MLS Review - Parasitology - Protozoans

MEDICAL LABORATORY SCIENCE REVIEW • CLINICAL PARASITOLOGY: PROTOZOANS OUTLINE • Protozoa o Stages seen in Amoeba o Intestinal Amoeba o Free-living Opportunistic Amoeba o Ciliata o Flagellates o Apicomplexa PROTOZOA • • • • Sporozoans - rare in Philippines; small Protozoa/Sarcodina/Amoeba - equipped with locomotory organelle pseudopodia Intestinal Amoeba o Has cystic (infective stage) and trophozoite stage o Most are non-pathogenic o Entamoeba histolytica – only and most pathogenic ▪ Causative agent of amoebiasis o E. dispar o E. coli o E. polecki o E. hartmanni o E. gingivalis – Trophozoite Only o Endolimax nana o I. butschlii Free Living Amoeba – “Opportunistic” o Has the ability to migrate to brain o Can be observed in diverse locations o Naegleria fowleri o Acanthamoeba • • INTESTINAL AMOEBA Entamoeba histolytica • • • • • • STAGES SEEN IN AMOEBA • Cyst o Active State – “infective stage“ o Non-Tissue Invader ▪ Cuboidal or spherical shape hinders ability to penetrate intestinal wall o Resistant to damage: Covered with thick cell wall o Can be stained by iodine ▪ I. butschlii - iodine cyst; has iodine pool o Immotile o Smaller as compared to trophozoite o Can be Multinucleated o Usually in Formed Feces Permanent Stain: Iron Hematoxylin and Trichrome Stain Transitory Stages Seen in Amoeba: reproduction process o Excystation = Cyst → Trophozoite = Metacystic trophozoite o Encystation = Trophozoite → Cyst = Precyst (possess more than one nuclei) ▪ Stage prior to encystment Amoebic dysentery “Intraintestinal” or “ *Extraintestinal* ” Intraintestinal o Stomach pains → hyperacidity → loose bowel movement → vomiting → dehydration o In cases of children, it may lead to severe complications due to severe dehydration Extraintenstinal: Hepatic migration Trophozoite o Cytoplasm: ▪ Outer ectoplasm - clear, transparent, & refractile. ▪ Inner endoplasm - finely granular, having a ground glass appearance. The endoplasm contains ingested RBC o Nucleus: Spherical in size and contains central karyosome, surrounded by clear halo and anchored to nuclear membrane by fine radiating fibrils cartwheel appearance. Cyst: o Chromatoidal bodies which are cigar/bacilli/rod shape ▪ Chromatoidal bodies - genetic material remnants o Quadrinucleated Distinguishing Feature o Uninucleated o Clear Cytoplasm (Fine Granules) o Bull’s Eye karyosome o Finger Like Pseudopodia o Rapid unidirectional progressive motility o With Ingested RBC Trophozoite o Vegetative State – “feeding and motile stage” ▪ Stores food/nutrients o Tissue Invader ▪ Attaches to intestinal walls/other surface o Susceptible to damage ▪ Pseudopodia is sensitive o Can only be stained by permanent stain o Motile: Possess pseudopodia o Larger as compared to cyst o Uninucleated o Watery Feces ▪ Must be free of urine contamination 4 • Morphologically Similar to Pathogenic Entameoba histolytica o Entameoba dispar - with different RNA and DNA content compared to E. histolytica ▪ Most morphologically similar to E. histolitca o Entamoeba polecki - amoeba of pigs ▪ Cyst is uninucleated o Entamoeba hartmanni - small race of amoeba ▪ Similar to E. histolytica in size E. histolytica vs. E. coli E. histolytica Trophozoite Size (um) 12-60 Motility Unidirectional, progressive Pseudopodia Long and finger like Cytoplasm Ground glass; slightly clear Inclusion RBC Nucleus Central but indistinct Bull’s eye karyosome Cyst Size (um) 10-15 # of nucleus 4 Shape Spherical Chromatoidal Cigar/rod-shaped bodies Karyosome E. coli 20-50 Sluggish non directional Blunt and rounded Trashy FREE-LIVING OPPORTUNISTIC AMOEBA Naegleria fowleri • • Bacteria and debris Eccentric Large 10-30 8 Spherical or oval Broomstick • • COMMENSAL INTESTINAL AMOEBA Size (um) E. nana I. butschlii Trophozoite 6-15 6-20 • E. gingivalis 5-25 Pseudopodia Bluntly rounded Bluntly rounded Multiple bluntly rounded Nuclear Blot-like Achromatic Central Karyosome granules karyosome Movement Sluggish non-directional Cyst Size (um) 5-14 5-18 ABSENT # of nucleus 4 1 Shape Oval Irregular Chromatoidal Small and bodies Spherical Sample is CSF o Collection through aspiration Associated with primary meningoencephalitis from stagnant water through inhalation o One of the most pathogenic free-living parasite o Capable of migration to meninges o Characterized by terrible headache Amoebic Trophozoite o Elongated with broad anterior and tapered posterior o Only amoeba seen in CSF Amoebic Cyst o Uninucleated with thick cell wall Flagellate o Pear shape flagellate with two whiplike structure; o Jerky or spinning movement Acanthamoeba • • • • Sample is CSF or Corneal Scrapings from contaminated contact lens, solution Causative agent of primary amoebic keratitis Amoebic Trophozoite o Spine like pseudopods known as acanthopodia o Large Eccentric karyosome o Seen only in CSF Amoebic cyst o Double cell wall with outer ragged edges o Large central karyosome 5 CILIATA • Giardia intestinalis trophozoite o Presence of sucking disk, two axonemes and two parabasal bodies ▪ 4 pairs o Pear-shaped or teardrop which is bilaterally symmetrical o Falling leaf motility o 4 pairs of Flagella (8 flagella) ▪ One pair anterior ▪ One pair posterior ▪ Two pairs central extending lateral • Giardia intestinalis cyst o Bilaterally symmetrical o Ovoid in Shape o Two – immature cyst Four –mature o Cytoplasm retracted in cell wall o Median bodies (2-4) ▪ Immature - 2 ▪ Mature - 4 o Uses Giemsa stain Balantidium Coli • • • • • • • • Can only cause intraintestinal lesion Largest intestinal Protozoa o Can be observed in LPO Only pathogenic ciliata to human Presence of two nucleus o Macronucleus: vegetation; horseshoe appearance o Micronucleus: reproduction; Presence of contractile vacuoles Trophozoite: o Cytosome: cell mouth/opening found anteriorly o Cytopyge: cell anus found posteriorly Trophozoite exhibits directional tumbling motility Mode of locomotion: cilia o Small hair-like structure surrounding the B. coli Trophozoite Cyst FLAGELLATES Amoeba Lumen Giardia lamblia Dwelling/ Chilomastix mesnili Atrial Flagellate Enteromonas hominis Retortamonas intestinalis Dientamoeba fragilis Trichomonas tenax Trichomonas hominis Trichomonas vaginalis Hemoflagellates Leishmania • • Habitat Small intestine Large Intestine Large Intestine Large Intestine Large Intestine Mouth Large Intestine Vagina and urethra Reticuloendothelial cells Trypanosoma brucei Connective tissue Trypanosoma cruzi BOTH Flagellates may migrate to other parts of the body D. fragilis may be pathogenic or non-pathogenic depending on the immunity of the individual Giardia lamblia or Giardia intestinalis • • • • • Most common protozoan pathogen worldwide o First discovered in USA; from a group who swam in a lake and after which they presented diarrhea and watery stool and it was discovered that they possess the parasite Causes Traveller’s diarrhea due to contaminated water in undeveloped countries ONLY flagellate that inhabits the small intestine NOT a tissue invader but attaches to mucosal linings by means of sucking disks Associated with Gay bowel syndrome and malabsorption syndrome (steatorrhea - lipid/FAT in stool) o Gay bowel syndrome - sexually transmitted by perianal and rectal disease, and trauma ▪ Seen in men who had sex with men Trichomonas • • No cystic stage Trichomonas tenax o Non-Pathogenic o Specimen: buccal scrapings, o Pear shape; Five anterior flagella and one posterior flagellum ▪ One flagellum is coiled and joining the body o Nervous jerky (non-pathogenic); o One ovoid Nucleus filled with chromatin granules o Undulating membrane extending 2/3 of body length ▪ Costa – distinguishing feature of trichomonas • Makes one side to be thiclk o Thick axostyle curved around nucleus o Small cytostome 6 • • Trichomonas hominis o Non-pathogenic o Specimen: stool o Pear shape; Five anterior flagella and one posterior flagellum o Nervous jerky ( non-pathogenic) o Spherical nucleus with central karyosome o Full body length undulating membrane o Axostyle extends beyond the posterior part o Conical cytostome cleft Trichomonas vaginalis o Pathogenic o Specimen: urine, vaginal discharge, semen o Pear shape; Five anterior flagella and one posterior flagellum o Rapid jerky (pathogenic) o Ovoid nucleus filled with chromatin granules o Undulating membrane half of the body o Axostyle is coiled around the nucleus o Absence of cytostome but granules along axostyle is present o Women affected are symptomatic ▪ Intense itchiness ▪ T. vaginalis can attach to vaginal wall causing irritation and mucus production ▪ Pap smear confirms presence of parasite o Vaginitis – intense itchiness and foul smelling vaginal discharge ▪ Examination is done through vaginal and cervical secretions for presence of trophozoites (Trichomonas have no cyst stage) ▪ Unstained wet dropped method or Giemsa/Acridine orange/Pap stain/Romanovsky stain or Diamond medium culture can be used ▪ Acquired through sexual contact ▪ Non-venereal transmission is possible ▪ Can be transmitted in toilet seats, linens, and towels o May cause strawberry cervix ▪ Severe cases lead to this o Males can be affected but are asymptomatic carriers o Medication: vaginal suppository medications OTHER INTESTINAL FLAGELLATE • Chilomastix mesnili o Can be mistaken with G. lamblia o Found in large intestine o MOT: ingestion of cyst from contaminated food/water o Trophozoite: ▪ Pear-shaped ▪ Stiff rotary movement ▪ 4 flagella • 3 anterior extending • 1 extending to posterior along body ▪ Prominent cytosome with fibrils ▪ Spiral groove ▪ Curved posterior ▪ Corkscrew motility o Cyst: ▪ Ovoid in shape ▪ Lemon shape with clear hyaline knob on its anterior end ▪ Well defined cytosome at side of nucleus • Enteromonas hominis o Exhibits jerky motion o 7-9 um (same size as RBCs) o Distributed worldwide in warm temperatures o Asymptomatic transmission o Infective stage: cyst o Trophozoite: ▪ Body flattened on one side ▪ Four flagella: 3 anterior; 1 posterior ▪ Well-defined nuclear membrane o Cyst: ▪ Quadrinucleated with two at each opposite side • Retortamonas intestinalis o 3-5 um o Reported ins tool examination o Found in warm temperatures and crowded areas (psychiatric hospitals or areas with poor sanitation) o Non-pathogenic 7 o o • Trophozoite: ▪ Two anterior flagella ▪ Cytostome extending halfway with well-defined fibril border Cyst: ▪ Lemon-shaped ▪ Two fused fibrils resembling bird’s beak near nucleus • o Dientamoeba fragilis o No cystic stage o Only amoeboflagellate protozoan o Can be pathogenic or non-pathogenic o Pathogenic: Abdominal pain, LBM o Trophozoite: ▪ Irregularly round ▪ Can be mistaken as E. histolytica ▪ Broad hyaline leaflike pseudopodia ▪ Fragmented karyosome (4-8): Tetrakaryosome ▪ Binucleate condition • Only binucleate amoeboflagellate ▪ Progressive directional motility o • HEMOFLAGELLATES • • • Requires two host to survive Definitive host: man Intermediate Host: vectors/arthropods Trypanosoma Trypanosoma brucei cruzi Intermediate Phlebotomus Glossina Reduviid bug/ Host (sand fly) (tse tse fly) Triatomine/ Kissing Bug/ Assasin Bug Developmental 1 Amastigote (man 3 Epimastigote (vector) Stage intracellular;) 4 Trypomastigote (man) 2 Promastigote 5 Metacystic trypomastigote (vector) 6 Amastigote (T. cruzi only) Infective Stage Promastigote Metacystic trypomastigote to MAN (found in fly saliva) Leishmania Forms o Leishmania – mammalian parasite o Amastigote (Human) ▪ Lives intracellularly (monocytes, PMNs, endothelial cells) ▪ Round to oval ▪ 2-3 um ▪ No flagella ▪ Kinetoplast is present with dot like blepharoblast ▪ Parabasal body adjacent to blepharoblast ▪ Usually found in cells ▪ Promastigote (Vector) ▪ Aka leptomonas ▪ Seen in intermediate hosts ▪ Long and Slender ▪ One near center ▪ Kinetoplast present ▪ Free single flagella in anterior ▪ Undulating membrane is absent ▪ Trypanosoma gambiensi and rhodesiense forms o Epimastigote (Vector) ▪ Aka crithidia ▪ Long and Wider than promastigote ▪ Undulating membrane half of body length ▪ Free flagellum o ▪ Trypomastigote (Human) ▪ Long and Slender (C, S or U shape) ▪ Undulating membrane whole body length ▪ Free flagellum Leishmania ▪ 8 • • Trypanosomes o Trypanosome gambiensi ▪ MOT: salivary skin inoculation by vector ▪ West African Sleeping Sickness ▪ Winterbottom sign and Kerandel sign o Trypanosoma rhodesiensi ▪ MOT: salivary skin inoculation ▪ East African Sleeping Sickness o Trypanosoma cruzi ▪ MOT: fecal transmission after skin inoculation ▪ Most pathogenic (Consists of 4 stages) ▪ South American Trypanosomiasis/Chaga ▪ Chagoma and Romana Sign Pathogenecity of Leishmania o Leishmania donovani ▪ Spx of choice: aspiration ▪ Visceral leishmaniasis/ Kalazar fever/ Dumdum fever/ Black disease/ Tropical Splenomegaly o Leishmania tropica ▪ Spx of choice: skin biopsy ▪ Most superficial ▪ Cutaneous leishmaniasis/ Oriental sore/ Aleppo/ Baghdad boil/ Delhi ulcer o Leishmania braziliensis ▪ Spx of choice: tissue biopsy ▪ Invades mucocutaneous layer ▪ Mucocutaneous leishmaniasis/ Espundia/ Chiclero ulcer/ American leishmaniasis • May cause deformity • Plasmodium falciparum o Rings: double chromatin dots; accole forms; multiple infections in same red cell o Trophozoites: compact (rarely seen in peripheral blood) o Gametocytes: mature (M) and immature (I) forms (I is rarely seen in peripheral blood) o Schizonts: 8-24 merozoites (rarely seen in peripheral blood) o RBC inclusion: Maurer’s dots • Plasmodium vivax o Trophozoite: amoeboid, deforms the erythrocyte o Schizonts: 12-24 merozoites o Gametocytes: round-oval o RBC inclusion: Schuffner’s granules/dots Apicomplexa • • • • No definite locomotory organelle Arthropod-transmitted blood-borne causative agents Most common worldwide: P. vivax Infective Stages o Man: intermediate host ▪ Infective Stage: sporozoite o Mosquito (Vector): definitive host ▪ Infective: gametocyte ▪ Transfer sporozoite to healthy individual Plasmodium 9 • Plasmodium malariae o RBC inclusion: Zieman’s dots • Plasmodium ovale o Trophozoite: compact o Schizonts: 6-14 merozoites; dark pigment (rosette) o Gametocytes: round-oval o RBC inclusion: Schuffner’s/James’s dots • • • • Plasmodium knowlesi • Notes To Remember: o Sickle Cell Trait: resistant to P. falciparum o Relapse: P. vivax and P. ovale o Duffy Negative ( Fy (a-b-)): esistant to P. vivax o P. falciparum - can cause fatal cases of malaria ▪ Can affect 10-40% circulatory RBCs P. P. vivax P. malariae P. ovale falciparum RBC preference All sizes Reticulocytes Senescent/old Retics/ (↑ RBC size) RBCs (↓ RBC young RBC size) (↑ RBC size) Hypnozoite Absent Present Absent Present Stages in PBS Gametocyte, All stages are present ring, trophozoite Microgametocyte Spherical, pale blue cytoplasm Macrogametocyte Spherical deep blue cytoplasm • Blood o Thick smear: for rapid diagnosis ▪ Done in emergency cases of paroxysm ▪ Must be hemoglobinized prior to staining o Thin Smear: for identification of species ▪ Fixation by alcohol prior to staining Stains: Giemsa, Wright’s, Delafield hematoxylin ▪ Wright’s - fixation of thin smear not required dur to use of alcohol ▪ Hematoxylin - dehemoglobinize sample with 2% formalin with 1% HAc o For demonstration of malaria, blood should be collected during the peak of fever (paroxysm) ▪ Paroxysm - chills, high body temps, profuse sweating o Rule: Smear for suspicion & another smear after out of fever o Smears should be done before antimalarial treatment o Stains ▪ Giemsa: commonly used stain • Buffered ( H2O) + Fixed in Methanol ▪ Leishman Stain • No Fixation Needed: Alcohol Other Methods o Quantitative Buffycoat Method ▪ Capillary tube is used and broken at the middle o Para-sight F test - dipstick test for simple and rapid examination of P. falciparum o Serological tests - IHA, IFA, ELISA ▪ Expensive ▪ Used in epidemiological studies and cannot be differentiated with current and previous infections Malaria can be transmitted through vector bites, blood transfusion, and transplacental transfer Stages of Malarian Parasites o Cryptozoite - stage initially developed in hepatic cells ▪ Trophozoite initially migrates to hepatic cells ▪ Will burst due to numerous schizonts affecting RBCs o Trophozoite/Ring form - earliest form after invasion of RBCs ▪ Aka as signet/ruby ring stage • Due to small dot present in circular area ▪ Vegetative stage containing one nucleus and develops within RBCs o Schizonts - trophozoite with nucleus has divided o Merozoites - several cryptozoites/schizonts within RBCs ▪ Released when RBC lyse making them ready to attack or penetrate other healthy RBCs o Hypnozoite - dormant stage that persist in liver cells but only in cases of P. vivax & P. ovale o Gametocytes - large part ▪ Sexually differentiated immature cells ▪ Only time Anopheles can suck blood • Matures inside the Anopheles • Develops into zygote (cell form union male and female gametocyte ▪ Ookinete - motile zygote ▪ Oocyst - encysted form of ookinete ▪ Sporocyst - oocyst where sporozoites have developed and infect salivary glands of mosquito ▪ Sporozoite - already in salivary gland • Injected in an healthy individual o Gametes - mature sex cells Schizogony cycle - occur in body of human; asexual cycle (no union of gametocyte inside the human) Sporogony cycle - occur in vector; sexual cycle (union of macro and micorgametocytes) Extracellular infection - infect hepatic or parenchymal cells Intracellular infection o • • • • 10 OTHER Apicomplexa • Babesia microti o Intraerythrocytic parasite transmitted by bites o Can be mistaken as malarial parasites ▪ Similar to P. malariae ▪ Confused with ring forms fo P. falciparum ▪ B. microti lack of blood pigments in schizogonic stage in infective RBCs • Also lack large growing trophozoite and gametocytes • Many ring forms are only observed (4-5) o Individual rings are much smaller than those found in malarial infection • Trophozoite is pear shaped o Transmitted through blood transfusion o Due to tick bites (Ixodes) o Merozoite - four trophozite attached with maltese cross appearance o Use blood smear using Wright’s & Giemsa stain o Babesiosis / piroplasmosis - disease o May also cause hemolytic anemia and jaundice ▪ Fever, hepatomegaly after 1-2 weeks onset o Non-malarial parasite but similar to malarial parasites due to formation of 4-5 ring forms in RBCs • Cryptosporidium parvum o MOT: ingested oocyst from fecally contaminated food and water o Infective stage: Oocyst ▪ Thick cell wall 1-6 dark granules ▪ 4-6 um, roundish ▪ 4 sporozoites • Blastocystis hominis o Centrally located large vacuole o 2-4 nuclei at the periphery o Infective stage: Vacuolated form o MOT: from contaminated food and water o Pathogenicity: ▪ Immunocompromised persons (full-blown AIDS) • Found with C. parvum ▪ Sever diarrhea, fever, nausea, vomiting • Microsporidia o Infective stage: Spore o Extruding polar filaments (tubules) o 1-5 um o Initiates infection by injecting sporoplasm (infectious material) to the host o MOT: accidental ingestion/injection of sporoplasm from feces/stool o May be observed with full-blown AIDS o With enteritis, keratoconjuctivitis Toxoplasma gondii o Infective stage: ▪ Tachyzoite - cresent ▪ Bradzoite - enclose to form cyst o MOT: ingestion of oocyst, transplacental transmission ▪ Transmission from cats o Intracellular pathogens (RBCs, macrophage) o Toxoplasmosis ▪ May be asymptomatic ▪ Fatigue, chills, fever, headaches, myalgia SPOROZOANS • Isospora belli o MOT: ingestion of ripe oocyst from contaminated water and food o Small o Habitat: intestine o Pathogenesis: diarrhea, abdominal pain ▪ May be asymptomatic with LBM o Disease: human ccoccidiosis o Infective stage: Oocyst: transparent and oval ▪ Ovoidal ▪ Cell wall has two layers ▪ Development of sporoblast (young oocyst) ▪ Sporoblast will contain two sporocyt which would contain 4 sausage shaped sporocyst • • Sarcocystis lindemanni o MOT: ingestion of bradyzoites from infected muscle (uncooked pork and cattle) o Habitat: striated muscle o Sarcocystosis/Sarcosporidiosis ▪ Fever, diarrhea, abdominal pain o Infective stage: Oocyst: sausage shape sporocyst ▪ Ovoidal, transparent, double-layered ▪ Develop into 4 sporozoites 11 • Pneumocystis jirovecii o Infective stage: Trophozoite ▪ 4-8 unorganized nuclei in rosette ▪ Somewhat circular o MOT: unknown ▪ Suspected to be person-to-person o Invasion of alveolar spaces of lung tissue, spleen, lymph nodes and bone marrow o Found in children, full-blown AIDS, immunocompromised patients o • Laboratory Diagnosis for Sporozoan o Intestinal ▪ Direct fecal smear ▪ Concentration techniques ▪ Kinyoun’s Acid-Fast - P. jirovecci o Serologic ▪ PCR o Muscle biopsy - T. gondii o Recovery of sporozoans ▪ Serologic: IFAT, ELISA, IHA 12