Habitat: RES, cardiac muscle, CNS
• Intermediate host Vector: Reduviid Bug, (kissing bug)
• MOT: Feces of vector entering bite wound; blood
transfusion, organ transplants; transplacentally
• Exhibits all 4 stages of development
• Humans
o Trypomastigotes: bloodstream
o Amastigotes: tissue cells
• Vector
o Amastigote, Epimastigote, Promastigote:
midgut
o Metacyclic trypomastigote: hindgut
• Humans
o Trypomastigotes: bloodstream
o Amastigotes: tissue cells

TRYPANOSOMA CRUZI
PATHOGENESIS Acute phase Chronic phase
Non specific S/S: fever, malaise, nausea, • Enlargement of vital organs
vomiting, lymphadenopathy, cutaneous (myocardium, megaesophagus)
inflammation
• Chagomas
• Romaña’s sign
GALLERY

Chagas dx: Megacolon

Chagas dx: Megacardium
DIAGNOSIS ACUTE PHASE CHRONIC PHASE
• Thick and thin blood smear using Giemsa • ELISA
• CSF, tissue samples, lymph examination • Indirect hemagglutination
• Concentration methods • Indirect immunofluorescence
• Blood culture : Chang’s , NNN (Novy- • PCR
MacNeal Nicolle
Medium)
• PCR
• Xenodiagnosis
TREATMENT ACUTE PHASE CHRONIC PHASE – symptom specific management
• Nifurtimox • Pacemakers
• Benznidazole • Antiarrhythmic drugs (amiodarone)
 • Megasyndromes are managed w/ special diets,
laxatives, surgery
EPIDEMIOLOGY • Most cases : Latin America
• Other cases reported in Mexico, USA,Canada, France, Switzerland, Japan, and Australia
PREVENTION & • Vector control
CONTROL • Blood transfusion regulations
• No vaccines available

2. Trypanosoma brucei complex is the etiologic Agents of African Sleeping Sickness

a. Trypanosoma brucei rhodesiense causes Rhodesian or East African Sleeping Sicknessand Endemic in East and South
Africa
b. Trypanosoma brucei gambiense Causes Gambian or West African Sleeping Sickness and Endemic in West and Central
Africa
c. Trypanosoma brucei brucei affects wild and domestic animals

Vector: Tsetse fly (Glossina sp.)

Only epimastigote and trypomastigote are exhibited

Trypanosoma brucei morphological forms

• Epimastigote (crithidial form) in the insect
• Trypomastigote (trypanosomal form) in the mammalian host
Vector Transmitted Parasitic Infections
• Vector: Tsetse Fly (Glossina spp.)
o T. b. rhodesiense – G. pallidipes, G. morsitans
o T. b. gambiense – G. palparis
• Infective Stage to Humans: Metacyclic Trypomastigote

TRYPANOSOMA BRUCEI COMPLEX

PATHOGENESIS Initial lesion: local, painful, pruritic, Late HAT
erythematous chancre • Meningoencephalitic stage
Early HAT • Neurologic symptoms
• Hemolymphatic stage • Convulsions, tremors, speech and reflexes defects,
• Fever, joint and muscle pain, malaise paralysis
• Winterbottom’s sig • Kerandal’s sign
• Death
GALLERY

DIAGNOSIS • Demonstration of trypomastigote in chancre, lymph node aspirate, CSF

 • Thick and thin blood smear (Giemsa)
• Buffy coat concentration
• ELISA
• IFA
• Indirect hemagglutination
• Mini-anion exchange centrifugation technique
• PCR
• CATT
• Animal Inoculation and Culture
TREATMENT • Intravenous suramin solution: first stage of the disease
• Intravenous melarsoprol: if w/ CNS involvement
PREVENTION & • Vector control
CONTROL • Protective clothing
• Regulation and treatment of reservoir host (cattle and game animals)

3. Leishmania spp
• Divided into:
• Old World: L. tropica, L. aethiopica, L. major
• New World: L. mexicana, L. amazonensis, L. guyanensis, L. braziliensis, L. chagasi
• Vector-borne
o Old World: Phlebotomus sandfly
o New World: Lutzomyia
• Obligate intracellular parasite
• Primarily a zoonotic disease

A. Leishmania tropica
Habitat: Endothelial Cells of skin capillaries; phagocytic
monocytes
Vector: Phlebotomus papatasii; P. sergenti
B. Leishmania braziliensis
Habitat: Mucocutaneous junctions (nasal septum, mouth,
pharynx)
Vector: Phlebotomus peruensi; P. verrucarum
C. Leishmania donovani
Habitat: Endothelial Cells of the RES
Vector: Phlebotomus argentipes
• Phlebotomus
• MOT: congenitally, blood transfusion, contamination of bite wounds, direct contact with contaminated
specimens
• Infective stage: promastigote

Leishmania morphology

• Amastigote (leishmania) seen in the mammalian host

• Promastigote (leptomonad) seen in sand fly

TRYPANOSOMA BRUCEI COMPLEX

PATHOGENESIS 4 categories Cutaneous Leishmaniasis (Old World
• Cutaneous Leishmaniasis (CL) Leishmaniasis, Aleppo Button, Delhi Boil, Baghdad
• Diffuse Cutaneous Leishmaniasis (DCL) Boil, Jericho Boil)
• Mucocutaneous Leishmaniasis (MCL) • Etiology: Leishmania tropica
• Visceral Leishmaniasis (VL) • Disease Manifestation
• Elevated Skin Ulcers (Painless)
• Leaves an Ugly Scar; Highly Disfiguring
Mucocutaneous Leishmaniasis (American, New
World Leishmaniasis)
• Etiologic Agent: Leishmania braziliensis
• Disease manifestation
Spread to Oronasal and Pharyngeal Mucosa
• ESPUNDIA, TAPIR NOSE, CHICLERO ULCER
Visceral Leishmaniasis (VL)
• L. donovani complex (L. donovani, L. chagasi, L.
infantum)
• Acute phase: may be mistaken for malaria
• Post- Kala-azar dermal leishmaniasis: cutaneous
eruption resulting in hypopigmented macules, malar
erythema, nodules, and ulcerations
GALLERY

DIAGNOSIS • Microscopic demo of Leishmania form lesions and Immunologic assays

tissue scrapings, aspirates, and biopsy • ELISA
• Culture: NNN medium, Schneider’s medium • rk39 Ag test (for VL)
• Animal inoculation (hamster) • Direct agglutination
• Montenegro skin test • Urine Ag test
• Flow cytometry
• PCR
• RFLP analysis

TREATMENT • Antimony compounds: IM or IV for up to 4 weeks; primary treatment

• IV Amphotericin B
• Antineoplastic drug miltefosine
• Pentimidine: second-line drug for CL & VL
• Combination therapy
EPIDEMIOLOGY • Primarily a disease of poverty
• VL is an important opportunistic infection in AIDS
PREVENTION & • Use of insect repellant (DEET & permethrin)
CONTROL • Insecticide-treated clothing
• Fine-mesh bed nets
• Screens and sprays in houses
• Regulation of reservoir hosts
APICOMPLEXA • Other S/S
o Anemia
Taxonomy of Protozoans
o Splenomegaly
Kingdom Protista
o head ache
• Phylum Sarcomastigophora o body pains
o Subphylum Sarcodina o nausea
o Subphylum Mastigophora o vomiting
• Phylum Ciliophora o pallor
• Phylum Apicomplexa • Occurrence of Relapse and Recrudescence
• Phylum Microspora (fungi) • Pathogenesis
o P. falciparum
GENERALITIES ▪ Presence of “sticky knobs” on infected
• Intracellular Protozoans RBCs (cytoadhesion)
• Possess Apical Complex - PfEMP-1 (most adhesive
• May require an intermediate host to complete the protein)
life cycle • Complications
• Undergoes both sexual and asexual reproduction o Cerebral Malaria
o Blackwater Malaria
Classification o Nephrotic Syndrome
• Present in Blood
o Plasmodium
o Babesia
• Intestinal Coccidians
o Partially Acid-Fast Intestinal Coccidians
• Tissue Coccidians (extraintestinal)
o Toxoplasma
o Sarcocystis

Plasmodium
Malarial Parasites
• Plasmodium falciparum
• Plasmodium vivax
• Plasmodium ovale
• Plasmodium malariae
• Plasmodium knowlesi – zoonotic

Generalities
• Intracellular Parasite
• Undergoes alternating sexual (sporogony) and
asexual stages (schizogony) in its life cycle
Epidemiology
• Vector-Borne Disease
o Female Anopheles minimus flavirostris – • As of 2017, there are 219 million cases worldwide
primary vector of malaria in the country (WHO Malaria Report)
o Most cases are still coming from: WHO
• Intermediate Host: MAN
African Region (92%)
• Habitat of Parasite: Liver and Red Blood Cells
• Widest distribution:
(Humans)
o P. falciparum (in Africa, SEA, Western
• Infective Stage to Mosquito: Gametocytes
Pacific Region)
• Infective Stage to Man (Transmission Stage):
o P. vivax (in Americas)
Sporozoites
• Common species of Plasmodium in the
• Mode of Transmission: Mosquito Bite; Blood
Philippines include:
Transfusion; Congenital
o P. falciparum- most prevalent
Malaria – remains the leading parasitic disease that
o P. vivax
cause mortality worldwide
• Endemic areas: As of 2009, 58 out of 80
• Classical Paroxysms – characteristic periodicity
provinces are endemic
o Chills (15-60 mins)
• High Endemicity: Palawan, Kalinga-Apayao,
o Fever (2-6 hours)
Ifugao and Agusan del Sur
o Sweating (2-4 hours)
Plasmodium knowlesi
• Malaria of Monkeys (macaques)
• Zoonotic Infection
• Important Aspects:
o Early Trophozoites similar to P. falciparum
o Other Morphologic forms are similar to P.
malariae
o 24 hour erythrocytic cycle
• Quite difficult to identify
• Molecular Methods are used to confirm the
diagnosis
• Endemic areas
o South East Asia

Laboratory Diagnosis
• Sample: Capillary Blood
• Gold Standard: Microscopy
o Preparation of Thick and Thin Smears
o Stain Used: Giemsa (pH 7.2)

Thick and Thin Smear

• Thick Smear
o Dehemoglobinize using water prior to staining
o Let it Dry
o Stain Immediately; NO
NEED TO FIX with
Methanol
• Thin Smear
o Fix with MEOH
o Stain with Giemsa
• Thick Smear – used for parasite count
• Thin Smear – used for species ID
 • Quantitative Reporting (Thick Smear) • Other drugs
o Quinine
o Primaquine
o Prophylactic Drugs – mefloquine,
Lab Diagnosis doxycycline, atovaquone/proguanil
• Other methods:
o Quantitative Buffy Coat Prevention and Control
o Rapid Diagnostic Tests • Early diagnosis and Treatment
o Serology (IFAT) • Personal protection measures (use of insecticide
o Molecular Methods treated nets, insect repellants)
o Culture • Use of Chemoprophylaxis
• Larvicides (use of B. thuringiensis)
Quantitative Buffy Coat (QBC) • Larviparous fish
• capillary tube with acridine orange stain • Health Education
• (+) bright green and yellow under fluorescence • Vaccine – no effective vaccine yet
microscope • Zooprophylaxis

Babesia microti / Babesia spp.

• Apicomplexans that cause malaria-like symptoms
• Zoonotic infection
• Tick (Ixodes) borne parasite
• Definitive Host: Tick (Ixodes)
• Intermediate Hosts: White footed
mouse, deer, livestock, cattle,
humans (accidental host)
• Deer – primary reservoir host
• MOT: Bite of an infected tick; blood transfusion;
vertical transmission
• Infective Stage: Sporozoite
RDTs
• (Immunochromatographic Mtds)
• Antigen detection
o HRP-II (Ag produced by P. falciparum
trophozoite and gametocytes)
▪ ex. Paracheck PF ; ParaHIT f
o pLDH (produced by viable parasites)
▪ -can distinguish Pf from nonPf species
▪ ex. Optimal Brand
• Other Panmalarial Ag such as Aldolase

Other Methods
• Serology
o ELISA
o IHA
o IFAT Disease Manifestation and Pathogenesis
• Molecular: PCR for low parasitemia and mixed • Diseases: Babesiosis / Piroplasmosis / Nantucket
infections Fever/ Redwater Fever/ Tick Fever/ Texas Cattle
• Culture: RPMI 1640 Fever
• Most cases are asymptomatic and usually self-
Treatment limiting
• Chloroquine: main stay treatment; • Signs and symptoms mimic malaria:
• Arthemether-based combination treatment (ACTs) o Mild chills and fever
such as Arthemether- Lumefantrine (Coartem) o Hemolytic anemia
o WHO recommended drug for falciparum o Jaundice
malaria o Hepatomegaly
o combination therapy o No malarial paroxysm
• Doxycycline – prophylaxis
Babesia morphology
• Similar to malarial parasite, but no
schizonts or gametocytes
• Up to four trophozoites per cell

Diagnosis
• Examination of Giemsa stained smears
• Serology
o IFAT
o Inoculation of Animals (Gold Hamster or Gerbil)
• History of Tick Bite
• Molecular Methods/PCR: gold standard

Treatment
• Oocysts
• Combination of Clindamycin and Quinine or
Azithromycin and atovaquone

Epidemiology
• Reported Cases in Europe and North America • IS: tachyzoites, bradyzoites and oocyst
(North East), cases also reported in West Coast
• Human Infections usually occur during spring and
summer
• No human infections reported yet in the country
• Parasite Stages found in humans
o Tachyzoites
▪ Rapidly multiply and infect cells of the
COCCIDIANS
intermediate hosts and non-intestinal
1. Toxoplasma gondii epithelial cells of cats
• Tissue Coccidians o Bradyzoites
• Found worldwide and can infect a variety of animals ▪ Multiply slowly;
including humans ▪ Develop mostly in neural and muscular
• Definitive Host: Members of the Felidae Family tissues
• Intermediate Host: Birds, Rodents, Pigs, Man ▪ May also develop in visceral organs
• Accidental or Dead-End Host: Humans
• Infective Stages: Oocysts and Tissue Cysts
• MOT:
o Ingestion of infected undercooked meat
o Consumption of food or water contaminated •
• Disease Manifestation and Pathogenesis
with cat feces
o Toxoplasmosis
o Blood transfusion or Organ Transfusion
o Usually asymptomatic in healthy individuals
o Vertical Transmission
o Immunocompromised
▪ Encephalitis is usually manifested
▪ Other manifestations include
retinochoroiditis, lymphadenopathy,
splenomegaly
o Congenital Defects
• Congenital Infections
o Stillbirth, abortion
o Triad of Toxoplasmosis
▪ Hydrocephalus
▪ Chorioretinitis
▪ Intracranial Calcification
o Microcephaly may also occur
• Toxoplasma gondii retinitis
 • Toxoplasma gondii hydrocephalus o Rare in humans
• Diagnosis and Treatment
o History of Recent ingestion of raw or
undercooked meat
o Muscle Biopsy – definitive diagnosis
o Stool Exam – detection of sporocyst
• Diagnosis ▪ Flotation Techniques
o Serology -usually done o PCR
▪ Sabin-Feldman Test – uses methylene o Treatment: Rarely Required (asymptomatic)
blue dye ▪ May use albendazole, metronidazole; co-
- Sensitive and Specific trimoxazole
▪ Enzyme Immunoassay, Hemagglutination o Prevention: Thorough Cooking of Meat;
Test, FAT Freezing of meat
▪ IgM antibody detection
▪ TORCH Testing Partially Acid-Fast Intestinal Coccidians
o Examination of Giemsa stained tissue sections • Inhabit the small intestine
or imprints • Infects humans as well as other hosts (animals)
o Examination of CSF smears, buffy coat smears • Undergo alternating sexual and asexual stages in its
o Molecular Methods life cycle
• Treatment • Implicated in outbreaks of diarrhea
o Pyrimethamine and Sulfadiazine • MOT: ingestion of oocysts
• Prevention
o Thoroughly cook meat; proper hygiene;  Cryptosporidium hominis
disinfect and clean daily cat litter pans; • Formerly known as Cryptosporidium parvum
pregnant women should avoid contact with • MOT: Oral-Fecal
cats • Infective Stage: Ingestion of Sporulated Oocyst
2. Other Tissue Coccidians: Sarcocystis • Habitat: Small Intestine (Jejunum)
• Intracellular protozoans infecting humans and
animals
• Major species affecting humans
o Sarcocystis hominis
o Sarcocystis suihominis
• IH: pigs and cattle Disease Manifestation and Pathology
• IS: Sarcocyst (containing mature sporozoites) • Healthy Immunocompetent Patients
o Self-Limiting Watery Diarrhea (5-10 frothy
bowel movements)

Immunocompromised patients
• Chronic Diarrhea and may have extraintestinal
infections
o Severe and life-threatening

• Disease and Pathogenesis

o Sarcosporidiosis or Sarcocystosis
▪ Forms
- Invasive form – rare
o Vasculitis and myositis
- Intestinal form
o Nausea, abdominal pain and
diarrhea
o Usually mild (48 hours)
 Cyclospora cayetanensis • Diagnosis
• Formerly known as cyanobacterium-like body o Concentration Techniques such as
• Maybe mistaken for Cryptosporidium Sheather’s Sugar Flotation, FECT
• Bigger in size (8-10 um) o Staining using Modified-Kinyoun Method
• Disease manifestation o Molecular Methods
o Intermittent Watery Diarrhea o Serologic Tests
o Development of D-xylose malabsorption ▪ NB: The consistency of the stool
o Usually self-limiting specimen is important; the more
• Diagnosis watery or diarrheic, better recovery of
o Same with Cryptosporidium oocysts
o Take note of the size of the oocyst (8-10 ▪ Size of Oocyst is also important (4-6
um); larger um) for Cryptosporidium

 Blastocystis hominis

 Cystoisospora belli

• Formerly classified as a yeast

• Formerly known as Isospora belli • Currently considered as a commensal of the GI
• Least common Intestinal coccicidian infecting tract
man • Classified Member of Stramenopiles
• MOT: Oral-Fecal • Zoonotic
• Infective Stage: Oocysts • MOT: ingestion of thick-walled cysts
• Disease Manifestation and Pathogenesis • Morphologic Forms
o Infects intestinal cells of humans o Classic Vacuolated Form (Central-Body
(duodenum) Form)
o Usually asymptomatic o Granular Forms
o May manifest as intermittent diarrhea that o Multivacuolar
can occur in months with fever, malaise, o Avacuolar
anorexia, abdominal pain and flatulence. o Ameboid Form
o Cyst