[NOTES] PROTOZOA (2nd Year BS Medical Technology) PDF

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CLINICAL PARASITOLOGYModule 5 - ProtozoaTranscribed by PORPION References: Canvas introduction, PowerPoint presentation, and video discussionTABLE OF CONTENTSIntroduction Taxonomy Locomotory Organ Distinctions Phylum Sarcomastigophora Subphylum Sarcodina ✓ Amoeba ✓ Commensal Amoeba Free-Living Amoeb...

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CLINICAL PARASITOLOGY Module 5 - Protozoa Transcribed by PORPION References: Canvas introduction, PowerPoint presentation, and video discussion TABLE OF CONTENTS Introduction Taxonomy Locomotory Organ Distinctions Phylum Sarcomastigophora ✓ Amoeba – Subphylum Sarcodina ✓ Commensal Amoeba ✓ Naeglaria fowleri Free-Living Amoebas ✓ Acanthamoeba spp. ✓ Other Free-living Amoeba ✓ Balantidium coli Phylum Ciliophora Phylum Sarcomastigophora ✓ Atrial Flagellates – Subphylum Mastigophora ✓ Giardia lamblia ✓ Trichomonas vaginalis ✓ Non-Pathogenic Atrial Flagellates ✓ Blood and Tissue Flagellates ✓ Trypanosoma spp. ✓ Leishmania spp. ✓ Pathogenesis of the Leishmania spp. Phylum Apicomplexa – ✓ Plasmodium spp. Plasmodium spp. and ✓ Life Cycle ✓ Pathology: Classical Babesia spp. Malaria Paroxysms ✓ Diagnosis ✓ Treatment ✓ Resistance to Malaria ✓ Plasmodium knowlesi ✓ Babesia spp. ✓ ✓ ✓ ✓ ✓

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INTRODUCTION Need a microscope to view, unlike metazoans The science of the study of the protozoa is called protozoology They are the first group of organized but simplest animals in the animal kingdom They are unicellular without any tissue grade of organization They generally multiply asexually • By binary or multiple fission, budding, and in some cases, sexual reproduction by conjugation. • May switch to sexual method or undergo cystic change to its host • Parasitic protozoa perform asexual and sexual reproduction in different hosts (alternation of generation) They are very minute and microscopic animals The structure of a protozoan “cell” consists of • Nucleus • Cytoplasm body o Differentiated into two parts Outer hyaline portion, the ectoplasm (locomotive, for protection, and for sensory purposes) Inner granular portion, the endoplasm (nutritive and reproductive function) Generally possess definite locomotory organs • Flagella, cilia, or pseudopodia • A contractile vacuole is present in many protozoans • Some form cysts in extreme conditions Can be free-living, parasitic, or symbiotic in nature Monogenetic protozoa • Require one host to complete their life cycle • Amoeba, intestinal flagellates, and B. coli

May adopt in a different host and transfer themselves passively in the cystic stage Digenetic protozoa • Require two hosts to complete their life cycles • Leishmania spp, Trypanosoma spp., and Plasmodium spp. o In malarial infection, man is the secondary/intermediate host, but the mosquito is the primary/definitive host o In trypanosomiasis, the tsetse fly is the intermediate host and man is the definitive host o In leishmaniasis, the sandfly is the intermediate host and man is the definitive host •

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TAXONOMY Kingdom Protista -> Subkindgom Protozoa -> Phlyum Sarcomastigaphora, Phylum Ciliophora, Phylum Apicomplexa, and Phylum Microscopa

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LOCOMOTORY ORGAN DISTINCTIONS Differences in locomotory organs • Subphylum Sarcodina has pseudopodium (false feet) • Subphylum Mastigophora has flagella • Phylum Ciliophora has cilia

PHYLUM SARCOMASTIGAPHORA – SUBPHYLUM SARCODINA All organisms under Subphylum Sarcodina are known as ‘amoebas’

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AMOEBA Have pseudopodium (hyaline foot-like protrusion for locomotion) All amoebas are commensal (non-pathogenic unless the host is immunocompromised) except for Entamoeba histolytica All are intestinal dwellers (specifically the large intestine) except Entamoeba gingivalis (found in the mouth) All undergoes encystation (trophozoite -> cyst stage which occurs in the large intestine) except for Entamoeba gingivalis and Dientamoeba fragilis Multiplies through binary fission (trophozoite) Life cycle (Process of encystation) 1. Trophozoite form 2. Pre-cystic form 3. Cystic form (with hyaline cystic wall) 4. Metacystic form

NOTE: Excystation is cyst -> trophozoite which occurs in the small intestine Entamoeba histolytica ✓ ✓ ✓

First described by Loesch in 1880 Direct life cycle Multiplies by binary fission

NOTE: Refer to the PowerPoint presentation for the life cycle Infective stage Mode of transmission Phoretic vectors Pathology Treatment

Cyst Ingestion of food/water with E. histolytica cyst Cockroach and flies Amoebiasis Metronidazole

Comparison of E. histolytica Trophozoite and Cyst Trophozoite Motile NOTE: Because trophozoites pseudopods

Cyst Non-motile only have

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Feeding stage Vegetative state Found in watery, soft, or semi-formed stool Fragile

NOTE: They are destroyed by gastric juices hence they are the non-infective stage Cannot encyst if excreted from body

Non-feeding stage Infective stage Found in soft to formed stool

NOTE: Due to its cystic wall which contains chitin and lectins (Jacob and Jessie lectins)

NOTE: Once the cyst is in the small intestine, it undergoes excystation which involves nuclear division. The nuclei of the cyst divides from 4 to 8 nucleus. After nuclear division, it undergoes cytoplasmic division. Excystation yield 8 trophozoites due to E. histolytica cyst being quadri-nucleated (it can have 4 nuclei). COMMENSAL AMOEBA Entamoeba coli Harmless inhabitant of the colon Entamoeba dispar

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The 1st image shows the I. butschlii cyst while not stained with iodine, the 2nd image shows the cyst stained with iodine

Resistant to acidic pH

NOTE: Encystation only occurs in the large intestine

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Morphologically similar with E. histolytica but with a different genome Distinguished from E. histolytica by PCR

Morphologic Comparison of E. histolytica and E. coli

Movement Pseudopodi a shape Release of pseudopodi a Nucleus

TROPHOZOITE STAGE E. histolytica E. coli Unidirectional, active Sluggish, nonprogressive progressive Finger-like Blunt Explosive/one at a time Uninucleated with central karyosome

Cycoplasm (endoplasm ) Inclusion

Clean-looking

Size

Bigger (1260micrometers)

RBCs

Slow manner/several at a time Uninucleated with eccentric karyosome Dirty-looking

Bacteria, yeast, cell debris Smaller (1550micrometers)

Image

Entamoeba hartmanni ✓ ✓ ✓

Similar with E. histolytica but smaller and has no RBC inclusion Known as the “small race” of E. histolytica Their cysts are quadrinucleated with coarse cytoplasm; immature cyst has chromatoidal bars

Entamoeba polecki ✓ ✓ ✓

Parasite of pigs and monkeys; rarely infect humans Distinguished from E. histolytica by Cyst Their cysts are consistently uninucleated

Entamoeba gingivalis ✓ Can be found in the mouth (gum and teeth surface) ✓ Abundant in cases of oral disease ✓ No cyst stage ✓ Transmitted through kissing, droplet spray, sharing utensils Entamoeba moshkovskii ✓ Morphologically similar with E. histolytica and E. dispar ✓ Classified as free-living amoeba (rarely infect humans) ✓ Osmotolerant ✓ Able to survive at temperature range of 0-41°C Endolimax nana ✓ ✓ ✓

Smallest protozoa Its trophozoite form is sluggish and mononuclear Its cyst form is quadrinucleated with eccentric karyosome (Cross-eyed cyst)

NOTE: The four morphologically similar amoebas are the E. histolytica, E. dispar, E. moshkovskii, and E. 2angladeshi.

Number of nuclei Chromatoid al bars Nuclear membrane Size Image

CYST STAGE E. histolytica Quadrinucleated (< or = 4) Cigar-shaped/rodshaped/Coffin-lod

E. coli > 4 (mature: 8)

Thin

Witchbroom/splintered/ needle-like Thick

10-20micrometers

8-35micrometers

Iodamoeba butschlii ✓ Has an iodine cyst (cyst which has affinity to iodine) ✓ Has a large glycogen vacuole/body which stains deeply ✓ with iodine ✓ Uninucleated

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o Entamoeba spp. stained blue Saline + Lugol’s Iodine o Only nucleus of E. histolytica cyst can be observed Concentration Techniques (FECT, MIFT) Culture: • To isolate E. histolytica • Robinson’s & Inoki medium • Balameth Monophasic medium • Boeck & Dorbolav’s Diphasic medium • TYS-S-33 Serologic Tests (ELISA, IHAT, CIE, AGD, IFAT) Liver aspirate biopsy • Checks for presence of E. histolytica trophozoite in case of extraintestinal infections Radiographic techniques (X-Ray, MRI, CT -Scan) Molecular Tests (PCR) • Differentiates morphologically similar amoeba •

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Pathogenesis (Amoebiasis) ✓ Intestinal amoebiasis • Most common pathology • Amoebic dysentery o Caused by E. histolytica o Differentiated from bacillary dysentery (caused by the Shigella dysenteriae bacteria) by the presence of Charcot-Leyden crystals • Amoebic colitis o Ulceration of intestine (cecum, rectosigmoid) o Caused by E. histolytica o The ulcer is flask-shaped

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NOTE: IHAT can detect past infections up to 10 years. In terms of Lugol’s iodine, only cysts can be observed because iodine destroys trophozoites due to their fragility Other Intestinal Amoeba

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Blastocystis hominis • No exact taxonomic classification (stramenophile) • Ovoid or spherical, vacuole-like structure • May cause diarrhea to some patient (immunocompromised) • Commensal

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Dientamoeba fragilis • Commensal • Ameboflagellate • No cyst stage • Rosette-shaped nuclei (12); no peripheral chromatin • Can be transported by pinworm to man • Chronic infection may mimic Irritable Bowel Syndrome

Occurs due to its motility Hepatic amoebiasis • Most common extraintestinal amoebiasis • Amoebic liver abscess has an anchovy sauce-like appearance Pulmonary amoebiasis Cerebral amoebiasis Amebic pericarditis Cutaneous amoebiasis Genital amoebiasis

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Pathogenic determinants Gal/Gal Nac Lectin • Galactose/N-acetyl galactosamine • Cytoadherence o Attachment to large intestine Caspase-3 • Causes apoptotic death of mucosal cells Myosin IB • Ingestion of RBC • Results in presence of RBCs in E. histolytica Amebapores • Pore-formers • Causes ulceration of tissues/cells of the host Cysteine proteinase • The most virulent factor • Tissue invasiveness o Leads to extraintestinal amoebiasis

Laboratory Diagnosis ✓ Direct Fecal Smear • Saline solution (0.85-0.95 NSS) o Trophozoite motility is observed • Saline + Methylene Blue

FREE-LIVING AMOEBAS

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Naeglaria fowleri Causative agent of Primary Amoebic Meningoencephalitis (PAM) Thermophilic organisms (up to 30°C) Multiplies through promitosis (intracellular mitosis) • The nuclear membrane doesn’t break down

Mode of Transmission Oral and intranasal routes while swimming in contaminated lakes, pools, and rivers Diagnosis ✓ ✓ ✓ ✓

Uses cerebrospinal fluid • Collected through spinal tap Culture • Non-nutrient medium with Page’s saline seeded with living Escherichia coli PCR ELISA

Ways to Eliminate ✓ 1mg/L chlorine solution ✓ pH 8-8.01 ✓ Place in 38°C water | 23-3-2021 |

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Morphologic Forms CYST

Uninucleated with rounded chromatoidal bars

TROPHOZOITE AMOEBOID FLAGELLATE D ✓ Only form ✓ With two recognized flagella at in humans broad end ✓ With single ✓ Spinning or pseudopod jerky motility ✓ Uninucleate d with large ✓ (directional) central ✓ Does not divide karyosome without peripheral nuclear chromatin

OTHER FREE-LIVING AMOEBA Balamuthia mandrillaris Pathology is similar with GAE

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CYSTS Spherical Three layered cystic walls (visible through electron microscopy) Two layers (visible through light microscopy)

TROPHOZOITES Finger-like pseudopodia

Sappinia diploidea ✓

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Acanthamoeba spp. Most common is A. castellani

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Granulomatous Amebic Encephalitis (GAE) • Destructive encephalopathy and meningeal irritation Amebic Keratitis (AK) • Associated with improperly disinfected contact lenses (when cleaned with tap water or impure lens solution)

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Mode of Transmission ✓ Intranasal ✓ Entrance of pathogen through cuts, wounds. Diagnosis ✓ Specimen • Discharges, exudates, tissue secretions. ✓ Culture • PYGC medium with antibiotics o Peptone yeast glucose cysteine o The antibiotics remove unwanted bacterial growth Morphologic Forms ✓ ✓

CYSTS Double-walled With ostioles (pores in between the cyst walls)

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TROPHOZOITES With large nucleus with centrally located nucleolus With acanthopodia (spiny-filaments or “thorn-like”) appendages for locomotion Replicates by mitosis

Newly recognized human pathogen causing amoebic encephalitis Trophozoite • With two nuclei attached together by connecting perpendicular filaments

PHYLUM CILIOPHORA Known as ciliates

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Balantidium coli Only member of the Phylum Ciliophora Largest intestinal protozoan

First name Former name New name Infective stage Reservoir host Mode of transmission Morphologic Forms CYSTS ✓ Ovoid, rounded with well- defined cystic wall (cilia enclosed in cystic wall) ✓ Binucleated

Paramecium coli B. coli Neobalantidium coli Cyst Pigs Ingestion of infective stage

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TROPHOZOITES “Thrown ball/Rolling” motility Ovoidal, bean-shaped Prominent cytostome (for food entry) Indented cytopyge (for waste exit) 2 nuclei Macronuclei (vegetative)(bigger/kidneyshaped) Micronuclei (reproductive)(smaller/roundshaped)

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Balantidiasis/Balantidial Dysentery/Ciliary Dysentery • Similar with Amoebic dysentery • Characteristic of the diarrhea is blood stool with mucous

Virulent Factors ✓ Hyaluronidase (lytic enzyme that causes ulceration) • Lytic enzymes cause cell destruction ✓ Ulceration (rounded base and wide neck) Laboratory Diagnosis ✓ Specimen used is stool ✓ Direct Fecal Smear ✓ Concentration techniques

Morphologic Forms ✓ ✓ ✓ ✓ ✓

Treatment Tetracycline or Metronidazole

Chronic infection is seen with steatorrhea (passage of fats in stool) • Fats are nonpolar so they float in water

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CYSTS Ovoid, football shaped Quadrinucleated (mature cyst) Young cyst has 2 nucleus Thick shell (doublewalled) Flagella retracted to axoneme (median body) Deeply stained curved fibril

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NOTE: Refer to the PowerPoint presentation for the life cycle Old name Other names

New name Habitat

Diagnostic stage Infective stage Mode of transmission Treatment

G. lamblia G. intestinalis, Lamblia duodenalis, L. intestinalis, Cercomonas intestinalis Giardia duodenalis Duodenum (where it performs excystation for ~30mins.), jejunum, and upper ileum Trophozoite and cyst Mature cyst Ingestion of infective stage Metronidazole

Pathology ✓ Giardiasis, Traveller’s Diarrhea, Gay-bowel syndrome ✓ Acute infection has a “rotten egg” odor of stool

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Flagella (locomotor apparatus) Kinetoplast (provides energy) • Blepharoplast • Parabasal Body Cytostome (cell mouth) • For food entry Undulating membrane (membrane laterally projecting from the body of certain flagellates, participates in active motility of the flagella) Axostyle/axial rod (supports locomotion) Costa (rib-like structure within the cytostome)

Giardia lamblia First described by Antoine van Leeuwenhoek using his own stool Multiplies by binary fission

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ATRIAL FLAGELLATES General Structure

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PHYLUM SARCOMASTIGOPHORA – SUBPHYLUM MASTIGOPHORA

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TROPHOZOITES Bilaterally symmetrical Pear/tear drop shaped, pyriform With Large ventral sucking disc (for attachment) “Old man with eyeglasses” 4 pairs of flagella - One pair – anterior - One pair – posterior - 2 pairs – central, extending laterally 2 nuclei with distinct karyosome (bilaterally symmetrical) Present axostyle with parabasal body (energy) “Falling-leaf motility” Multiplies by Longitudinal Binary Fission Covered with variantspecific surface proteins (VSPs)

NOTE: VSPs are surface antigens present around the trophozoite Diagnosis ✓ ✓ ✓ ✓ ✓ ✓

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Specimen used are stool and duodenal aspirates Stool Exam Duodenal Aspirates Beale String Test/Entero-Test Immunochromatography • For antigen detection in stool • Utilizes the VSPs Direct Fluorescent Antibody Assay • Gold standard for detection Trichomonas vaginalis The ONLY pathogenic Trichomonas spp. Most prevalent non-viral sexually transmitted infection No cyst stage Can be transferred from the pregnant woman to their child through the birth canal Multiplies by binary fission | 23-3-2021 |

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NOTE: Refer to the PowerPoint presentation for the life cycle Diagnostic stage Infective stage Mode of transmission Habitat

Treatment

Trophozoites Trophozoites Sexual intercourse ✓ Urogenital tract - Female (Vulva/vagina where pH is 5.2-6.4) - Male (prostate gland) ✓ Oral Metronidazole ✓ Acidic douche (10% vinegar)

Pathology ✓ Female (Trichomoniasis, Ping-pong disease) • Non-specific vaginitis • Itchiness, burning sensation • Vulvovaginitis • Greenish-yellow leukorrheic discharge • Inflammation of cervix (“strawberry cervix”) ✓ Male (often asymptomatic) • Non-specific prostatitis, UTI

“Boring/spiral” or “corkscrew” motility

Retortamonas intestinalis ✓ ✓ ✓

CYSTS Lemon-shaped/Pearshaped Uninucleated with central karyosome With two fused fibrils anterior to the nuclear region

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TROPHOZOITES Single large nucleus in the anterior portion of the organism Cytostome opposite to the nucleus

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Trophozoite: • Pear-shaped/pyriform shaped • 4 anterior flagella • Short Undulating membrane (with one flagella) • “Rapid Jerky/Jerky-tumbling motility”

Enteromonas hominis ✓ ✓ ✓

Diagnosis ✓ Microscopy (can be stained with Giemsa or Wright) • Female: Urine, Vaginal Discharge, Vaginal Scrapping • Male: Urine, Prostatic Fluid, Seminal Fluid ✓ Culture: • Medium: o Diamond’s Modified Culture Medium o Feinberg-Whittington Culture Medium o Trypticase Liver Serum Medium NON-PATHOGENIC ATRIAL FLAGELLATES Chilomastix mesnili ✓ ✓ ✓

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TROPHOZOITE Asymmetrical Pear shaped 3 anterior flagella 1 flagellum inside cytostome – cytostomal fibril “shepherd’s crook” (safety-pin appearance) Prominent spiral groove in the midportion of the body

TROPHOZOITES Single nucleus with large central karyosome 4 flagella: 3 directed anteriorly, 1 directed posteriorly

Trichomonas hominis ✓ Usually found in the intestines ✓ Commensal ✓ Relatively smaller than T. vaginalis CYSTS NO CYST STAGE

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Largest flagellate in man CYSTS “Nipple-like cyst” Uninucleated with distinct anterior protuberance

CYSTS One to four nuclei located at the opposite ends With central karyosome With well defined cystic wall

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TROPHOZOITES Pear-shaped Full body-length undulating membrane Uninucleated with small central karyosome Motility: Jerky motility

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Trichomonas tenax

Trypanosoma cruzi

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Usually found in the mouth

CYSTS NO CYST STAGE

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TROPHOZOITES Four anterior flagella One posterior flagella