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This document provides information on laboratory methods for diagnosing parasitic infections. It discusses examination of blood, fecal, and other specimens. For blood examination, it details methods for malaria diagnosis including thick and thin blood films and concentration techniques. It provides information on morphological stages of Plasmodium species. The document also briefly mentions Babesiosis diagnosis and symptoms.

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PART I
laboratory methods protozoa blood and tissue intestinal and urogenital
 examination of blood  examination of fecal specimens  examination of urogenital and other specimens (sputa, aspirates, biopsies)  immunodiagnostic methods  molecular diagnostic methods laboratory methods
examination of blood  malaria, babesiosis, trypanosomiasis, leishmaniasis, filariasis  thick and thin blood films  concentration techniques QBC blood parasite detection method Knott’s concentration or membrane filtration
examination of blood species ID parasite detection GIEMSA stain - host cell & parasite chromatin; Hb in rbc stains pale red - only stain that allows visualization of the rbc stippling
examination of blood species ID parasite diagnosis at least 200 fields (requiring at least 15 mins) using 100x objective at least 100 oil immersion fields (requiring at least 5 mins)
examination of blood  concentration techniques QBC blood parasite detection method uses fluorochrome acridine orange in a microhematocrit centrifuge format more sensitive than traditional thick/thin smears
examination of blood  Centrifugation (Knott’s technique)  Prepare 2% formaldehyde (2 ml of 37% formaldehyde + 98 ml H2O).  9 ml 2% formaldehyde + 1 ml of patient’s venous blood  centrifuge at 500 × g for 10 minutes  discard supernatant.  sediment = WBCs and microfilariae (if present).  temporary wet mounts.  thick and thin smears  allow to dry  dip in absolute methanol before Giemsa staining to enhance staining of microfilariae
examination of blood  Filtration  Millipore® or Nucleopore® membrane filter (5 µm pore) in filter holder with syringe attachment  1 ml of venous blood (in EDTA) + 10 ml of 10% Teepol® 610 (Shell Co.); allow to stand for several minutes to allow lysis;  transfer to a 10 ml Luer-Loc® syringe  attach the filter apparatus.  force the solution through the 5 µm pore filter, followed by several syringes of water to wash out the remaining blood, then 1 or 2 syringes full of air to clear excess fluid.  temporary wet mount: remove the filter and place it on a glass slide, adding a drop of stain or dye and a coverslip  permanent slide: pass 2 to 3 ml of methanol through the filter while it is still in the holder; remove filter and dry it on a glass slide; then stain it with Giemsa stain, horizontally (so that the filter does not wash off the slide); coverslip filter before examining.
examination of fecal specimens  specimen collection, handling & preservation  macroscopic examination  microscopic examinations direct wet mount concentration techniques permanent stains wheatley’s trichrome stain iron hematoxylin stain modified acid-fast stains stains for microsporidia additional techniques for examination of enteric parasites cellulose tape technique for pinworms egg studies nematode culture & recovery techniques
examination of fecal specimens  specimen collection, handling & preservation  fresh specimens: examined w/n 1 hr of passage  liquid specimens: w/n 30 minutes or placed immediately in preservatives  clean containers w/ tight-fitting lids  preservatives: 2-vial technique: 5-10% buffered formalin polyvinyl alcohol (PVA) fixative merthiolate-iodine-formalin (MIF) sodium acetate-formalin (SAF) zinc-sulfate-based PVA
examination of fecal specimens  specimen collection, handling & preservation  adequate O & P evaluation: 3 specimens collected every other day purgation  sodium sulfate, buffered phosphosoda  specimens collected in separate containers and submitted w/n minutes of collection G. lamblia Strongyloides E. histolytica
examination of fecal specimens  macroscopic examination consistency  formed  soft  loose  watery + mucus, blood, larval or adult worms & proglottids CYSTS TROPHOZOITES
examination of fecal specimens  microscopic examinations  direct wet mounts of fresh or preserved material fresh: allow detection & observation of motile protozoan trophozoites & helminth larvae preserved: allow detection of parasites that do not concentrate well  concentration techniques more sensitive increase the examiner’s ability to detect protozoan cysts & helminth eggs & larvae unsatisfactory for detecting protozoan trophozoites  permanent stains useful for detection & morphologic examination of protozoan trophozoites & cysts
examination of fecal specimens  microscopic examination  direct wet mount small amt of stool + drop of 0.85% saline (NaCl), covered w/ coverslip 10x, 40x or 1:5 dilution of Lugol’s iodine loss of trophozoite motility & cyst refractility difficulty in recognizing chromatoid bodies organisms: very pale and transparent (refractile objects) low light intensity
examination of fecal specimens  microscopic examination  concentration techniques  zinc sulfate centrifugal flotation technique of Faust  fresh/formalinized stool  parasites are recovered from the surface film of the solution following centrifugation  yields a cleaner preparation  unreliable for the recovery for nematode larvae, infertile eggs of ascaris, & the eggs of most trematodes & large tapeworms
examination of fecal specimens  microscopic examination  concentration techniques  formalin-ether/ethyl acetate sedimentation of Ritchie  efficient in recovering most protozoan cysts & helminths eggs & larvae, including operculate eggs  moderately effective for schistosome eggs  easiest to perform and least subject to technical error  less distortion of protozoal cysts than flotation technique
examination of fecal specimens  microscopic examination  permanent stains [100x] WHEATLEY’S TRICHROME STAIN IRON HEMATOXYLIN STAIN MODIFIED ACID-FAST STAINS STAINS FOR Microsporidia  depend on the age of the specimen, proper smear preparation & fixation, & quality of the reagents
examination of fecal specimens  microscopic examination  permanent stains [100x] WHEATLEY’S TRICHROME STAIN  most common: simple, reliable, cost-effective  stool specimens + Schaundinn’s or PVA fixatives
examination of fecal specimens  microscopic examination  permanent stains [100x] IRON HEMATOXYLIN STAIN  superior in enhancing key nuclear & cytoplasmic characteristics  preferred stain for SAF fixative
examination of fecal specimens  microscopic examination  permanent stains [100x] MODIFIED ACID-FAST STAINS  MODIFIED KINYOUN METHOD  MODIFIED ACID-FAST DIMETHYL SULFOXIDE  AURAMINE-O  sensitive & cost-effective  lack specificity
examination of fecal specimens  microscopic examination  permanent stains [100x] STAINS FOR Microsporidia  *Modified Trichrome Stain  Fluorescent Staining Method using whitening agents, Uvitek-2B & Calcofluor white
examination of fecal specimens  additional techniques for examination of Enteric Parasites CELLULOSE TAPE TECHNIQUE (PINWORMS) EGG STUDIES NEMATODE CULTURE & RECOVERY TECHNIQUES
examination of urogenital & other specimens [sputa, aspirates, biopsies]  Direct Wet mount  10X: Trichomonas vaginalis - jerky movement  Acid-fast or specific antibody-based stains  Cryptosporidium  Modified trichrome or fluorochrome stains  Microsporidia  same methods used in sputa  Giemsa stain (hemoflagellates) vaginal & urethral discharges, prostatic secretions, urine sputa aspirates
Immunodiagnostic methods
Molecular Diagnostic methods
blood & tissue protozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
Plasmodium species Plasmodium vivax Plasmodium ovale Plasmodium malariae Plasmodium falciparum female Anopheles  .  .
Plasmodium spp morphologic stages:  GROWING forms  trophozoites  DIVIDING forms  schizonts  SEXUAL forms  gametocytes
Plasmodium spp morphologic stages:  GROWING forms  trophozoites  DIVIDING forms  schizonts  SEXUAL forms  gametocytes
Plasmodium spp morphologic stages:  GROWING forms  trophozoites  DIVIDING forms  schizonts  SEXUAL forms  gametocytes
Plasmodium spp  DX  history of travel  [+] thick and thin blood films blood specimens are collected just prior to the next anticipated fever spike or at the outset of a fever  shaking chills  fever (≥400C)  generalized diaphoresis  resolution of fever  synchronous to the rupture of RBCs with the release of new infectious blood stage forms …..MEROZOITES
Plasmodium spp  DX  febrile periodicity tertian : P. vivax/ovale/falciparum quartan : P. malariae tropical & temperate areas: P. vivax tropical areas: P. falciparum worldwide: P. malariae
Plasmodium spp  THICK blood films  presence of the parasites  THIN blood films  appearance of infected RBCs  appearance of parasites  stages
 2-6: Young trophozoites (ring stage parasites);  7-18: Trophozoites; ameboid  19-27: Schizonts;  28 and 29: Macrogametocytes (female);  30: Microgametocyte (male)
 1: Normal red cell;  2-5: Young trophozoites (Rings);  6-15: Trophozoites;  compact  16-23: Schizonts;  24: Macrogametocytes (female);  25: Microgametocyte (male)
 1: Normal rbc  2-5: Young trophozoites (rings);  6-13: Trophozoites;  compact  band form  14-22: Schizonts;  23: Developing gametocyte;  24: Macrogametocyte (female);  25: Microgametocyte (male)
 1: Normal red cell;  2-18: Trophozoites  2-10 correspond to ring-stage trophozoites;  19-26: Schizonts  26: ruptured schizont;  27, 28: Mature macrogametocytes (female);  29, 30: Mature microgametocytes (male) size: 1/6 the diameter of RBC double chromatin dots applique or accole forms doubly infected cells
blood & tissue protozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
Babesiosis  Babesia microti : I. scapularis  Babesia divergens: I. ricinus  WA1 : I. pacificus Ixodes sp.  fever, chills, malaise, anemia  no febrile periodicity
Babesiosis
Babesiosis  fever, chills, malaise, anemia  no febrile periodicity  Hx of residence in or travel to endemic areas  HX of a recent tick bite  presence of parasites in blood films  IFA Ixodes sp.  DX
Babesiosis  multiple rings in 1 RBC  may form a tetrad  NO hemozoin pigment in infected cells  NO growing trophozoites & gametocytes
blood & tissue protozoa Malaria Babesiosis Hemoflaggelates: KINOPLAST – large mitochondrion Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
Hemoflagellates  morphologic forms  depending on their presence in vertebrate hosts (human) or their insect vectors
 T. brucei rhodesiense EAST AFRICA rapidly progressive acute febrile illness with LAD CNS involvement is not prominent  T. brucei gambiense WESTERN AFRICA CLASSIC AFRICAN SLEEPING SICKNESS chronic: intermittent fever, night sweats, malaise Winterbottom’s sign: cervical LAD Trypanosoma brucei Tsetse fly
Trypanosoma
Trypanosoma brucei  geographical Hx  clinical mx  high total IgG  BLOOD  CSF  pleocytosis - 50-500 mononuclear cells per microliter  presence of parasites  thick/thin bld films  buffy coat prep  LN/BM aspirates  spun CSF (Giemsa stain)  DX
Trypanosoma brucei Thin blood smear stained w/ Giemsa trypomastigotes  30 um long  small kinetoplast  graceful movement
Trypanosoma cruzi  AMERICAN TRYPANOSOMIASIS OR CHAGAS DISEASE Kissing bug/ REDUVIID
Trypanosoma cruzei
Trypanosoma cruzi  AMERICAN TRYPANOSOMIASIS OR CHAGAS DISEASE  Acute  mc: <5 y.o.  malaise, chills, fever, hepatomegaly, myocarditis  Romana’s sign  Chagoma  Chronic  destruction of the effector cells of the parasympathetic system by autoantibodies  megaesophagus  megacolon  alterations of cardiac conduction system
Trypanosoma cruzi  Acute stage  thick/thin blood films  buffy coat prep  aspirates of chagomas or enlarged LN  culture: Novy-MacNeal- Nicolle medium  Chronic stage  MOC: Serodiagnosis  EIA, IFA, CF  DX: trypomastigotes
Trypanosoma cruzi Blood Smear. Giemsa stain.  20 um shorter  S and C shapes  larger kinetoplast
blood & tissue protozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
Leishmania sp.  LEISHMANIASIS: disease of RE System  clinical forms  CUTANEOUS old world or oriental sore new world  MUCOCUTANEOUS espundia: L. braziliensis  VISCERAL old world: L. donovani new world: L. chagasi KALA-AZAR Sandfly
Leishmania
Leishmania  amastigotes  smears  imprints  biopsies  growth of promastigotes in culture  DX
Leishmania…… amastigotes skin touch imprint inside the macrophage BM smear. Giemsa stain. 2-4um delicate cytoplasm nucleus kinetoplast
Leishmania….promastigotes Culture
blood & tissue protozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
Toxoplasma gondii Toxoplasma gondii • organ transplantation •blood transfusion •transplacental
Toxoplasma gondii  Tachyzoites/tissue cyst  bld/bloody fluids: acute  tissues : chronic  H&E stain  fluorescent or immunoperoxidase stains  Giemsa stain  growth of promastigotes in culture  DX
Toxoplasma gondii tachyzoites  3x7 um  crescent-shaped or oval  central nucleus
blood & tissue protozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae NAEGLERIA ACANTHAMOEBA BALAMUTHIA
 dx  autopsy tissue sections: brain  antemortem dx: CSF • direct wet mounts • stained prep • culture: • non-nutrient agar seeded w/ lawn of heat-killed or living Escherichia coli
NAEGLERIA  trophozoites ameboid 10-35 um large round central karyosome CSF. Trichrome stain. Culture from CSF.
NAEGLERIA Histopathology of amebic meningoencephalitis due to Naegleria fowleri. CDC. Direct fluorescent antibody stain.
 dx  (autopsy) brain tissue  culture • Acanthamoeba • same w/ Naegleria • Balamuthia • tissue culture using mammalian cell lines
Acanthamoeba CSF. trophozoites: acanthopodia double-walled cysts
Acanthamoeba & Balamuthia  DX antigenically distinct specific monoclonal or polyclonal antisera in immunofluorescence or immunoperoxidase assays
blood & tissue protozoa
intestinal & urogenital protozoa Amebae Entamoeba histolytica Nonpathogenic amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
Diagnosis:  MICROSCOPIC EXAMINATION  direct wet mount  permanent stain  Stool exam  Aspirates  LIVER ABSCESS  last material aspirated  Tissue sections  PAS stain  CULTURE  E. histolytica/dispar  EIA ANTIGEN DETECTION TESTS  PCR  DNA probes  Extraintestinal infections  Serologic tests  EIA, ID, IHA
trophozoites cysts
NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin Appearance Inclusions Entamoeba histolytica 10-60 mm. Usual range, 15-20 mm commensal form.1 Over 20 mm invasive form.2 Progressive with hyaline, finger-like pseudopods. 1 Not visible in unstained preparations. Fine granules. Usually evenly distributed and uniform in size. Small, discrete. Usually centrally located, but occasionally is eccentric. Finely qranular. Red blood cells occasionally. Noninvasive organisms may contain bacteria. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Entamoeba histolytica 10-20 mm Usual range, 12-15 mm. Usually spherical. 4 in mature cyst. Immature cysts with 1 or 2 occasionally seen. Peripheral chromatin present. Fine, uniform granules, evenly distributed. Small, discrete, usually centrally located. Present. Elongated bars with bluntly rounded ends. Usually diffuse. Concentrated mass often present in young cysts. Stains reddish brown with iodine.
NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin Appearance Inclusions Entamoeba hartmanni 5-12mm. Usual range, 8-10 mm. Usually non- progressive but may be progressive occasionally. 1 Not visible in unstained preparations. Similar to E. histolytica. Small, discrete, often eccentric. Finely granular. Bacteria. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Entamoeba hartmanni 5-10 mm Usual range, 6-8 mm. Usually spherical. 4 in mature cyst. Immature cysts with 1 or 2 often seen. Similar to E. histolytica. Similar to E. histolytica. Present. Elongated bars with bluntly rounded ends. Similar to E. histolytica.
NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin Appearance Inclusions Entamoeba coli 15-50mm. Usual range, 20-25 mm. Sluggish, non- progressive, with blunt pseudopods. 1 Often visible in unstained preparations. Coarse granules, irregular in size and distribution. Large, discrete, usually eccentric. Coarse, often vacuolated. Bacteria, yeasts, other materials. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Entamoeba coli 10-35 mm Usual range, 15-25 mm. Usually spherical. Occasionally oval, triangular, or other shapes. 8 in mature cyst. Occasionally super- nucleated cysts with 16 or more are seen. Immature cysts with 2 or more occasionally seen. Peripheral chromatin present. Coarse granules irregular in size and distribution, but often appear more uniform than in trophozoites. Large, discrete, usually eccentric but occasionally centrally located. Present, but less frequently seen than in E. histolytica. Usually splinter-like with pointed ends. Usually diffuse, but, occasionally well defined mass in immature cysts. Stain reddish brown with iodine.
NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin Appearance Inclusions Endolimax nana 6-12 mm. Usual range, 8-10 mm. Sluggish, usually non- progressive with blunt pseudopods. 1 Visible occasionally in unstained preparations. None. Large, irregularly shaped, blot-like. Granular, vacuolated. Bacteria. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Endolimax nana 5-10 mm. Usual range, 6-8 mm. Spherical, ovoidal, or ellipsoidal. 4 in immature cysts. Immature cysts with less than 4 rarely seen. None Large (blot-like), usually central. Occasionally granules or small oval masses seen, but bodies as seen in Entamoeba spp. are not present. Usually diffuse. Concentrated mass seen occasionally in young cysts. Stains reddish brown with iodine.
NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin Appearance Inclusions Iodamoeba buetschlii 8-20 mm. Usual range, 12-15 mm. Sluggish, usually non- progressive. 1 Not usually visible in unstained preparations. None. Large, usually central. Surrounded by refractile, achromatic granules. These granules are often not distinct even in stained slides. Coarsely granular, vacuolated. Bacteria, yeasts, or other material. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Iodamoeba buetschlii 5-20 mm. Usual range, 10-12 mm Ovoidal, ellipsoidal, triangular, or other shapes. 1 in mature cyst. None Large, usually eccentric. Refractile, achromatic granules on one side of karyosome. Indistinct in iodine preparations. Occasionally granules present, but chromatoid bodies as seen in Entamoeba spp. are not present. Compact, well-defined mass. Stains dark brown with iodine.
intestinal & urogenital protozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
Blastocystic hominis
Blastocystic hominis Size Shape Motility Number of Nuclei Other Features 5-30 mm. Usual range, 8-10 mm. Spherical, oval, or ellipsoidal Nonmotile 1, usually, but 2-4 may be present. Located in "rim" of cytoplasm. In binucleated organisms, the 2 nuclei may be at opposite poles. In quadrinucleated forms, the 4 nuclei are evenly spaced around periphery of cell. Cell contains large central body, or "vacuole" with a thin band, or "rim" of cytoplasm around the periphery. Occasionally a ring of granules may be seen in cytoplasm and the cell appears to have a "beaded rim."  may predominate in heavy infections
Blastocystic hominis trichrome stain
intestinal & urogenital protozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
Dientamoeba fragilis MULTIPLE Stool examinations  [last portion of the stool evacuated]
Dientamoeba fragilis NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin Appearance Inclusions Dientamoeba fragilis3 5-15 mm. Usual range, 9- 12 mm. Pseudopods are angular, serrated, or broad lobed, and hyaline, almost transparent. 2 (In approximatel y 20% of organisms only 1 nucleus is present.) Nuclei invisible in unstained preparation s. None. Large cluster of 4-8 granules. Finely, qranular. Bacteria: occasional ly red blood cells.
Giardia lamblia
Giardia lamblia  DX  Microscopic examinations direct wet mount concentration techniques (cyst) permanent stain  Small bowel aspirates or String test specimens  Antigen detection methods
Giardia lamblia Size (Length) Shape Motility Number of Nuclei Number of Flagella* Other Features 10-20 mm. Usual range, 12-15 mm. Pear shaped. “Falling leaf.” 2 Not visible in unstained mounts. 4 lateral. 2 ventral. 2 caudal. Sucking disk occupying 1/2-3/4 of ventral surface. Median bodies lying horizontally or obliquely in lower part of body.
Giardia lamblia Size (Length) Shape Number of Nuclei Other Features 8-19 mm. Usual range. 11-12 mm. Oval or ellipsoidal. Usually 4. Not distinct in unstained preparations. Usually located at one end. Fibrils or flagella longitudinally in unstained cysts. Deep staining fibers or fibrils may be seen lying laterally or obliquely across fibrils in lower part of cyst. Cytoplasm often retracts from a portion of cell wall.
Chilomastix mesnili Chilomastix mesnili
Chilomastix mesnili Size (Length) Shape Motility Number of Nuclei Number of Flagella* Other Features 6-24 mm. Usual range, 10-15 mm. Pear shaped. Stiff, rotary. 1 Not visible in unstained mounts. 3 anterior. 1 in cytosome. Prominent cytostome extending 1/3-1/2 length of body. Spiral groove across ventral surface.
Chilomastix mesnili Species Size (Length) Shape Number of Nuclei Other Features 6-10 mm. Usual range, 8-9 mm. Lemon shaped with anterior hyaline knob. 1. Not visible in unstained preparations. Cytostome with supporting fibrils. Usually visible in stained preparations.
Trichomonas vaginalis asymptomatic
Trichomonas vaginalis  DX:  vaginal fluid, prostatic fluid, sediments of freshly passed urine Direct wet mount Culture Immunofluorescent & EIA techniques monoclonal antibodies  Papanicolaou-stained gyne smears
Trichomonas vaginalis
Pentatrichomonas/Trichomonas hominis Size (Length) Shape Motility Number of Nuclei Number of Flagella* Other Features 8-20 mm. Usual range. 11- 12 mm. Pear shaped. Nervous, jerky. 1 Not visible in unstained mounts. 3-5 anterior. 1 posterior. Undulating membrane extending length of body.
Trichomonas tenax  nonpathogenic  occasionally recovered from the oral cavity
Enteromonas hominis Retortamonas intestinalis
Enteromonas hominis Species Size (Length) Shape Motility Number of Nuclei Number of Flagella* Other Features Enteromonas hominis 4-10 mm. Usual range, 8-9 mm. Oval. Jerky. 1 Not visible in unstained mounts. 3 anterior. 1 posterior. One side of body flattened. Posterior flagellum extends free posteriorly or laterally.
Enteromonas hominis Species Size (Length) Shape Number of Nuclei Other Features 4-10 mm. Usual range, 6-8 mm. Elongated or oval. 1-4, usually 2 lying at opposite ends of cyst. Not visible in unstained mounts. Resembles E. nana cyst. Fibrils or flagella are usually not seen.
Retortamonas intestinalis Species Size (Length) Shape Motility Number of Nuclei Number of Flagella* Other Features Retortamon as intestinalis 4-9 mm. Usual range, 6-7 mm. Pear shaped or oval. Jerky. 1 Not visible in unstained mounts. 1 anterior. 1 posterior. Prominent cytostome extending approximately 1/2 length of body.
Retortamonas intestinalis Species Size (Length) Shape Number of Nuclei Other Features 4-9 mm. Usual range, 4-7 mm. Pear shaped or slightly lemon shaped. 1. Not visible in unstained mounts. Resembles Chilomastix cyst. Shadow outline of cytostome with supporting fibrils extends above nucleus.
intestinal & urogenital protozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
Balantidium coli
Balantidium coli Species Size Shape Motility Number of Nuclei Other Features Trophozoite 50-70 mm or more. Usual range, 40- 50 mm. Ovoid with tapering anterior end. Rotary, boring. 1 large, kidney shaped macronucleus. 1 small micronucleus immediately adjacent to macronucleus. Macronucleus occasionally visible in unstained preparations as hyaline mass. Body surface covered by spiral, longitudinal rows of cilia. Contractile vacuoles are present. Cyst 45-65 mm. Usual range, 50-55 mm. Spherical or oval. 1 large macronucleus visible in unstained preparations as hyaline mass. Macronucleus and contractile vacuole are visible in young cysts. In older cysts, internal structure appears granular.
intestinal & urogenital protozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
Isospora belli
Isospora belli  DX  UNSPORULATED OOCYSTs direct wet mounts concentration techniques acid-fast stain
Isospora belli Size Shape Motility Number of Nuclei Other Features Oocyst: 25-30 mm. Usual range, 28- 30 mm. Ellipsoidal Nonmotile Usual diagnostic stage is immature oocyst with single granular mass (zygote) within. Mature oocyst contains 2 sporocysts with 4 sporozoites each.
Sarcocystis sp.
Sarcocystis sp.  DX  SPORULATED SPOROCYSTS direct wet mounts acid-fast stains
Sarcocystis sp. Species Size Shape Motility Number of Nuclei Other Features Sporocyst1 Oval Nonmotile Mature oocysts with thin wall collapsed around 2 sporocysts or free fully mature sporocysts with 4 sporozoites inside are usually seen in feces. hominis 13-17 mm. Usual range, 14-16 mm. suihominis 11-15 mm. Usual range, 12-13 mm.
Sarcocystis sp. wet mount viewed in uv microscopy. cyst in undercooked meat
Cryptosporidium sp. Cryptosporidium
Cryptosporidium sp.  DX  concentration methods  acid-fast stain: MODIFIED COLD KINYOUN METHOD deep fuscia  commercial immunofluorescent & EIA reagents
Cryptosporidium sp. Species Size Shape Motility Number of Nuclei Other Features Cryptosporidium Oocyst: 3-6 mm. Usual range, 4-5 mm. Spherical or oval. Nonmotile Mature oocyst contains 4 "naked" sporozoites. No sporocysts are present.
Cyclospera cayetanensis Cyclospera cayetanensis
Cyclospera cayetanensis  DX: unsporulated oocysts  direct wet mount  trichrome stain  UV epifluorescence  modified acid-fast stain  auramine-o stain
intestinal & urogenital protozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
Microsporidia NO reservoir hosts identified! merogony
Microsporidia  DX  tissue examination routine light & electron microscopy  stool specimens modified trichrome staining method (WEBER) Fluorochrome stain Uvitex 2B, Calcoflour white
Microsporidia  Enterocytozoon bieneusi  Light microscopy photographs of stained stool smears Quick-Hot Gram Chromotrope stain Weber stain
Microsporidia  Enterocytozoon bieneusi mature spore.  double rows of polar tubule coils in cross section electron micrograph.  Encephalitozoon intestinalis spores and developing forms inside septated parasitophorous vacuole.
Microsporidia Scanning electron micrograph of a microsporidian spore with an extruded polar tubule inserted into a eukaryotic cell. The spore injects the infective sporoplasms through its polar tubule.

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CM Parasitic Infections 1.pdf

  • 1.
  • 2.
    laboratory methods protozoa blood andtissue intestinal and urogenital
  • 3.
     examination ofblood  examination of fecal specimens  examination of urogenital and other specimens (sputa, aspirates, biopsies)  immunodiagnostic methods  molecular diagnostic methods laboratory methods
  • 4.
    examination of blood malaria, babesiosis, trypanosomiasis, leishmaniasis, filariasis  thick and thin blood films  concentration techniques QBC blood parasite detection method Knott’s concentration or membrane filtration
  • 5.
    examination of blood speciesID parasite detection GIEMSA stain - host cell & parasite chromatin; Hb in rbc stains pale red - only stain that allows visualization of the rbc stippling
  • 6.
    examination of blood speciesID parasite diagnosis at least 200 fields (requiring at least 15 mins) using 100x objective at least 100 oil immersion fields (requiring at least 5 mins)
  • 7.
    examination of blood concentration techniques QBC blood parasite detection method uses fluorochrome acridine orange in a microhematocrit centrifuge format more sensitive than traditional thick/thin smears
  • 8.
    examination of blood Centrifugation (Knott’s technique)  Prepare 2% formaldehyde (2 ml of 37% formaldehyde + 98 ml H2O).  9 ml 2% formaldehyde + 1 ml of patient’s venous blood  centrifuge at 500 × g for 10 minutes  discard supernatant.  sediment = WBCs and microfilariae (if present).  temporary wet mounts.  thick and thin smears  allow to dry  dip in absolute methanol before Giemsa staining to enhance staining of microfilariae
  • 9.
    examination of blood Filtration  Millipore® or Nucleopore® membrane filter (5 µm pore) in filter holder with syringe attachment  1 ml of venous blood (in EDTA) + 10 ml of 10% Teepol® 610 (Shell Co.); allow to stand for several minutes to allow lysis;  transfer to a 10 ml Luer-Loc® syringe  attach the filter apparatus.  force the solution through the 5 µm pore filter, followed by several syringes of water to wash out the remaining blood, then 1 or 2 syringes full of air to clear excess fluid.  temporary wet mount: remove the filter and place it on a glass slide, adding a drop of stain or dye and a coverslip  permanent slide: pass 2 to 3 ml of methanol through the filter while it is still in the holder; remove filter and dry it on a glass slide; then stain it with Giemsa stain, horizontally (so that the filter does not wash off the slide); coverslip filter before examining.
  • 10.
    examination of fecalspecimens  specimen collection, handling & preservation  macroscopic examination  microscopic examinations direct wet mount concentration techniques permanent stains wheatley’s trichrome stain iron hematoxylin stain modified acid-fast stains stains for microsporidia additional techniques for examination of enteric parasites cellulose tape technique for pinworms egg studies nematode culture & recovery techniques
  • 11.
    examination of fecalspecimens  specimen collection, handling & preservation  fresh specimens: examined w/n 1 hr of passage  liquid specimens: w/n 30 minutes or placed immediately in preservatives  clean containers w/ tight-fitting lids  preservatives: 2-vial technique: 5-10% buffered formalin polyvinyl alcohol (PVA) fixative merthiolate-iodine-formalin (MIF) sodium acetate-formalin (SAF) zinc-sulfate-based PVA
  • 12.
    examination of fecalspecimens  specimen collection, handling & preservation  adequate O & P evaluation: 3 specimens collected every other day purgation  sodium sulfate, buffered phosphosoda  specimens collected in separate containers and submitted w/n minutes of collection G. lamblia Strongyloides E. histolytica
  • 13.
    examination of fecalspecimens  macroscopic examination consistency  formed  soft  loose  watery + mucus, blood, larval or adult worms & proglottids CYSTS TROPHOZOITES
  • 14.
    examination of fecalspecimens  microscopic examinations  direct wet mounts of fresh or preserved material fresh: allow detection & observation of motile protozoan trophozoites & helminth larvae preserved: allow detection of parasites that do not concentrate well  concentration techniques more sensitive increase the examiner’s ability to detect protozoan cysts & helminth eggs & larvae unsatisfactory for detecting protozoan trophozoites  permanent stains useful for detection & morphologic examination of protozoan trophozoites & cysts
  • 15.
    examination of fecalspecimens  microscopic examination  direct wet mount small amt of stool + drop of 0.85% saline (NaCl), covered w/ coverslip 10x, 40x or 1:5 dilution of Lugol’s iodine loss of trophozoite motility & cyst refractility difficulty in recognizing chromatoid bodies organisms: very pale and transparent (refractile objects) low light intensity
  • 16.
    examination of fecalspecimens  microscopic examination  concentration techniques  zinc sulfate centrifugal flotation technique of Faust  fresh/formalinized stool  parasites are recovered from the surface film of the solution following centrifugation  yields a cleaner preparation  unreliable for the recovery for nematode larvae, infertile eggs of ascaris, & the eggs of most trematodes & large tapeworms
  • 17.
    examination of fecalspecimens  microscopic examination  concentration techniques  formalin-ether/ethyl acetate sedimentation of Ritchie  efficient in recovering most protozoan cysts & helminths eggs & larvae, including operculate eggs  moderately effective for schistosome eggs  easiest to perform and least subject to technical error  less distortion of protozoal cysts than flotation technique
  • 18.
    examination of fecalspecimens  microscopic examination  permanent stains [100x] WHEATLEY’S TRICHROME STAIN IRON HEMATOXYLIN STAIN MODIFIED ACID-FAST STAINS STAINS FOR Microsporidia  depend on the age of the specimen, proper smear preparation & fixation, & quality of the reagents
  • 19.
    examination of fecalspecimens  microscopic examination  permanent stains [100x] WHEATLEY’S TRICHROME STAIN  most common: simple, reliable, cost-effective  stool specimens + Schaundinn’s or PVA fixatives
  • 20.
    examination of fecalspecimens  microscopic examination  permanent stains [100x] IRON HEMATOXYLIN STAIN  superior in enhancing key nuclear & cytoplasmic characteristics  preferred stain for SAF fixative
  • 21.
    examination of fecalspecimens  microscopic examination  permanent stains [100x] MODIFIED ACID-FAST STAINS  MODIFIED KINYOUN METHOD  MODIFIED ACID-FAST DIMETHYL SULFOXIDE  AURAMINE-O  sensitive & cost-effective  lack specificity
  • 22.
    examination of fecalspecimens  microscopic examination  permanent stains [100x] STAINS FOR Microsporidia  *Modified Trichrome Stain  Fluorescent Staining Method using whitening agents, Uvitek-2B & Calcofluor white
  • 23.
    examination of fecalspecimens  additional techniques for examination of Enteric Parasites CELLULOSE TAPE TECHNIQUE (PINWORMS) EGG STUDIES NEMATODE CULTURE & RECOVERY TECHNIQUES
  • 24.
    examination of urogenital& other specimens [sputa, aspirates, biopsies]  Direct Wet mount  10X: Trichomonas vaginalis - jerky movement  Acid-fast or specific antibody-based stains  Cryptosporidium  Modified trichrome or fluorochrome stains  Microsporidia  same methods used in sputa  Giemsa stain (hemoflagellates) vaginal & urethral discharges, prostatic secretions, urine sputa aspirates
  • 26.
  • 27.
  • 28.
    blood & tissueprotozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
  • 29.
    Plasmodium species Plasmodium vivax Plasmodiumovale Plasmodium malariae Plasmodium falciparum female Anopheles  .  .
  • 31.
    Plasmodium spp morphologic stages: GROWING forms  trophozoites  DIVIDING forms  schizonts  SEXUAL forms  gametocytes
  • 32.
    Plasmodium spp morphologic stages: GROWING forms  trophozoites  DIVIDING forms  schizonts  SEXUAL forms  gametocytes
  • 33.
    Plasmodium spp morphologic stages: GROWING forms  trophozoites  DIVIDING forms  schizonts  SEXUAL forms  gametocytes
  • 34.
    Plasmodium spp  DX history of travel  [+] thick and thin blood films blood specimens are collected just prior to the next anticipated fever spike or at the outset of a fever  shaking chills  fever (≥400C)  generalized diaphoresis  resolution of fever  synchronous to the rupture of RBCs with the release of new infectious blood stage forms …..MEROZOITES
  • 35.
    Plasmodium spp  DX febrile periodicity tertian : P. vivax/ovale/falciparum quartan : P. malariae tropical & temperate areas: P. vivax tropical areas: P. falciparum worldwide: P. malariae
  • 36.
    Plasmodium spp  THICKblood films  presence of the parasites  THIN blood films  appearance of infected RBCs  appearance of parasites  stages
  • 37.
     2-6: Youngtrophozoites (ring stage parasites);  7-18: Trophozoites; ameboid  19-27: Schizonts;  28 and 29: Macrogametocytes (female);  30: Microgametocyte (male)
  • 38.
     1: Normalred cell;  2-5: Young trophozoites (Rings);  6-15: Trophozoites;  compact  16-23: Schizonts;  24: Macrogametocytes (female);  25: Microgametocyte (male)
  • 39.
     1: Normalrbc  2-5: Young trophozoites (rings);  6-13: Trophozoites;  compact  band form  14-22: Schizonts;  23: Developing gametocyte;  24: Macrogametocyte (female);  25: Microgametocyte (male)
  • 40.
     1: Normalred cell;  2-18: Trophozoites  2-10 correspond to ring-stage trophozoites;  19-26: Schizonts  26: ruptured schizont;  27, 28: Mature macrogametocytes (female);  29, 30: Mature microgametocytes (male) size: 1/6 the diameter of RBC double chromatin dots applique or accole forms doubly infected cells
  • 41.
    blood & tissueprotozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
  • 42.
    Babesiosis  Babesia microti: I. scapularis  Babesia divergens: I. ricinus  WA1 : I. pacificus Ixodes sp.  fever, chills, malaise, anemia  no febrile periodicity
  • 43.
  • 44.
    Babesiosis  fever, chills,malaise, anemia  no febrile periodicity  Hx of residence in or travel to endemic areas  HX of a recent tick bite  presence of parasites in blood films  IFA Ixodes sp.  DX
  • 45.
    Babesiosis  multiple ringsin 1 RBC  may form a tetrad  NO hemozoin pigment in infected cells  NO growing trophozoites & gametocytes
  • 46.
    blood & tissueprotozoa Malaria Babesiosis Hemoflaggelates: KINOPLAST – large mitochondrion Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
  • 47.
    Hemoflagellates  morphologic forms depending on their presence in vertebrate hosts (human) or their insect vectors
  • 48.
     T. bruceirhodesiense EAST AFRICA rapidly progressive acute febrile illness with LAD CNS involvement is not prominent  T. brucei gambiense WESTERN AFRICA CLASSIC AFRICAN SLEEPING SICKNESS chronic: intermittent fever, night sweats, malaise Winterbottom’s sign: cervical LAD Trypanosoma brucei Tsetse fly
  • 49.
  • 50.
    Trypanosoma brucei  geographicalHx  clinical mx  high total IgG  BLOOD  CSF  pleocytosis - 50-500 mononuclear cells per microliter  presence of parasites  thick/thin bld films  buffy coat prep  LN/BM aspirates  spun CSF (Giemsa stain)  DX
  • 51.
    Trypanosoma brucei Thin bloodsmear stained w/ Giemsa trypomastigotes  30 um long  small kinetoplast  graceful movement
  • 52.
    Trypanosoma cruzi  AMERICANTRYPANOSOMIASIS OR CHAGAS DISEASE Kissing bug/ REDUVIID
  • 53.
  • 54.
    Trypanosoma cruzi  AMERICANTRYPANOSOMIASIS OR CHAGAS DISEASE  Acute  mc: <5 y.o.  malaise, chills, fever, hepatomegaly, myocarditis  Romana’s sign  Chagoma  Chronic  destruction of the effector cells of the parasympathetic system by autoantibodies  megaesophagus  megacolon  alterations of cardiac conduction system
  • 55.
    Trypanosoma cruzi  Acutestage  thick/thin blood films  buffy coat prep  aspirates of chagomas or enlarged LN  culture: Novy-MacNeal- Nicolle medium  Chronic stage  MOC: Serodiagnosis  EIA, IFA, CF  DX: trypomastigotes
  • 56.
    Trypanosoma cruzi Blood Smear.Giemsa stain.  20 um shorter  S and C shapes  larger kinetoplast
  • 57.
    blood & tissueprotozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
  • 58.
    Leishmania sp.  LEISHMANIASIS:disease of RE System  clinical forms  CUTANEOUS old world or oriental sore new world  MUCOCUTANEOUS espundia: L. braziliensis  VISCERAL old world: L. donovani new world: L. chagasi KALA-AZAR Sandfly
  • 59.
  • 60.
    Leishmania  amastigotes  smears imprints  biopsies  growth of promastigotes in culture  DX
  • 61.
    Leishmania…… amastigotes skin touchimprint inside the macrophage BM smear. Giemsa stain. 2-4um delicate cytoplasm nucleus kinetoplast
  • 62.
  • 63.
    blood & tissueprotozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae
  • 64.
    Toxoplasma gondii Toxoplasma gondii •organ transplantation •blood transfusion •transplacental
  • 65.
    Toxoplasma gondii  Tachyzoites/tissuecyst  bld/bloody fluids: acute  tissues : chronic  H&E stain  fluorescent or immunoperoxidase stains  Giemsa stain  growth of promastigotes in culture  DX
  • 66.
    Toxoplasma gondii tachyzoites  3x7um  crescent-shaped or oval  central nucleus
  • 67.
    blood & tissueprotozoa Malaria Babesiosis Hemoflaggelates: Trypanosoma Leishmania Toxoplasma gondii Opportunistic Free-living Amebae NAEGLERIA ACANTHAMOEBA BALAMUTHIA
  • 68.
     dx  autopsytissue sections: brain  antemortem dx: CSF • direct wet mounts • stained prep • culture: • non-nutrient agar seeded w/ lawn of heat-killed or living Escherichia coli
  • 69.
    NAEGLERIA  trophozoites ameboid 10-35 um largeround central karyosome CSF. Trichrome stain. Culture from CSF.
  • 70.
    NAEGLERIA Histopathology of amebicmeningoencephalitis due to Naegleria fowleri. CDC. Direct fluorescent antibody stain.
  • 71.
     dx  (autopsy)brain tissue  culture • Acanthamoeba • same w/ Naegleria • Balamuthia • tissue culture using mammalian cell lines
  • 72.
  • 73.
    Acanthamoeba & Balamuthia DX antigenically distinct specific monoclonal or polyclonal antisera in immunofluorescence or immunoperoxidase assays
  • 74.
  • 75.
    intestinal & urogenitalprotozoa Amebae Entamoeba histolytica Nonpathogenic amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
  • 77.
    Diagnosis:  MICROSCOPIC EXAMINATION  directwet mount  permanent stain  Stool exam  Aspirates  LIVER ABSCESS  last material aspirated  Tissue sections  PAS stain  CULTURE  E. histolytica/dispar  EIA ANTIGEN DETECTION TESTS  PCR  DNA probes  Extraintestinal infections  Serologic tests  EIA, ID, IHA
  • 79.
  • 80.
    NUCLEUS CYTOPLASM Species Size(Length) Motility Number Peripheral Chromatin Karyosomal Chromatin Appearance Inclusions Entamoeba histolytica 10-60 mm. Usual range, 15-20 mm commensal form.1 Over 20 mm invasive form.2 Progressive with hyaline, finger-like pseudopods. 1 Not visible in unstained preparations. Fine granules. Usually evenly distributed and uniform in size. Small, discrete. Usually centrally located, but occasionally is eccentric. Finely qranular. Red blood cells occasionally. Noninvasive organisms may contain bacteria. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Entamoeba histolytica 10-20 mm Usual range, 12-15 mm. Usually spherical. 4 in mature cyst. Immature cysts with 1 or 2 occasionally seen. Peripheral chromatin present. Fine, uniform granules, evenly distributed. Small, discrete, usually centrally located. Present. Elongated bars with bluntly rounded ends. Usually diffuse. Concentrated mass often present in young cysts. Stains reddish brown with iodine.
  • 82.
    NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin AppearanceInclusions Entamoeba hartmanni 5-12mm. Usual range, 8-10 mm. Usually non- progressive but may be progressive occasionally. 1 Not visible in unstained preparations. Similar to E. histolytica. Small, discrete, often eccentric. Finely granular. Bacteria. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Entamoeba hartmanni 5-10 mm Usual range, 6-8 mm. Usually spherical. 4 in mature cyst. Immature cysts with 1 or 2 often seen. Similar to E. histolytica. Similar to E. histolytica. Present. Elongated bars with bluntly rounded ends. Similar to E. histolytica.
  • 84.
    NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin AppearanceInclusions Entamoeba coli 15-50mm. Usual range, 20-25 mm. Sluggish, non- progressive, with blunt pseudopods. 1 Often visible in unstained preparations. Coarse granules, irregular in size and distribution. Large, discrete, usually eccentric. Coarse, often vacuolated. Bacteria, yeasts, other materials. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Entamoeba coli 10-35 mm Usual range, 15-25 mm. Usually spherical. Occasionally oval, triangular, or other shapes. 8 in mature cyst. Occasionally super- nucleated cysts with 16 or more are seen. Immature cysts with 2 or more occasionally seen. Peripheral chromatin present. Coarse granules irregular in size and distribution, but often appear more uniform than in trophozoites. Large, discrete, usually eccentric but occasionally centrally located. Present, but less frequently seen than in E. histolytica. Usually splinter-like with pointed ends. Usually diffuse, but, occasionally well defined mass in immature cysts. Stain reddish brown with iodine.
  • 86.
    NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin AppearanceInclusions Endolimax nana 6-12 mm. Usual range, 8-10 mm. Sluggish, usually non- progressive with blunt pseudopods. 1 Visible occasionally in unstained preparations. None. Large, irregularly shaped, blot-like. Granular, vacuolated. Bacteria. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Endolimax nana 5-10 mm. Usual range, 6-8 mm. Spherical, ovoidal, or ellipsoidal. 4 in immature cysts. Immature cysts with less than 4 rarely seen. None Large (blot-like), usually central. Occasionally granules or small oval masses seen, but bodies as seen in Entamoeba spp. are not present. Usually diffuse. Concentrated mass seen occasionally in young cysts. Stains reddish brown with iodine.
  • 88.
    NUCLEUS CYTOPLASM Species Size (Length) Motility Number Peripheral Chromatin Karyosomal Chromatin AppearanceInclusions Iodamoeba buetschlii 8-20 mm. Usual range, 12-15 mm. Sluggish, usually non- progressive. 1 Not usually visible in unstained preparations. None. Large, usually central. Surrounded by refractile, achromatic granules. These granules are often not distinct even in stained slides. Coarsely granular, vacuolated. Bacteria, yeasts, or other material. NUCLEUS CYTOPLASM Species Size (Diameter or length) Shape Number Peripheral Chromatin Karyosomal Chromatin Chromatoid Bodies Glycogen Iodamoeba buetschlii 5-20 mm. Usual range, 10-12 mm Ovoidal, ellipsoidal, triangular, or other shapes. 1 in mature cyst. None Large, usually eccentric. Refractile, achromatic granules on one side of karyosome. Indistinct in iodine preparations. Occasionally granules present, but chromatoid bodies as seen in Entamoeba spp. are not present. Compact, well-defined mass. Stains dark brown with iodine.
  • 91.
    intestinal & urogenitalprotozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
  • 92.
  • 93.
    Blastocystic hominis Size ShapeMotility Number of Nuclei Other Features 5-30 mm. Usual range, 8-10 mm. Spherical, oval, or ellipsoidal Nonmotile 1, usually, but 2-4 may be present. Located in "rim" of cytoplasm. In binucleated organisms, the 2 nuclei may be at opposite poles. In quadrinucleated forms, the 4 nuclei are evenly spaced around periphery of cell. Cell contains large central body, or "vacuole" with a thin band, or "rim" of cytoplasm around the periphery. Occasionally a ring of granules may be seen in cytoplasm and the cell appears to have a "beaded rim."  may predominate in heavy infections
  • 94.
  • 95.
    intestinal & urogenitalprotozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
  • 96.
    Dientamoeba fragilis MULTIPLE Stoolexaminations  [last portion of the stool evacuated]
  • 97.
    Dientamoeba fragilis NUCLEUS CYTOPLASM Species Size (Length) MotilityNumber Peripheral Chromatin Karyosomal Chromatin Appearance Inclusions Dientamoeba fragilis3 5-15 mm. Usual range, 9- 12 mm. Pseudopods are angular, serrated, or broad lobed, and hyaline, almost transparent. 2 (In approximatel y 20% of organisms only 1 nucleus is present.) Nuclei invisible in unstained preparation s. None. Large cluster of 4-8 granules. Finely, qranular. Bacteria: occasional ly red blood cells.
  • 98.
  • 99.
    Giardia lamblia  DX Microscopic examinations direct wet mount concentration techniques (cyst) permanent stain  Small bowel aspirates or String test specimens  Antigen detection methods
  • 100.
    Giardia lamblia Size (Length)Shape Motility Number of Nuclei Number of Flagella* Other Features 10-20 mm. Usual range, 12-15 mm. Pear shaped. “Falling leaf.” 2 Not visible in unstained mounts. 4 lateral. 2 ventral. 2 caudal. Sucking disk occupying 1/2-3/4 of ventral surface. Median bodies lying horizontally or obliquely in lower part of body.
  • 101.
    Giardia lamblia Size (Length)Shape Number of Nuclei Other Features 8-19 mm. Usual range. 11-12 mm. Oval or ellipsoidal. Usually 4. Not distinct in unstained preparations. Usually located at one end. Fibrils or flagella longitudinally in unstained cysts. Deep staining fibers or fibrils may be seen lying laterally or obliquely across fibrils in lower part of cyst. Cytoplasm often retracts from a portion of cell wall.
  • 102.
  • 103.
    Chilomastix mesnili Size (Length)Shape Motility Number of Nuclei Number of Flagella* Other Features 6-24 mm. Usual range, 10-15 mm. Pear shaped. Stiff, rotary. 1 Not visible in unstained mounts. 3 anterior. 1 in cytosome. Prominent cytostome extending 1/3-1/2 length of body. Spiral groove across ventral surface.
  • 104.
    Chilomastix mesnili Species Size(Length) Shape Number of Nuclei Other Features 6-10 mm. Usual range, 8-9 mm. Lemon shaped with anterior hyaline knob. 1. Not visible in unstained preparations. Cytostome with supporting fibrils. Usually visible in stained preparations.
  • 105.
  • 106.
    Trichomonas vaginalis  DX: vaginal fluid, prostatic fluid, sediments of freshly passed urine Direct wet mount Culture Immunofluorescent & EIA techniques monoclonal antibodies  Papanicolaou-stained gyne smears
  • 107.
  • 108.
    Pentatrichomonas/Trichomonas hominis Size (Length) ShapeMotility Number of Nuclei Number of Flagella* Other Features 8-20 mm. Usual range. 11- 12 mm. Pear shaped. Nervous, jerky. 1 Not visible in unstained mounts. 3-5 anterior. 1 posterior. Undulating membrane extending length of body.
  • 109.
    Trichomonas tenax  nonpathogenic occasionally recovered from the oral cavity
  • 110.
  • 111.
    Enteromonas hominis Species Size(Length) Shape Motility Number of Nuclei Number of Flagella* Other Features Enteromonas hominis 4-10 mm. Usual range, 8-9 mm. Oval. Jerky. 1 Not visible in unstained mounts. 3 anterior. 1 posterior. One side of body flattened. Posterior flagellum extends free posteriorly or laterally.
  • 112.
    Enteromonas hominis Species Size(Length) Shape Number of Nuclei Other Features 4-10 mm. Usual range, 6-8 mm. Elongated or oval. 1-4, usually 2 lying at opposite ends of cyst. Not visible in unstained mounts. Resembles E. nana cyst. Fibrils or flagella are usually not seen.
  • 113.
    Retortamonas intestinalis Species Size(Length) Shape Motility Number of Nuclei Number of Flagella* Other Features Retortamon as intestinalis 4-9 mm. Usual range, 6-7 mm. Pear shaped or oval. Jerky. 1 Not visible in unstained mounts. 1 anterior. 1 posterior. Prominent cytostome extending approximately 1/2 length of body.
  • 114.
    Retortamonas intestinalis Species Size(Length) Shape Number of Nuclei Other Features 4-9 mm. Usual range, 4-7 mm. Pear shaped or slightly lemon shaped. 1. Not visible in unstained mounts. Resembles Chilomastix cyst. Shadow outline of cytostome with supporting fibrils extends above nucleus.
  • 115.
    intestinal & urogenitalprotozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
  • 116.
  • 117.
    Balantidium coli Species SizeShape Motility Number of Nuclei Other Features Trophozoite 50-70 mm or more. Usual range, 40- 50 mm. Ovoid with tapering anterior end. Rotary, boring. 1 large, kidney shaped macronucleus. 1 small micronucleus immediately adjacent to macronucleus. Macronucleus occasionally visible in unstained preparations as hyaline mass. Body surface covered by spiral, longitudinal rows of cilia. Contractile vacuoles are present. Cyst 45-65 mm. Usual range, 50-55 mm. Spherical or oval. 1 large macronucleus visible in unstained preparations as hyaline mass. Macronucleus and contractile vacuole are visible in young cysts. In older cysts, internal structure appears granular.
  • 118.
    intestinal & urogenitalprotozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
  • 119.
  • 120.
    Isospora belli  DX UNSPORULATED OOCYSTs direct wet mounts concentration techniques acid-fast stain
  • 121.
    Isospora belli Size ShapeMotility Number of Nuclei Other Features Oocyst: 25-30 mm. Usual range, 28- 30 mm. Ellipsoidal Nonmotile Usual diagnostic stage is immature oocyst with single granular mass (zygote) within. Mature oocyst contains 2 sporocysts with 4 sporozoites each.
  • 122.
  • 123.
    Sarcocystis sp.  DX SPORULATED SPOROCYSTS direct wet mounts acid-fast stains
  • 124.
    Sarcocystis sp. Species SizeShape Motility Number of Nuclei Other Features Sporocyst1 Oval Nonmotile Mature oocysts with thin wall collapsed around 2 sporocysts or free fully mature sporocysts with 4 sporozoites inside are usually seen in feces. hominis 13-17 mm. Usual range, 14-16 mm. suihominis 11-15 mm. Usual range, 12-13 mm.
  • 125.
    Sarcocystis sp. wet mountviewed in uv microscopy. cyst in undercooked meat
  • 126.
  • 127.
    Cryptosporidium sp.  DX concentration methods  acid-fast stain: MODIFIED COLD KINYOUN METHOD deep fuscia  commercial immunofluorescent & EIA reagents
  • 128.
    Cryptosporidium sp. Species SizeShape Motility Number of Nuclei Other Features Cryptosporidium Oocyst: 3-6 mm. Usual range, 4-5 mm. Spherical or oval. Nonmotile Mature oocyst contains 4 "naked" sporozoites. No sporocysts are present.
  • 129.
  • 130.
    Cyclospera cayetanensis  DX:unsporulated oocysts  direct wet mount  trichrome stain  UV epifluorescence  modified acid-fast stain  auramine-o stain
  • 131.
    intestinal & urogenitalprotozoa Amebae Blastocystic hominis Flagellates Dientamoeba fragilis Giardia lamblia Chilomatrix mesnili Pentatrichomonas hominis Trichomonas vaginalis Other flagellates: Enteromonas hominis Retortamonas intestinalis Trichomonas tenax Ciliates Balantidium coli Coccidia Isospora belli Sarcocystis sp. Cryptosporidium sp. Cyclospera cayetanensis Microsporidia
  • 132.
  • 133.
    Microsporidia  DX  tissueexamination routine light & electron microscopy  stool specimens modified trichrome staining method (WEBER) Fluorochrome stain Uvitex 2B, Calcoflour white
  • 134.
    Microsporidia  Enterocytozoon bieneusi Light microscopy photographs of stained stool smears Quick-Hot Gram Chromotrope stain Weber stain
  • 135.
    Microsporidia  Enterocytozoon bieneusi maturespore.  double rows of polar tubule coils in cross section electron micrograph.  Encephalitozoon intestinalis spores and developing forms inside septated parasitophorous vacuole.
  • 136.
    Microsporidia Scanning electron micrographof a microsporidian spore with an extruded polar tubule inserted into a eukaryotic cell. The spore injects the infective sporoplasms through its polar tubule.