Clinical Parasitology - 05 Protozoa

• CLINICAL PARASITOLOGY PROTOZOA OUTLINE • Free-Living Amoebas • Kingdom Protozoa o Introduction o Naegleria fowleri o Taxonomy o Acanthamoeba Spp • Phylum Sarcomastigophora o Other Free-Living Amoeba Subphylum Sarcodina • Phylum Ciliophora (Ciliates) (Amoebae) o Balantidium coli o Amoeba • Phylum Sarcomastigophora o Entamoeba histolytica Subphylum Mastigophora o Commensal Amoeba o Atrial Flagellates o Morphologic Comparison o Non-Pathogenic Atrial Between E. histolytica and E. Flagellates coli o Pathogenesis - Amoebiasis o Laboratory Diagnosis o Other Intestinal Amoeba • • • Entamoeba histolytica • • • KINGDOM PROTOZOA INTRODUCTION • • Protozoans are generally different from metazoan Protozoans are microscopically visible o Requires microscope for morphology and structure TAXONOMY All undergoes encystation except for Entamoeba gingivalis and Dientamoeba fragilis o Encystation - amoeba transform from trophozoite to cyst stage; occurs in large intestine Multiplies through Binary fission (trophozoite stage) Life cycle: 1. Trophozoite form - capable of encystation 2. Pre-cystic form 3. Cystic form – with hyaline cystic wall 4. Metacystic form Excystation - from cyst to trophozoite; occur in the small intestine • • • Infective Stage: Cyst Mode of Transmission: Ingestion of food/water with E. histolytica cyst Phorectic Vectors: Cockroach and Flies o Only function to transport E. histolotica; no development occur Pathology: Amoebiasis Treatment: Metronidazole First described by Loesch in 1880 LIFE CYCLE TROPHOZOITE Motile (due to pseudopods) Feeding Stage Vegetative state Found in watery, soft or semi-formed stool Fragile (destroyed by gastric juices) Cannot encyst if excreted from the body • • • • Subphyla are differentiated based on locomotory organs Subphylum Sarcodina - has pseudopods (false feet) Subphylum Mastigophora - has flagella Phylum Ciliophora - has cilia PHYLUM SARCOMASTIGOPHORA SUBPHYLUM SARCODINA (AMOEBAE) AMOEBA • • • Have pseudopodium – hyaline foot-like protrusion for locomotion o Pseudopodium - false feet All amoebas are commensal except for Entamoeba histolytica o Commensal - do not infect host unless immuncompromised (w/ HIV-AIDS, cancer, neonates, geriatric, autoimmunity. All are intestinal dwellers (in large intestine) except Entamoeba gingivalis (in mouth) CYST Non-motile Non-feeding Stage Infective Stage Found in soft to formed stool Resistant to acidic pH (due to cystic wall with chitin & lectin-Jacob & Jessie lectins) • • • Reproduction through binary fission Once cyst is in small intestine, it will undergo excystation o Nuclear division (nucleus divides to 4-8 nucleus) o Also under Cytoplasmic division o Result to 8 trophozoite 1 cyst = 8 trophozoite o Cyst - quadrinucleated (up to 4 nucleus) ▪ When division, can be divided up to 8 nucleus ▪ Cytoplasmic division then happens • 8 nucleus will go to different divisions Points of Entamoeba Differentiation histolytica Movement Unidirectional, active progressive Shape of Finger-like Pseudopodia Release of Explosive/ Pseudopodia one at a time • • • • • • • • Entamoeba coli – harmless inhabitant of the colon o Normally found in colon of man o Important to be differentiated by E. histolytica Entamoeba dispar – morphologically similar with E. histolytica but with different genome o Distinguished from E. histolytica by PCR Entamoeba hartmanni – similar with E. histolytica but smaller and no RBC inclusion. (“small race” of E. histolytica) o Cyst: quadrinucleated with coarse cytoplasm; immature cyst has chromatoidal bars Entamoeba polecki – parasite of pigs and monkeys; rarely infect humans (incidental host) o Distinguished from E. histolytica by Cyst o Cyst: consistently uninucleated Entamoeba gingivalis – can be found in the mouth (gum and teeth surface) o Only non-intestinal dweller o Abundant in cases of oral disease o No cyst stage - cannot undergo encystation o Only trophozoite form o Transmission: kissing, droplet spray, sharing utensils Entamoeba moshkovskii – morphologically similar with E. histolytica and E. dispar. o Classified as free-living amoeba (rarely infect humans) o Osmotolerant o Able to survive at temperature range of 0-41˚C Endolimax nana – smallest protozoa of man o Trophozoite: Sluggish, mononuclear o Cyst: quadrinucleated with eccentric karyosome –crosseyed cyst Iodamoeba butschlii o Iodine cyst – has (strong) affinity to iodine o Cyst - Large glycogen vacuole/body which stains deeply with iodine ▪ Uninucleated Morphologically similar: o E. histolytica, E. dispar, E. moshkovski, E. bangladeshi MORPHOLOGIC COMPARISON BETWEEN E. histolytica AND E. coli TROPHOZOITE Points of Differentiation Pathogenicity Entamoeba histolytica Only pathogenic amoeba Entamoeba coli Normal inhabitant in man Slow manner/ Several at a time (hardly seen) Nucleus COMMENSAL AMOEBAE • Entamoeba coli Sluggish, nonprogressive Blunt Cytoplasm (Endoplasm) Inclusion Size Trophozoite Uninucleated with central karyosome (nucleolus) Clean-looking Uninucleated with eccentric karyosome (on side) Dirty-looking RBC Bacteria, yeast, cell debris Smaller (15-50 µm) Bigger (12-60 µm) CYST Points of Entamoeba Differentiation histolytica Number of Quadrinucleated (≤4) Nuclei Chromatoidal Cigar-shaped/ bars Rodshaped/Coffin-lid Nuclear Thin membrane Size 10-20 µm Entamoeba coli > 4 (mature: 8) Witchbroom/ splintered/needlelike Thick 8-35 µm PATHOGENESIS - AMOEBIASIS • Intestinal amoebiasis – most common pathology o Amoebic dysentery ▪ Caused by E. histolytica ▪ blood and mucus in stool (dysentery) ▪ Differentiated from bacillary dysentery by the presence of charcot-leyden crystal (eosinophil degradation product) ▪ Bacillary dysentery - from Shigella dysenteriae o Amoebic colitis – ulceration of intestine (cecum, rectosigmoid) caused by E. histolytica ▪ Characteristic of ulcer: Flask-shaped OTHER INTESTINAL AMOEBA • Amoebic colitis • Amoebic Liver Absess Extraintestinal amoebiasis o Hepatic amoebiasis ▪ Most common extraintestinal amoebiasis ▪ Amoebic Liver Abscess: Anchovy sauce-like appearance ▪ Occur due to motility of trophozoite through hepatic portal vein o Pulmonary amoebiasis o Cerebral amoebiasis o Amebic pericarditis o Cutaneous amoebiasis o Genital amoebiasis • Blastocystis hominis o No exact taxonomic classification (stramenophile) o Ovoid or spherical, vacuole-like structure o Typically commensals o May cause diarrhea to some patient (immunocompromised) Dientamoeba fragilis o Ameboflagellate o No cyst stage o Rosette-shaped nuclei (1-2); no peripheral chromatin o Can be transported by pinworm (E. vermicularis) to man o Chronic infection may mimic Irritable Bowel Syndrome FREE-LIVING AMOEBAS VIRULENCE FACTORS • • • • • • • Naegleria fowleri Pathogenic determinants Properties that can cause disease Gal/Gal Nac Lectin: cytoadherence o Gal/Gal - Galactose/N-acetylgalactosamine o Responsible for attachment to cells of large intestine Caspase-3: apoptotic death of mucosal cells Myosin IB: ingestion of RBC Amebapores: pore-formers; can cause ulcerations of tissues or cells of host Cysteine proteinase: tissue invasiveness – the most virulent o Causes extraintestinal amoebiasis (more severe) LABORATORY DIAGNOSIS • • • • • • • Direct Fecal Smear o Saline solution (0.85-0.95 NSS): Trophozoite motility o Saline + Methylene Blue: Entamoeba spp. stained blue o Saline + Lugol’s Iodine: nucleus of E. histolytica cyst can be observed ▪ Iodine - destroys trophozoites (due to fragility) Concentration Techniques: FECT, MIFT o FECT - Formalin Ether Concentration Culture: o Robinson’s & Inoki medium o Balameth Monophasic medium o Boeck & Dorbolav’s Diphasic medium o TYS-S-33 Serologic Tests: ELISA, IHAT, CIE, AGD, IFAT o ELISA - Enzyme-linked immunosorbent assay o IHAT - Indirect hemagglutination test ▪ Can detect past infection from 10 years o CIE - Counterimmunoelectrophoresis o AGD - Agar cell diffusion o IFAT - indirect fluorescence antibody test Liver aspirate biopsy: presence of E. histolytica trophozoite; for extraintestinal amoebiasis Radiographic techniques: X-Ray, MRI, CT-Scan Molecular Tests: PCR; to genotypically differentiate • • • • • • • Causative agent of Primary Amoebic Meningoencephalitis (PAM) - infects meninges of the brain Thermophilic organisms (up to 30˚C) Multiplies through promitosis (intranuclear mitosis) o Nuclear membrane do not breakdown Mode of Transmission: oral and intranasal routes while swimming in contaminated lakes, pools, rivers Do not survive in sea water Can be killed by: o 1 mg/L chloride solution o pH: 8.0-8.01 o extreme heat (up to 38°C) Diagnosis: Cerebrospinal fluid (Specimen) o Collection - by spinal tap o Culture: Non-nutrient medium with page’s saline seeded with living Escherichia coli o PCR, ELISA MORPHOLOGIC FORMS • • Cyst o Uninucleated with rounded chromatoidal bars Trophozoites (2) : o Amoeboid – only form recognized in humans ▪ With single pseudopod ▪ Uninucleated with large central karyosome without peripheral nuclear chromatin o Flagellated – with two flagella at broad end ▪ Flagella - aid motility ▪ Motility: Spinning or Jerky motility (directional) ▪ Do not divide Cyst stage Trophozoite stage Flagellated stage Acanthamoeba SPP. • • • • Acanthamoeba castellani - most common Pathology: o Granulomatous Amebic Encephalitis (GAE) ▪ Destructive encephalopathy and assoc. meningeal irritation o Amebic Keratitis (AK) ▪ Associated with improperly disinfected contact lenses (rinsing with tap water or contaminated lens solution) Mode of Transmission: o Intranasal o Entrance of pathogen through cuts, wounds. Diagnosis: o Specimen: discharges, exudates, tissue secretions. o Culture: PYGC medium with antibiotics ▪ PYGC - Peptone Yeast Glucose Cysteine ▪ Antibiotics - remove unwanted growth of bacteria MORPHOLOGIC FORMS • • Trophozoite: o With Large nucleus with centrally located nucleolus o With acanthopodia - spinyfilaments or “thorn-like” appendages for locomotion o Replicates by mitosis Cyst: double-walled (with ostiolespores in between walls) • • • • • PHYLUM SARCOMASTIGOPHORA SUBPHYLUM MASTIGOPHORA OTHER FREE-LIVING AMOEBA • • Balamuthia mandrillaris o Pathology: Similar with GAE o Trophozoite: finger-like pseudopodia o Cyst: spherical ▪ Three layered cystic walls (electron microscopy) ▪ Two layers (light microscopy) Sappinia diploidea o Newly recognized human pathogen causing amoebic encephalitis o Trophozoite: with two nuclei attached together by connecting perpendicular filaments PHYLUM CILIOPHORA (CILIATES) Balantidium coli • • • • • • • • First name: Paramecium coli Former name: Balantidium coli New Name: Neobalantidium coli Only member of Phylum Ciliophora Largest intestinal protozoan Infective Stage: Cyst Reservoir Host: Pigs o Additional source of nutrients o Not essential for development Mode of Transmission: Ingestion of Infective Stage (cyst) Morphologic forms: o Trophozoite: ▪ Ciliated ▪ Motility: “Thrown ball/Rolling” ▪ Ovoidal, bean-shaped ▪ Prominent cytostome – entry of food ▪ Indented cytopyge – exit of waste ▪ 2 nuclei: ▪ Macronuclei (vegetative) - big/kidney shaped ▪ Micronuclei (reproductive) - small/round-shaped o Cyst: ▪ Ovoid, rounded with well defined cystic wall (cilia enclosed in cystic wall) ▪ Binucleated Pathology: o Balantidiasis/Balantidial Dysentery/Ciliary Dysentery ▪ Similar with Amoebic dysentery ▪ Bloody stool with mucus Virulent Factors: o Hyaluronidase: lytic enzyme that causes ulceration ▪ Lytic enzymes - causes destruction of cells o Ulceration: Rounded base and wide neck Laboratory Diagnosis: o Specimen: Stool o Direct Fecal Smear o Concentration Techniques Treatment: Tetracycline or Metronidazole ATRIAL FLAGELLATES GENERAL STRUCTURE • • • • • • Flagella – locomotor apparatus; aids locomotion o Number of flagella differs in species Kinetoplast – provides energy o Blepharoplast o Parabasal Body Cytostome – cell mouth; entry of food 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 • • • • • • • • • Other Names: Giardia intestinalis, Giardia duodenalis, Lamblia duodenalis, Lamblia intestinalis, Cercomonas intestinalis o Giardia duodenalis - new name First described by Antoine van Leeuwenhoek o In his own stool specimen Habitat: Intestine - duodenum, jejunum and upper ileum Diagnostic Stage: Trophozoite and Cyst Infective Stage: Mature Cyst Mode of Transmission: Ingestion Pathology: o Giardiasis, Traveller’s Diarrhea, Gay-bowel syndrome o Acute infection: “rotten egg” odor of stool o Chronic infection: Steatorrhea – passage of fats in stool (stool floats) Treatment: Metronidazole Life cycle • • Cyst: o Ovoid, football shaped o Quadrinucleated (mature cyst) ▪ Young cyst: 2 nucleus o Thick shell (double walled) o Flagella retracted to axoneme (median body) o Deeply stained curved fibril Laboratory Tests: o Specimen: Stool, Duodenal aspirates o Stool Exam - presence of cyst/trophozoites o Duodenal Aspirates o Beale String Test/Entero-Test o Immunochromatography – antigen (VSP) detection in stool o Direct Fluorescent Antibody Assay – Gold Standard Trichomonas vaginalis • • • • • • • • o Trophozoite multiply through binary fission o Excystation - occur in duodenum within 30 minutes Trophozoite: o Bilaterally symmetrical o Pear/tear drop shaped, pyriform o With Large ventral sucking disc (for attachment) o “Old man with eyeglasses” o 4 pairs of flagella ▪ One pair – anterior ▪ One pair – posterior ▪ 2 pairs – central, extending laterally o 2 nuclei w/ distinct karyosome (bilaterally symmetrical) o Present axostyle with parabasal body (energy) o Motility: “Falling-leaf motility” o Multiplies by Longitudinal Binary Fission o Covered with variant-specific surface proteins (VSPs) ▪ Surface antigens • • The only pathogenic Trichomonas Most prevalent non-viral sexually transmitted infection No cyst stage Diagnostic Stage: Trophozoite Infective Stage: Trophozoite Mode of Transmission: Sexual intercourse Habitat: Urogenital Tract o Female: vulva (vagina) (pH 5.2-6.4) ▪ Slight acidity is favorable o Male: prostate gland Pathology: o Female: Trichomoniasis, Ping-pong disease ▪ Non-specific vaginitis (vaginal inflammation) ▪ Itchiness, burning sensation ▪ Vulvovaginitis (inflammation of vulva) ▪ Greenish-yellow leukorrheic discharge ▪ Inflammation of cervis – “strawberry cervix” ▪ If not treated in pregnant, can be passed in newborn through birth canal o Male (often asymptomatic): non-specific prostatitis, UTI Trophozoite: Strawberry Cervix o Pear-shaped/pyriform shaped o 5 flagella ▪ 4 anterior flagella ▪ Short Undulating membrane (with one flagella) o Motility: “Rapid Jerky/Jerky-tumbling motility” o • • • • Life Cycle Laboratory Diagnosis: o Microscopy: can be stained with Giemsa or Wright ▪ Female: Urine, Vaginal Discharge, Vaginal Scrapping ▪ Male: Urine, Prostatic Fluid, Seminal Fluid o Culture: ▪ Medium: • Diamond’s Modified Culture Medium • Feinberg-Whittington Culture Medium • Trypticase Liver Serum Medium o Youngest patient was 15 y.o. female (by Sir Victorino) Treatment: o Oral Metronidazole o Acidic douche (10% vinegar) NON-PATHOGENIC ATRIAL FLAGELLATES • Chilomastix mesnili o Largest flagellate in man o 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. ▪ Motility: “Boring/Spiral” or “Cork-screw motility” Cyst: ▪ “Nipple-like cyst” ▪ Uninucleated with distinct anterior protuberance Retortamonas intestinalis o Trophozoite: ▪ Single large nucleus in the anterior portion of the organism ▪ Cytostome opposite to the nucleus o Cyst: ▪ Lemon-shaped/Pear-shaped ▪ Uninucleated with central karyosome ▪ With two fused fibrils anterior to the nuclear region • Enteromonas hominis o Trophozoite: ▪ Single nucleus with large central karyosome ▪ 4 flagella: 3 directed anteriorly, 1 directed posteriorly o Cyst: ▪ One to four nuclei located at the opposite ends ▪ With central karyosome ▪ With well defined cystic wall • Trichomonas hominis o Usually found in the intestines o Commensal o Relatively smaller than T. vaginalis o No cyst stage o Trophozoite: ▪ Pear-shaped ▪ Full body-length undulating membrane ▪ Uninucleated with small central karyosome ▪ Motility: Jerky motility Trichomonas tenax o Usually found in the mouth o Trophozoite: Four anterior flagella, One posterior o No cyst stage • PHYLUM SARCOMASTIGOPHORA SUBPHYLUM MASTIGOPHORA Trypanosoma cruzi • BLOOD AND TISSUE FLAGELLATES MORPHOLOGIC FORMS Amastigote Shape Round to Oval Location of Off center Nucleus Kinetoplast W/ dotlike blepharoplast & parabasal body adjacent to it Flagella None (with axoneme) Undulating Membrane Shape Location of Nucleus Kinetoplast none Epimastigote Long & slightly wider than promastigote Posterior end Promastigote Long and Slender In or near center • • Located in anterior end One (extends from anterior end) none Trypomastigote C, S or U shape Anterior to the kinetoplast Located in the posterior end One (extends from anterior end) Extending entire body length Located anterior to the nucleus Flagella One (extends from anterior end) Undulating Extending half of body Membrane length • All are nucleated • Kinetoplast - structure that provides energy • Trophozoites - diagnostic stage for blood and tissue flagellates o Not all blood and tissue flagellates have these morphologic forms • • • Trypanosoma spp. ETIOLOGIC VECTOR AGENT T. cruzi Cone-nose Bug Assassin Bug Reduviid Bug Triatomine Bug Triatoma rubrofasciata Rhodnius Panstrongylus T. Tsetse fly gambiense (Glossina spp.) T. rhodesiense • DISEASE ASSOCIATION Chaga’s Disease or American Trypanosomiasis STAGES EXHIBITED All forms Gambian or West Epimastigote African and Sleeping Trypomastigote Sickness only Rhodesian or East African Sleeping Sickness T. gambiense & rhodesiense - member of T. brucei complex causing African sleeping sickness • Infective Stage: o Man: metacyclic trypomastigote o Vector: trypomastigote Mode of Transmission: Vector borne Pathology: Chagas’ Disease or American Trypanosomiasis o Fatal in young children o “Chagoma” – inflammation at the site of inoculation ▪ Hallmark of Chagas’ disease 1. Acute Trypanosomiasis ▪ Generalized lymphadenopathy ▪ Romaña’s Sign – edema of eyelid and conjunctiva; hallmark 2. Chronic Trypanosomiasis ▪ No characteristic symptoms and may last for 20 years or more ▪ Cardiomyopathy, megaesophagus & megacolon ▪ These advanced conditions can lead to death Treatment: Nifurtimox and Benznidazole Life Cycle Laboratory Diagnosis: o Specimen: Blood, CSF, fixed lymph nodes tissues and lymph juices. ▪ CSF - when CNS is involved Laboratory Tests: o Stained Smear – Giemsa staining (demonstration of trypomastigote) o Blood Cultures: NNN Medium (Novey-McNealNicolle) o Xenodiagnosis: use of laboratory animal o Serologic Test (IFA, IHAT, ELISA) ▪ IFA - Immunofluorescence assay o Molecular Testing: PCR ▪ Amplification of DNA from kinetoplast Trypanosoma brucei complex • • T. gambiense & T. rhodesiense Pathogenesis: o Chancre ▪ earliest sign of African Trypanosomiasis ▪ Hard, painful lesion at the site of inoculation o • • • Gambian trypanosomiasis ▪ Caused by T. gambiense ▪ Also known as ‘West African sleeping sickness’ ▪ Acute: fever, headache, joint and muscle pain, tachycardia, dizziness and rashes • Winterbottom’s sign – enlargement of the posterior cervical lymph node and have a ripe plum consistency; hallmark symptom ▪ Chronic: with CNS invasion • Severe headache, alternately morose and excitable, and lack interest in work • Tremors and “Kerandel’s sign” – hyperesthesia and inversion of sleep cycle can be observed. o Rhodesian Trypanosomiasis ▪ Caused by T. rhodesiense ▪ Also known as ‘East African sleeping sickness’ ▪ More rapid & fatal than Gambian Trypanosomiasis ▪ CNS involvement appear earlier ▪ Neurologic deterioration is rapid Laboratory Diagnosis: o Specimen: Blood, CSF, and lymph juices o Laboratory Tests: ▪ Wet Smear: presence of live parasite ▪ Stained Smear – giemsa staining (demonstration of trypomastigote) ▪ Serologic Test: IFA, ELISA, IHA • IHA - Immunohemaglobullin Assay Treatment: only effective on early stages o Suramin and Pentamidine o Melasorprol and Tryparsamide (w/ CNS involvement) o DL-alpha-difluoromethylornithine (DFMO, Eflornithine) Hallmark Symptoms Chancre Winterbottom’s Sign • Life Cycle • Pathogenesis: o Infective Stage: ▪ Man: promastigote ▪ Vector: amastigote o Mode of Transmission: vector-borne o Cutaneous Leishmaniasis: ▪ Caused by L. tropica ▪ Other Name: Jericho boils, Aleppo button, Baghdad boils, Oriental sore, Delhi fever ▪ Skin ulcer (leaving ugly scar on the skin) ▪ Painless lesions ▪ Diffuse case: resembles “Lepromatous leprosy” • Leprosy - caused by Myobacterium leprae ▪ Lesions do not heal spontaneously and tend to relapse after treatment o American or Mucocutaneous Leishmaniasis: ▪ Caused by L. braziliensis ▪ Espundia – metastatic spread of lesion to oronasal and pharyngeal mucosa ▪ Tapir Nose – disfiguring leprosy-like tissue destruction and swelling ▪ Chiclero Ulcer – erosion of the pinna of the ear o Visceral Leishmaniasis ▪ Caused by L. donovani ▪ Other Name: Kala-azar, Dumdum fever ▪ Twice daily elevation of fever (prominent finding) ▪ Splenomegaly and cachexia ▪ Parasites are numerous in reticuloendothelial cells of the spleen, liver, lymph nodes, bone marrow and other organs. ▪ FATAL Laboratory Diagnosis: o Specimen: Blood and Tissue Sample o Laboratory Tests: ▪ Biopsy: • Cutaneous (skin) • Visceral (bone marrow, spleen) ▪ Serologic Tests: • Complement Fixation Test • IFA • Counter current electrophoresis technique Leishmania spp ETIOLOGIC VECTOR DISEASE AGENT ASSOCIATION L. Tropica Sandfly Cutaneous (Old world vectors Leishmaniasis Leishmaniasis, (Phlebotomus Cutaneous spp.) Leishmaniasis, Jericho boil, Baghdad boil) L. braziliensis American or (New world Mucocutaneous Leishmaniasis) Leishmaniasis L. donovani Visceral Leishmaniasis or Kala-Azar or Dumdum fever STAGES EXHIBITED Human: Amastigotes (reticuloendothelial system) Vector: Promastigote (midgut and proboscis) • • Treatment: o Pentavalent antimonials: sodium stibogluconate & n-methyl-glucamine antimonite o Second-line Drugs: Amphotericin B, Pentamidine (Kala-azar), Metronidazole, Nifurtimox LIFE CYCLE PHYLUM APICOMPLEXA • • • • • • • • Isospora belli Cryptosporidium parvum Cryptosporidium hominis Cyclospora cayetanensis Toxoplasma gondii Sarcocystic spp. Plasmodium spp. Babesia spp COCCIDIAN • • • • Spore forming , single-celled obligate intracellular parasites Habitat: Intestine Pathology: Opportunistic pathogens (not all) Life Cycle: o Sporogony – sexual cycle producing oocysts (outside human host−vector) o Schizogony – asexual cycle producing merozoites (inside human host−intermediate host) o Gametogony – development of male and female gametocytes o These are generalized characteristics. o Plasmodium spp. - do not have all of these stages Plasmodium spp. • • • • • • • • SPOROGONY • • • • occurs inside mosquito Sexual cycle Process 1. Microgamete enters macrogamete 2. Zygote 3. Ookinete 4. Oocyst (filled with sporozoites) 5. Sporozoite When oocyst ruptures, sporozoites may be injected Causative agent of Malaria (Mal’aria = bad air) Leading parasitic disease that causes mortality worldwide 4 species (in order of frequency in the Philippines): o Plasmodium falciparum – most common in the Philippines o Plasmodium vivax – most common worldwide o Plasmodium malariae o Plasmodium ovale (exclusively rare) Possible mixed infection with P. vivax and P. falciparum Mode of Transmission: vector borne Vectors (Philippines): Female Anopheles mosquito o Principal Vector: Anopheles minimus var flavirostris o Secondary Vector: Anopheles litoralis, Anopheles maculates, Anopheles mangyamus Final Host: Female Anopheles mosquito (sporogony) Intermediate Host: Man (schizogony) PRE-ERYTHROCYTIC CYCLE VECTOR BIOLOGY • • Vectors (Philippines): Female Anopheles mosquito o Principal Vector: Anopheles minimus var flavirostris o Secondary Vector: Anopheles litoralis, Anopheles maculates, Anopheles mangyamus o Habitat: slow flowing shaded streams o Biting time: Night biting (indoor and outdoor) o Resting place: inside walls Infective Stage: o Man: Sporozoites o Mosquito: Gametocytes • • • • • occurs inside human Or Exo-erythrocytic cycle Occur in liver cells or hepatocytes Schizogony cycle Process 1. Sporozoites infects liver parenchymal cells 2. Schizont ▪ Balloon like with merozoites 3. Liver cells rupture releasing merozoites 4. Enters erythrocytic cycle PERIODICITY/FEBRILE CYCLE| Species Febrile cycle P. falciparum Malignant Tertian/Subtertian P. vivax Benign Tertian P. malariae Quartan P. ovale Ovale tertian Interval (hours) 36-48 Type of RBC infected All 48 72 48 Young Old (senescent) Young MORPHOLOGY Point of Difference Size of RBC infect Trophozoite Number of merozoites in schizont Stipplings (dots in RBC) ERYTHROCYTIC CYCLE • • occurs inside human Process o Merozoites invade RBC ▪ Through RBC glycophorins (glycoproteins present in RBC structure) ▪ RBC glycophorins - entrance for merozoites o Ring forms (young trophozoites) o Mature trophozoites o Schizont (RBC) o Rupture of RBC releasing merozoites o Develop into a micro or macrogametocyte Ring forms/RBC Chromatin dot Applique or accole Gametocytes Asexual stages in peripheral blood Size of RBC infect Trophozoite Number of merozoites in schizont Stipplings Ring forms Chromatin dot Applique or accole Gametocytes Asexual stages in peripheral blood P. falciparum Normal Usually not present 12-32 (24) P.vivax Enlarged Amoeboid 12-24 (16) Maurer’s, StephenChristopher, Cuneiform dots Single/usually multiple Single/double Present Macro - cresent-shaped Micro - banana/sausage shaped Not all stages Schuffer’s dots P. malariae Normal Band form P. ovale Normal to sl. enlarged Fimbriated, round, compact Usually 8 (6-12) 6-12 (8: rosetteformation/fruit-pie/ flowerlike/daisy head) Ziemann’s dots Single Single Not present Round/oval All stages Single Single/densed Not present Round/oval All stages Jame’s dots Single Single Not present Round/oval All stages Plasmodium falciparum • • • Most dangerous Plasmodium spp. o Can infect multiple RBC at the same time o Can invade old and/or young RBC Cause of: o Cerebral Malaria o Blackwater Fever – sudden massive intravascular hemolysis resulting to hemoglobinuria ▪ Hemolysis - bursting of RBC ▪ Hemoglobinuria - Hgb in urine ▪ RING FORMS GAMETOCYTE SCHIZONT Immature trophozoites o Can become gametocytes (there will be gender) ▪ Either male or female are indistinguishable SPOROGONY & SCHIZOGONY SPECIES P. falciparum P. vivax P. malariae P. ovale LENGTH OF SPOROGONY 9-10 days 8-9 days 15-20 days 14 days LENGTH OF SCHIZOGONY 36-48 hours 48 hours 72 hours 48 hours • Take note of neighboring RBC o Neighboring uninfected RBC is same size with infected RB • Plasmodium vivax RING FORMS TROPHOZOITES GAMETOCYTES SCHIZONTS • • • • • • • Ring form - single Trophozoites - amoeboid Gametocytes - round/oval Schizonts - 12-24 (16) RBC size are usually larger than uninfected RBC • Plasmodium malariae RING FORMS TROPHOZOITES GAMETOCYTES SCHIZONTS Recrudence – renewal of parasitemia or its clinical features arising from persistent undetectable asexual parasitemia in the absence of exo-erythrocytic cycle Relapse – Reinfection after treatment o Common to P. vivax and P. ovale infections, as a result of the reactivation of hypnozoites/cryptozoites form of the parasite in the liver Cerebral Malaria – diffuse symmetric encephalopathy, retinal hemorrhage, bruxism, mild neck stiffness. If left untreated, may lead to coma and death. Pathological Process of RBC: 1. Poikylocytosis and anisocytosis ▪ Poikylocytosis - variation in RBC shape ▪ Anisocytosis - variation in RBC numbers 2. Altered RBC membrane transport 3. RBC stiffness and cytoplasmic viscosity ▪ In Malaria, RBC become less stiff due to presence of parasite inside the RBC altering exoskeleton of RBC DIAGNOSIS • • • • • • Ring form - single Trophozoites - band form Gametocytes - round/oval Schizonts - 6-12 (8) in rosette Infected RBC are normal sized Plasmodium ovale RING FORMS • • • • • • • TROPHOZOITES GAMETOCYTES SCHIZONTS Rare Ringform - single per RBC; single chromatin dot Trophozoites - packed Gametocytes - Round/oval, RBC is small Schizont - 6-12 Infected RBC - normal sized to slightly enlarged Fimbriation of RBC PATHOLOGY • Classical Malaria Paroxysms o Cold Stage ▪ Sudden coldness and apprehension ▪ Mild shivering turns to teeth chattering and shaking of whole body ▪ May last for 50-60 minutes o Hot Stage/Flush Phase – best stage to collect blood sample ▪ High temperature (40-41˚C) • Results from RBC invasion of merozoites ▪ Patient is confused and delirious ▪ May last for 2-6 hours o Sweating Stage (Defervescence or Diaphoresis) ▪ Profuse sweating, temperature lowers and symptoms diminishes ▪ May last for 2-4 hours • • Microscopy (Gold Standard) o “Thick & Thin Blood Smear” o Stained with Giemsa or Wright’s Stain o Manner of Reporting ▪ Qualitative (Thick blood smear) + 1-10 parasites/100 thick field ++ 11-100 parasites/100 thick field +++ 1-10 parasites/thick field ++++ More than 10/thick field ▪ Quantitative (Thin blood smear) Malaria parasite per uL = # of parasites x 8000 200 WBC o Thick smear ▪ 1 gtt blood + 1 gtt H2O ▪ Emulsify bloofd & H2O ▪ Water - to dehemoglobinize RBC for easier visualization Quantitative Buffy Coat (QBC) o Uses special capillary tubes with acridine orange o POSITIVE: bright green and yellow under fluorescent microscope to observe flourescence Rapid Diagnostic Test (RDT) – detects plasmodiumspecific antigens o Specimen - capillary blood o Histidine-rich Protein II (HRP II) – water soluble protein produced by trophozoites and young gametocytes ▪ Specific for P. falciparum o Plasmodium LDH – produced by both sexual and asexual stages and can distinguish between P. falciparum and non-P. falciparum ▪ Produced by all species of malaria ▪ Distinguishing factor o Mixed infection may happen bet. P. falciparum & P. vivax resulting in 2 bands ▪ ▪ Due to structure or morphology resembling other Plasmodium, it is harder to differentiate visually PCR amplifies DNA of P. knowlesi Babesia spp. • • • Serologic Tests: IHA, IFAT, ELISA Molecular Methods: o PCR – for low cases and mixed infections TREATMENT • • Prophylactic: Mefloquine and Doxycycline o Used for person going to endemic areas o Malaria is common in Palawan o Prevents in getting infection from vector bite Therapeutic o Arthemether-Lumefantrine – first line drug for P. falciparum cases o Quinine (plus Tetracycline or Doxycycline) – 2nd line drug for P. falciparum if AL fail or not available. o Quinine IV Drip – drug of choice for complicated or severe P. falciparum malaria o Primaquine – given on the 4th day as single dose to prevent transmission. RESISTANCE TO MALARIA • • • • Most Africans and American Blacks o Duffy null Phenotype/Fy (a-b-) – no duffy antigen present on red blood cell surface. ▪ Duffy - blood group system ▪ Duffy antigen - glycophorins (RBC membrane structures for entrance of merozoites) o Resistant to P. vivax and P. knowlesi Hemoglobinopathies like sickle-cell anemia o Specifically HbC or HbS variants o Sickle-cell anemia - RBC is cresent/sickle shape o RBC shape is not favorable for malaria G6PD Deficiency – resistant to P. falciparum Infants (first year of life) – high level of HbF (fetal hemoglobin) o HbF - is not favorable for P. falciparum Plasmodium knowlesi • • • • • • A primate malarial parasite Causes malaria in long-tailed macaques (Macaca fascicularis) May also infect man (incidental host) Described in humans in Philippines and Southeast Asia Morphology: o Early Trophozoite – similar with P. falciparum o Mature Trophozoite – similar with P. malariae Diagnosis: o PCR - most reliable method for detecting and diagnosing P. knowlesi infection Babesia microti o Blood parasites that cause malaria-like infections o “Babesiosis” – pathology due to B. microti o Parasites divide through binary fission or budding o Cycle in the stick is still uncertain o Vector: Ticks (Ixodes scapularis) ▪ Life cycle is not yet studied o Infective Stage: Sporozoites o Diagnostic Stage: ▪ “Maltese cross” arrangement of merozoites ▪ Ring-form trophozoites